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101. Comparative cold-hardiness capacities of South American lizards in the genus Liolaemus
Warner, Jason R.
The ability to survive freezing temperatures is of paramount importance to organisms living at high elevations. Many ectothermic vertebrates have traits that allow them to cope with extreme cold, such as the ability to supercool and/or tolerate freezing of extracellular fluids. I studied several aspects of cold-hardiness of six lizard species in the genus Liolaemus (Iguania: Liolaemidae) living along a 2525-m elevation gradient (1550-4075 m) in northwestern Argentina. Specifically, I tested for supercooling, freeze tolerance, and the temperature of ice inoculation. I hypothesized that high-elevation (>3000 m) species would have greater capacities to supercool and tolerate freezing, while being more susceptible to ice inoculation, relative to their low-elevation congeners. Both conventional statistical analyses and those using independent contrasts with phylogenetic regressions revealed no correlation between supercooling capacity and elevation. Because of this, and the fact that all six species were able to supercool, this ability may be the ancestral condition for the lineage. This may have enabled independent invasions of cooler climates by members of this diverse genus. However, independent adaptation appears to explain freeze tolerance, as larger individuals were more susceptible to ice formation and high-elevation lizards survived longer during freezing events than their low-elevation congeners. Body size may partially explain freeze-tolerance capacity, as larger lizards have greater thermal inertia, which allows them to retain heat longer. Also, large-bodied Liolaemus generally occur at higher elevations. The cold-hardiness capacities characterized in this study also were correlated with seasonal low temperatures experienced in each environment. These results may explain the success of Liolaemus, which has numerous independent origins of species living in cold climates. Future studies should consider cold hardiness in a more holistic context of the biology of these lizards (alongside behavior, morphology, and biochemistry) and include comparisons among more species, including those from high latitudes, to further test cold hardiness as an adaptation to cold environments.
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102. DNA packaging by terminase large subunit in methanophage PG
Dang, Thomas
Recent studies have shown bacteriophages infect living organisms from all three domains of life, however, not much is known about phages that infect the Archaea. Of the approximate 50 reported archaeal phages, PG is one of only three known viruses that infects methanogens and falls within the order of tailed-bacteriophages, Caudovirales. Tailed bacteriophages have differing DNA replication strategies that is reflected by the various terminal chromosomal ends created by the terminase large subunit. Although PG's replication and packaging process is unknown, bioinformatic studies of PG's terminase large subunit could help identify potential DNA packaging strategies. Comparative analysis between other phages demonstrate that they cluster together according to the type of terminal ends they create. PG was shown to cluster with termini short direct terminal repeats and cohesive ends. Studying the terminase large subunit of PG could lead to a better understanding of its replication strategy, genetic history, and increase our understanding of viruses in general.
103. Intrahierarchical vocal communication of male domestic guinea pigs
Haley, Thomas Eugene
Male guinea pig vocalizations emitted in response to conspecific aggression were tape recorded. This was done by using an experimental method through which vocalizations of a starved individual could be recorded as it protected a single piece of food from the remainder of a four member hierarchy. In order to quantify results "silent," vocal units (A-type, B-type, squeal, shriek, and E-type) and behavioural categories (undisturbed, approached, and harassed) were defined using non-overlapping characteristics. The vocal responses were graded in unit type and in physical properties (maximum frequency and duration) as conspecific aggression increased. These graded responses were utilized differently by individuals of different hierarchical status. Positionally related vocal differences were explained in terms of positionally related ambivalences during the food defense period.
104. The physical properties of saline-based density gradient solutes
Milgrom, Etan
Certain physical properties (e.g. density, viscosity and refractive index) of saline-based solutions of the density gradient solutes sucrose, Ficoll, metrizamide, Ludox, Percoll and CsCl were determined at 4°C and 22°C. The values obtained were compared with the same solutes prepared in water. Data were used in a series of experimental studies in which the sedimentation rates of nylon beads and human red blood cells at unit gravity and in centrifugal fields were followed visually. Experimental results were compared with theoretical predictions of behavior.
105. From trees to octocorals: the role of phenotypic integration, shading, and self-thinning in underwater animals forests
Nelson, Hannah
While it is known that octocorals and trees share many similarities in form and function due to their sessile, modular nature, and octocoral communities create dense canopies termed "animal forests," there has been little quantification of whether these two groups are shaped by the same ecological and evolutionary processes. This thesis is comprised of two different studies that take classic concepts developed in the terrestrial plant literature (e.g. phenotypic integration, self-thinning, shading) and examines how well they can be applied in octocorals. In Chapter 2, I examined the extent to which variation in octocoral morphology is constrained by covariation among traits (i.e. integration), including how traits scale with overall colony size (i.e. allometry) by comparing five morphological traits among and within eight species of the common Caribbean octocoral Eunicea. I found that polyp, branch, and colony level traits covary with one another, but the pattern of trait covariation depends on whether covariation is investigated at an intra- or interspecific scale. Species with more branches tended to have shorter, thinner branches. This pattern of integration has been well established for many higher plants, suggesting modularity may have given rise to similar constraints in both groups. This study highlights the role of covariation among traits in constraining the morphology of modular organisms. In Chapter 3, I investigated whether self-thinning models can be applied to mixed-species octocoral assemblages by exploiting natural variation in the density of octocorals at two shallow reefs in St. John, U.S. Virgin Islands. I found a negative power relationship between mean colony width and the density of colonies, which suggests that mixed-species octocoral communities experience density-dependent mortality due to competition (e.g. self-thinning) for space, waterborne resources, and/or light. Light was reduced next to octocorals, but shading could not account for differences in spacing among species. A substantial fraction (50-73%) of the variation in average colony size was not explained by variation in octocoral density. These results suggest that self-thinning models can be applied to mixed-species assemblages, but the predictive power of these size-density relationships may be weakened by asymmetries in competitive abilities or resource use among species.
106. Effects of Environmental Change on the Eco-Evolutionary Dynamics of Species in Natural Microcosm Communities
Holdridge, Erica
Natural communities are expected to undergo shifts in composition and species interactions as a result of environmental change, such as changes to regional temperature regimes and increased nutrient input into ecosystems. Predicting how communities will respond is complicated by three factors: non-additive effects of multiple stressors, differences in response among trophic levels, and trait evolution leading to adaptation. This study addressed all three of these factors using a natural microcosm community. The purple pitcher plant, Sarracenia purpurea, is a carnivorous plant that retains rainwater inside of its cup-shaped leaves. Within this water, an inquiline community of microbes and invertebrate larvae forms. I used a subset of this community consisting of a single protist species, Colpidium sp., and a community of bacteria to explore how the interaction between temperature and increased nutrient input affects different trophic levels ecologically and evolutionarily. I factorially manipulated temperature and nutrient input and maintained these conditions for 28 days (~150 protozoan generations). I then performed reciprocal transplants to each treatment, resulting in four common garden environments to test how evolutionary effects may depend upon ecological context. I found that historic nutrient input levels affected two protist traits - cell size and peak density - but the magnitude of these effects depended on the contemporary environment. The combined effects of historical temperature and nutrients did not differ significantly from the expected additive effect. Protist and bacterial abundances differed in their response to treatments, with protist abundance affected by both historical and contemporary environments while bacterial abundance was only affected by contemporary environment. Finally, bacterial community composition was affected by treatments both directly and indirectly, through their effects on protist traits and abundance. The results of this study show that the ways in which communities respond to environmental change can differ in light of evolutionary responses, the trophic levels being considered, and additivity of multiple stressors. The future success of management and conservation of natural systems rests upon the best possible understanding of not only the ecological implications but also, and perhaps even more importantly, the evolutionary consequences of a changing abiotic environment.
107. Algorithm selected transcription factors used to initiate reprogramming and enhanced differentiation in somatic and pluripotent stem cells
Solomon, Aryeh
Direct reprogramming offers a promising alternative method in producing cells for regenerative medical applications. However, a major obstacle in this process is the selection of transcription factors best suited to initiate reprogramming. By comparing differential gene transcription levels across numerous tissue samples, the Core Expression Module Analysis (CEMA) developed by the Lowry lab identified and compiled compact sets of genes unique to a given cell lineage. With additional gene ontology analysis, a list of 3-10 transcription factor (TF) were generated for each cell type. We hypothesized that ectopic expression of these selected TF can induce direct reprogramming in terminally differentiated somatic cells and direct differentiation and maturation in pluripotent stem cells. To test this, we incorporated these lineage conversion factors into a YFP marker gene containing lentiviral polycistronic cassette that uses an rtTA Inducible Tet-On promoter system. For the direct reprogramming project, two plasmids were generated to produce two distinct cell lineages; tissue endothelial cells and mature neural progenitor cells. Fibroblast were infected with rtTA and TF cassette lentivirus and using puromycin and neomycin selection, with YFP expression, we were able to positively identify infected cells. Treatment with doxycycline over an extended period resulted in morphological changes appearing in a small subset of infected cells, with ectopic and endogenous up regulation of key genes measured via IF staining and qRT-PCR. Using a similar protocol with ESCs/iPSC, infected YFP positive colonies ectopically expressing early neural progenitor cell transcription factors were selected and differentiated towards the neural fate using lab established methods. After sorting and analyzing cells with FACS and qRT-PCR, cells that had received continuous doxycycline treatment while remaining YFP positive demonstrated a decrease in Lin28B expression, a microRNA preventing cell maturation, with cell and colony morphology comparative to fetal NPC samples. With additional refinement to CEMA and the reprogramming protocols, mature cells from all lineages may be generated, with applications ranging from disease modeling to cell replacement therapy.
108. Effects of off-road vehicles on vertebrates and habitat quality in the Mojave Desert
Shargo, Eric S.
The use of off-road vehicles (ORVs) in the California desert has become increasingly popular during the past 15 years. This study was designed to assess the impact of this activity on habitat quality and the small vertebrate fauna in the creosote bush community of the Mojave Desert. Two ORV-frequented sites in each of two localities and four control areas (one for each experimental site) were compared. Vegetation, small mammals and reptiles were sampled during late spring. Density and diversity of mammalian and reptilian vertebrates were significantly lower in all ORV-used areas, as was habitat quality. Moderately used areas contained 49% as much perennial plant cover, supporting 81% as many individuals and 76% as many species of vertebrates as the control areas. Where vehicular activity is concentrated, areas become denuded, supporting little or no animal life or vegetation. These "pit areas" averaged 5.3% as much plant cover as controls, while the number of individuals and species was 12.4% and 34.5% of the controls, respectively. These results indicate that ORVs have a detrimental effect on the desert habitat proportional to the degree of usage.
109. Quantitative evaluation of characterized alpha and beta glycosidase effects on sea urchin embryo cellular interactions
Idoni, Brian Anthony
Using the NIH designated sea urchin embryo model, we are studying the molecular basis of a set of cellular interactions that have interested investigators for over a century, archenteron organization/elongation/attachment to the blastocoel roof. Here we evaluate the effects of four commercially available glycosidases: alpha and beta amylase and alpha and beta glucosidase, that we independently characterized, on embryo development. In quantitative dose-response experiments we show an I-50 of 10-30 units/ml, in artificial sea water or low calcium artificial sea water, of beta amylase and alpha glucosidase in inhibiting archenteron organization/elongation/attachment to the blastocoel roof in living 48 hr Lytechinus pictus gastrula embryos using a microplate assay that allows quantitative assessment of embryo morphological characteristics. All concentrations tested (up to 5000 units/ml) of alpha amylase and beta glucosidase had no significant effects on archenteron morphology. All experiments involved assessing 3600-5600 embryos in at least 144 replicate wells for each enzyme. The differences in archenteron XVll morphology compared with controls were significant with p values of less than 0.05, while any differences observed with the ineffective enzymes had p values of greater than 0.05, using two-tailed t-tests. All enzymes used were tested for their activity on known substrates, and found to be active, and specific unit activities were established. Product inhibition studies suggested that the effective enzymes acted by their specific glycosidase activities and polyacrylamide gel electrophoresis suggested that there was no detectible protease activity in the enzyme samples. The results suggest that glycans are involved in these cellular interactions and we discuss possible conformations of these glycans.
110. Immunofluorescence localization of extracellular matrix component (s) in two species of sea urchin
Alikani, Mina
An indirect immunofluorescence method was used to localize and trace the expression of an aggregation-promoting factor(s) (S-2) in the early stage embryos (1, 2, 4, 8, and 16-cell) of two species of sea urchin, Strongylocentrotus purpuratus and Lytechinus pictus. The S-2 antigen(s) was isolated from the supernatant of calcium-magnesium-free sea water dissociated S. purpuratus blastulae. Whole embryos from which fertilization membranes had been removed were incubated with purified anti-S-2 antibody (AS-2). Bound AS-2 was labeled with a second antibody (goat anti-rabbit IgG) that had been conjugated with fluorescein isothiocyanate. Immunofluorescence was assessed and photographed using a fluorescence microscope. The fertilized eggs and early stage embryos of both species of urchin displayed peripheral fluorescence. The antibody did not bind to the surfaces of unfertilized eggs nor did it penetrate the hardened fertilization membrane of embryos. Embryos incubated with preimmune rabbit serum showed no staining. (See more in text.)
111. Identification of sigma / anti-sigma factor regulatory pairs in the cyanobacterium Nostoc punctiforme
Amir Ali, Arsalan
Nostoc punctiforme is a filamentous cyanobacterium that has an immense potential to be used as a platform to produce biofuels and other high-value compounds. Their ability to synthesize lipid droplets and other valuable metabolites while eliminating the requirement for land also considerably increases their commercial prospective. N. punctiforme grows using plant-like oxygenic photosynthesis and can also differentiate into spore-like akinetes, nitrogen-fixing heterocysts, or motile hormogonia in response to stress. This unique ability to adapt to environmental stresses by differentiation also makes them a valuable model for studying cellular differentiation. A subset of the genes involved in stress responses are hypothesized to be regulated by the twelve alternative sigma factor subunits of RNA polymerase, which were previously identified in the genome (along with the housekeeping sigma, SigA). The sigma factors are known to be post translationally regulated by the anti-sigma factors, that are hypothesized to sequester the sigma factors until stress induces their release. However, unlike the sigma factors, the anti-sigma factors are well conserved at the primary sequence level. Thus, not much is known about anti-sigma factors in general or about the specific sigma/anti-sigma interactions in N. punctiforme. Seven putative anti-sigma factors were identified in N. punctiforme by sequence homology. We screened for the interaction of all thirteen sigma factors with these putative anti-sigma factors using a bacterial 2-hybrid assay (BacTH). Each of the anti-sigma factors interacted with two or more sigma factors in the BacTH analysis, but no interactions were found with group 3 sigma factors or the highly divergent sigma factor SigI. Twenty-three positive interactions were identified using the BacTH system, which were then quantified by using beta-galactosidase assays. We then tried to confirm twelve of the interactions showing the highest beta-galactosidase activity by using GST-pulldown assays and Western blotting. Two novel sigma/anti-sigma interacting pairs were identified by this study. The sigma factor SigB2 was shown to interact with the anti-sigma factor SapB2 and the sigma factor SigD was shown to interact with the anti-sigma factor SapD. These findings will help increase our understanding of complex regulatory processes that occur at the transcriptional level during cellular differentiation, and following stresses associated with production of high-value compounds in N. punctiforme.
112. Effects of α-mannosidase in sea urchin embryo gastrulation
Aleksanyan, Heghush
The sea urchin embryo is a NIH designated model system to study mechanisms that may be involved in human health and disease. In order to determine if mannose residues are involved in sea urchin embryo cellular interactions at gastrulation, 6324 Lytechinus pictus embryos were incubated with varying concentrations of independently characterized (assayed for purity and activity) α-mannosidase for 24 hours using a quantitative microplate assay. The results showed a variety of morphological deformations even for α-mannosidase activity of as low as 0.06 U/ml. These effects did not occur using heat treated (that we call denatured) α-mannosidase. Thousands of replicates were statistically evaluated and the results suggest that α1-2-, α1-3-, and/or α1-6-linked mannose residues may play an important role during sea urchin gastrulation. Additionally, formaldehyde fixed, 48 hour old Lytechinus pictus embryos were microdissected to attempt to identify a specific site of α-mannosidase action within the embryo. The procedure involved separation of archenterons from the blastocoel roofs. Microdissected control groups maintained the adhering properties of the cells located on the tip of the archenteron to the roof of the blastocoel when the dissected pieces were placed back together. But, in these small sample preliminary experiments embryo pieces that were incubated with α-mannosidase lost their adhesive properties suggesting the tip of the archenteron and roof of the blastocoel are possible sites for the effect of alpha-mannosidase. Both the microplate and microdissection approaches used here independently suggest a role for D-(+)-mannose residues in sea urchin gastrula adhesive interactions.
113. Mitochondrial enzyme activity during the cell cycle of Polytomella agilis
Patterson, Nancy Diana
A population of Polytomella agilis was synchronized with hydroxyurea, which inhibits cells at the onset of DNA synthesis (the G1/S border). To gain information on the sites of mitochondrial enzyme synthesis (succinic dehydrogenase, NADH dehydrogenase and cytochrome c oxidase} or proteins involved in their assembly or incorporation into membranes, the effects of chloramphenicol (an inhibitor of mitochondrial protein synthesis) and cycloheximide, an inhibitor of extramitochondrial protein synthesis) on the specific activities of these enzymes were studied, through the cell cycle. In untreated cells, peak patterns of enzyme synthesis were observed. A mitochondrial site of succinic dehydrogenase is suggested since its activity declined in chloramphenicol-treated cells during the first cycle. Cycloheximide had no effect on its activity. NADH dehydrogenase is apparently synthesized under mitochondrial control since chloramphenicol results in decreased activity in the first cycle. In the subsequent cell cycle little, if any, activity was detected. Cycloheximide affected the pattern of activity during the cell cycle, suggesting the inhibition of some cytoplasmic components involved in the regulation of the assembly, membrane-integration or activity of the enzyme. In the presence of chloramphenicol, cytochrome c oxidase activity did not decline until the second cell cycle. Enzyme activity was not inhibited in either cycle by cycloheximide. This suggests a mitochondrial site of synthesis. The diminished activities of both cytochrome c oxidase and NADH dehydrogenase in the second cycle following the addition of chloramphenicol suggests that the mitochondrion of P. agilis arises by growth and division. Cell division was not inhibited by chloramphenicol after the first cell cycle, suggesting that a completely functional mitochondrion is not an absolute requirement for cell division. Cell division does depend on cytoribosomal protein synthesis, since it is blocked by cycloheximide.
114. Brain-Derived Neurotrophic Factor and Exercise-Induced Neuroprotection in the spastic Han-Wistar rat, a model of ataxia
Van Kummer, Brooke
Moderate aerobic exercise has been shown to enhance motor skills and protect the nervous system from injury and neurodegenerative diseases, like ataxia. Our lab uses the spastic Han-Wistar rat as a model of ataxia. Mutant rats develop fore limb tremor, hind limb rigidity and have a significantly decreased life span. Our lab has previously shown that moderate treadmill exercise reduced Purkinje cell degeneration in the cerebellum, delayed the onset of visible motor dysfunction, and significantly increased life span. This suggested that aerobic exercise ameliorates the observed cerebellar dysfunction in the sHW rat. The focus of this thesis is to understand the mechanism of how exercise may mediate neuroprotection by studying Brain-Derived Neurotrophic Factor (BDNF). BDNF is known to increase after exercise and has been reported to be crucial in Purkinje cell survival through activation of various signaling cascades. The actions of BDNF are mediated by the TrkB receptor. To link BDNF to exercise-induced neuroprotection, 28-day-old mutant and normal rats received surgically implanted Alzet osmotic pumps that chronically infused the TrkB antagonist K252a or the control vehicle (1% DMSO) into the third ventricle. During the 28 day infusion rats were subjected to five-day-per-week exercise regimens on a motorized treadmill. Mutant rats receiving K252a exhibited a 21.4% loss in Purkinje cell survival compared to control mutants. Cerebellar TrkB expression was evaluated using non-drug treated 30-day-old rats that were subjected to various regimens: 7 days of running, 30 days of running, or non-running controls for each age. Running animals expressed up to 3× more TrkB than sedentary animals. BDNF was quantified via Sandwich ELISA, and cerebellar expression was found to be 26.6% greater in mutant rats on 7-day treadmill exercise regimen compared with those subjected to 30 days of treadmill exercise. In normal animals, BDNF expression in acute runners was 10% greater than in chronic runners. Rats did not express significantly more BDNF than sedentary controls in either exercise duration group. In summary, these results suggest the neuroprotective benefits of aerobic exercise can be blocked using the K252a TrkB antagonist, and that BDNF activation of the TrkB receptor signaling pathway is involved in mediating exercise-induced neuroprotection.
115. Evolution of temperature-dependent sprint performance in nocturnal and secondarily diurnal geckos
Luken, Alissa
Temperature has a profound influence on most aspects of an ectotherm's life. Most of the world's 1500+ gecko species are nocturnal and therefore presumably active at body temperatures (Tbs) that are suboptimal for performance. However, several clades and numerous gecko species have independently reverted to diurnality and are therefore active at warmer Tbs allowing for comparisons of temperature-dependent sprint performance between nocturnal and secondarily diurnal species. Following the hotter-is-better hypothesis, which states that warm-adapted species will have better performance than cold-adapted species, we hypothesized that diurnal geckos would have faster maximum speeds than nocturnal species at their thermal optimum. Likewise, the optimality hypothesis predicts co-adaptation between environmental temperatures and optimal Tbs for performance. Thus, we hypothesized that diurnal geckos would sprint faster at warmer Tbs and slower at cooler Tbs relative to nocturnal geckos. We tested these hypotheses by comparing the temperature-dependent sprint performance of 27 gecko species over a range of ecologically relevant Tbs to determine how activity time (nocturnal/diurnal) affected sprint performance. Conventional statistics and those that accounted for the genetic distances among the species were used to compare the sprint performance of nocturnal and diurnal geckos. Additionally, two clades with closely related nocturnal and secondarily diurnal species were compared using conventional statistics. Nocturnal geckos had slower sprint speeds than diurnal geckos overall, which is consistent with the hotter-is-better hypothesis. In accord with the optimality hypothesis, diurnal species sprinted relatively faster at higher Tbs and slower at cooler Tbs relative to nocturnal species suggesting that there has been an evolutionary shift in the temperature-dependent sprint performance of diurnal species with the higher activity temperatures associated with diurnality. Phylogenetic history did not influence temperature-dependent sprint performance because diurnality has evolved independently several times among the species included in this study and clades with diurnal species are spread throughout the gekkotan tree. Our study provides insight into the ways in which an evolutionary change in activity time can affect the evolution of temperature-dependent sprint performance in geckos and illustrates the likely constraints on performance for other cold-adapted nocturnal species.
116. TCR engineering of Natural Killer Cells for melanoma treatment
Yan, Jun
Melanoma is the most dangerous form of skin cancer because it is resistant to most common cancer treatments, such as chemotherapy and radiation. In 2014, a novel immunotherapy based on the blockage of the PD-1/PDL-1 signaling pathway was approved for malignant melanoma. However, this treatment is only effective with patients with high-level expression of PD-1/PDL-1. Therefore, developing an alternative melanoma treatment is an urgent mission. The goal of this proof of principle study was to augment the ability of natural killer (NK) cells to effectively target and kill melanoma cells by engineering them to express a T cell receptor (TCR) specific for melanoma antigen Mart-1. TCRs are not normally expressed on NK cells, although the protein components necessary for TCR signaling exist in these cells. By using this approach, the NK and T cell anti-melanoma properties would be consolidated within one cell type. Furthermore, as melanoma cells generally use different strategies to suppress T and NK cells, the TCR expressing NK cells could compensate for the lack of T cell activity when these cells are absent or viii immunosuppressed. In this study, human embryonic stem cells (hESC) were used as the source of genetically modified NKs, as they are relatively easy to genetically manipulate, expand to clinically relevant quantities, and to differentiate into NK cells that are phenotypically and functionally indistinguishable from their blood derived counterparts. TCRs require the CD3 complex of proteins for transport to the cell surface and proper signaling. To provide all the elements needed to support normal TCR function, the coding sequences for the CD3 γ, ζ, δ and ε chains, along with the Mart-1-specific TCR genes, were introduced into H1 hESC. Individual transgenic hESC lines were selected, the expressions of both Mart-1 TCR and CD3 on their surfaces were confirmed, and then they were differentiated into TCR/CD3 NK cells. In the future, Mart-1-specific TCR and CD3 expressing cells will be used in in vitro and in vivo experiments to establish that the presence of the Mart-1 TCR should endow these transgenic NKs with a higher anti- melanoma cytotoxic potential. Importantly, this technology could be adapted and expanded for treatment of other tumors and malignancies.
117. Gene expression profilling in a family with a novel form of beta-thalassemia
Taghavifar, Forough
The genetic causes of β-thalassemia are largely well described. However, the disease is very heterogeneous at both the molecular and clinical levels. Studying the transcriptome profiles of β-thalassemia patients, especially individuals who carry novel mutations in the β-globin gene (HBB), may improve our understanding of the heterogeneity and molecular mechanisms of the disease and its possible treatment. Here, I characterized members of a family with β-thalassemia using whole genome expression analysis. I report a novel mutation in the exon 1 region of HBB (HBB:c.51C>T) that was associated with an unexpected phenotype of β-thalassemia in a heterozygote who also carries a typical β-thalassemia allele. I analyzed effects of the novel mutation at the transcriptome level by RT-qPCR and high-throughput RNA sequencing using an Illumina Hiseq2500 system. The results revealed that the novel mutation creates a cryptic donor splice site in the HBB, which causes alternative splicing from the site and down-regulates (~0.7) expression of the β-globin. Gene expression profiling analysis showed that there were more than 300 differentially expressed genes (DEGs) in β-thalassemic blood. The DEGs were enriched in pathways that are directly or indirectly related to β-thalassemia such as hemopoiesis, heme biosynthesis, response to oxidative stress, inflammatory responses, immune responses, controlling of Circadian rhythms, apoptosis, and other cellular activities. It was possible to compare these findings with published results of RNA-seq analysis of sickle cell disease and KLF1-null anemia, and recognize similarities and differences in their transcriptional expression patterns. While many DEGs involved in response to hemolysis, iron homeostasis, and anemia were in common between these three types of anemias, over 200 DEGs were unique to β-thalassemia. Although this study was limited by the small sample size of the patients, it provides a wealth of data on β-thalassemia because it is the first broad investigation of blood cell gene expression in this disease, and gives us novel insight that can be used in drug discovery to identify novel therapeutic approaches for the disease.
118. The multicomponent nature of teratoma cell adhesion factor
Meyer, James T.
The complex nature of the teratoma cell adhesion system has been demonstrated. Fractionation of crude ascites fluid on a DEAE cellulose ion exchange column made it possible to show that two or more components are involved in teratoma adhesion factor (TAF) activity. Glycoproteins in fractionated ascites fluid were localized on polyacrylamide gels. The possible role of these glycoproteins in teratoma cell adhesion and current hypotheses on the mechanism of carbohydrate involvement in intercellular adhesion are discussed.
119. The effects of mechanical habitat conversion on eight Southern California rodent species
Carnes, Michael Andrew
TIhe effects of mechanical habitat conversion on eight species of rodents were studied in the San Gabriel Mountains north of Pacoima, Los Angeles County, California, from November 1973 to December 1974. Two kinds of 'type-conversion', fuel break grassland and rejuvenated chaparral were live-trapped and comparisons were made between them and with immature and mature chaparral. Openland-preferring species were favored on type-conversions and species composition in rejuvenated chaparral were similar to that in immature chaparral. Rodent densities were approximately the same on both type-conversions and in both cases were greater than that in immature chaparral and less than that in mature chaparral. Rodent species diversity was high in rejuvenated chaparral and immature chaparral relative to grassland and mature chaparral. Rodent diversity was not correlated with density. (See more in text.)
120. An inquiry into high revertant frequency in Neurospora crassa
Thorne, Richard Charles
The serine auxotroph, ser (JBM 4-13), appeared to be highly unstable and displayed a highly variable revertant frequency of 1 x 10-6 to 2.8 x 10-1. The mutant's characteristics were examined with respect to the properties of unstable genes predicted by current models explaining genetic instability. After a sustained effort, no auxotrophic strain could be isolated with a non-variable revertant frequency. Time and temperature were tested for their ability to alter revertant frequency. A revertant was isolated for comparison with the auxotroph and with a nutritionally wild-type strain for the effects of time and serine concentration on growth. Both the auxotroph and the wild-type strain were exposed to UV irradiation to determine dose response curves as well as the effect of UV irradiation on the revertant frequency in the auxotroph. Mixed cultures of known ratios of revertants to auxotrophs were grown at various serine concentrations to determine the effect on the revertrant frequency. The results of these studies indicate that it is unlikely that the high revertant frequency is due to extrinsic mechanisms such as controlling elements, episome-like factors, mutator genes, virus particles or defective repair mechanisms. Genetic instability due to an intrinsic factor has not been excluded by the present study; however, considerable evidence was obtained indicating that the high revertant frequency is due to a selective advantage of the revertant over the auxotroph in mixed cultures grown on low concentrations of serine supplement. The selective advantage of the revertant over the auxotroph was not apparent at higher concentrations of serine. The variability in the revertant frequency between cultures could be a result of differences in the time at which the reversion event occurred in the individual cultures.
121. Optimization of cerebral organoid differentiation from different lines of human pluripotent stem cells
Vishlaghi, Neda
The cerebral cortex is a specialized region of the brain responsible for higher neurological functions such as sensory processing, learning and memory, cognition, and abstract thinking. This region is disproportionally enlarged in humans and other primates, and thought to be one of the factors that allows for our enhanced intellectual capacities compared to lower species such as rodents. Hence, rodent models are not ideal tools for studying the primate-specific features that drive brain growth and complexity. Several studies have shown that human embryonic (hESCs) and induced pluripotent stem cells (iPSCs) have the ability to form three-dimensional cortex-like structures called organoids that recapitulate distinct aspects of human brain development. However, our and others' results indicate that cortical organoid formation is variable and often not reproducible, differing between hESC and hIPSC lines and maintenance methods. One of the biggest influences that we have found is growth under mouse embryo fibroblast (MEF)-dependent vs. independent conditions. Maintenance with MEFs permitted robust and reproducible organoid differentiation from either hESC or hIPSC. Importantly, cells initially grown under feeder-free conditions can be entrained to produce organoids when passaged with MEFs. Moreover, the positive effects of MEFs on organoid differentiation require sustained MEF-dependent culture. Organoids differentiations have been checked by immunostaining for cortex main markers. The transcriptome of cells cultured under different conditions, analyzed by RNA-seq to identify hallmarks of successful cortical organoid differentiation. A better understanding of how these different maintenance conditions influence organoid development will help predict experimental outcomes and improve the application of these methods for modeling normal and diseased human brain development.
122. Proximate causes of variation in signaling among three sister species of Gryllus crickets
Gutierrez, Nicholas
Mating signals play an important role in both mate selection and species recognition. Acoustic mating signals have been shaped under various evolutionary constraints with remarkable malleability in response to variable conditions. These same traits have been shown to serve as indicators of species identity and mate quality. But how will sexually selected signal traits be expressed as environmental conditions change and as species diverge? Using three closely related Gryllus crickets (Gryllus lineaticeps, G. personatus, and G. sp15/"G. staccato"), I assessed how components of male song (chirp rate, pulse rate, frequency, and pulses per chirp) and morphology responded to varying environmental dietary conditions within and among genetically structured family groups (sibships). Experimental diets had some effects on morphology with differences in mass and pronotum width in some species, but the diets had no effects on body condition or components of calling song, save one trait in one species. In contrast, sibship consistently affected chirp rate and frequency but did not affect pulse rate and pulses per chirp either within or among species. Interestingly, the dynamic song traits, chirp rate and pulses per chirp, exhibited broad variation in all species, as expected, but responded differently from each other to sibship. Likewise, the static song traits, frequency and pulse rate, exhibited limited within-species variation, as expected, but had different contrasting effects of sibship. Overall genetic variation in song traits was most strongly associated with species divergence, and then with dam but not sire effects within species. Overall, these results suggest conservation across species of the major sources of within-species song variation; that is, divergence among species appears to be associated with changes in the average values of song traits, especially static ones, but not consistent divergence in the sources of within-species variation.
123. Geometric Morphometric Analysis of the Humerus in New and Old world Vultures
Holmes, Jacob
The vulture guild is comprised of two distinct groups, Old and New World, that provide a unique insight into how morphology varies among convergent species. All vultures are considered to be large birds of prey that utilize a style of flight called thermal soaring to search and feed primarily on already dead animal flesh. Even though this flight style is exhibited among all 23 species, slight variations in their skeletal morphology may relate to their differences in ecology. I hypothesized that vulture humeral morphology varies in relation to these organisms' habitat, average body mass, courtship displays, and migration capabilities. To address this hypothesis, I used three-dimensional geometric morphometrics to measure the overall shape differences of vulture humeri. Computer models of each bone were created to observe the crucial skeletal features that relate to muscular functions and were analyzed using discriminant function analyses. Humeral morphology was found to vary most by habitat preference and body mass. Vultures that inhabit forested areas have humeri that exhibit features that suggest increased flapping flight compared to those in open and mountainous regions. The results for the heaviest species allude to these birds enhancing their wingbeats in ways other than humeral morphology
124. Effects of NRG1 and NGF on neural crest and Schwann cell migration
Cornejo, Martha
Schwann cells are vital to sustaining the neurons and making myelin in the Peripheral Nervous System, and play a key role in some neurodegenerative illnesses such as the Charcot-Marie-Tooth disease. The Schwann cell is derived from a transient population of cells, the neural crest, in the early stages of embryogenesis of vertebrate organisms. Understanding the factors influencing NC and SC migration in reaching their final destinations during development would have fundamental applications to help neuropathies and degenerative diseases from the Peripheral Nervous System. Here we have investigated the effect on chemotaxis and chemokinesis that the protein ligands NRG1 and NGF have on chick Neural Crest cells and mouse Schwann cells. The findings from this research shows that NRG1 and NGF play a chemoattractant role in the migratory guidance for the NC and the SC.
125. Comparative ecomorphology of cormorants (Phalacrocoracidae) from three mediterranean climate regions
Harell, Allan
Cormorants (Phalacrocoracidae) are seabirds that depend on their hind limbs and beak to pursue and capture their prey underwater, rendering these apparatuses critical for their survival. Although the group is cosmopolitan, cormorant communities of three or more sympatric species are only found in mediterranean climate regions characterized by nutrient rich currents. I used morphological parameters (from museum specimens) related to feeding and locomotion in cormorants from three such regions- California, central Chile, and western S. Africa-to assess convergent functional patterns. Morphological designs were explained by feeding category in 8 of 9 focal species, and a higher resemblance in design pattern was seen between California and S. Africa. Discriminant Function Analysis (DFA) was used to create three feeding categories-generalist (G), pelagic (P), and benthic (B)-based on ecological and dietary information from three Californian feeding types (Phalacrocorax auritus, P. penicillatus, and, P. pelagicus) and three S. African types (P. carbo lucidus, P. capensis, and P. coronatus). All six species were classified to their consensus groups with probabilities ≥85%. Chilean species, P. olivaceous and P. bougainvillii, were assigned by the DFA to their predicted groups G and P, respectively with probabilities ≥82% and 100%, but P. gaimardi was not assigned to its predicted group B, but instead was assigned to P, suggesting a more pelagic feeding ecology. Proportional knee and tarsus lengths, and beak depth were found to be the strongest determinants of feeding category and may be useful tools in predicting feeding ecologies of other hind limb pursuit diving waterbirds.
126. Identifying spatial patterns of gene flow in the Blainville's horned lizard (Phrynosoma blainvillii) in urban Southern California
Wenner, Sarah
Landscape genetics combines principles of landscape ecology and population genetics to determine the effect of landscape characteristics on the functional connectivity of populations. Functional connectivity, the degree to which organisms move across the landscape, provides a proxy for gene flow and is limited by characteristics of the species and the environment. Identifying the landscape variables that promote or impede functional connectivity can provide a comprehensive look at the fine-scale genetic effects of natural and anthropogenic fragmentation and landscape change. Here, I examine population genetic structure and functional connectivity among populations of an ecological specialist, the Blainville's horned lizard (Phrynosoma blainvillii), in a fragmented landscape in southern California. I studied horned lizards from Los Angeles and Ventura counties at the boundary between evolutionary lineages. Using RADseq SNP data, I assessed population genetic structure, as inferred from Bayesian assignment tests. I interpreted the results in the context of broad-scale phylogeography, as well as at a fine-scale among habitat fragments in the highly urbanized region. Based on the ecological requirements of P. blainvillii, I developed a suite of hypotheses to test the at-site landscape variables that promote production of horned lizards and the between-site landscape variables that impede gene flow. I used gravity models to test which of the hypotheses best explained the estimates of functional connectivity. My results show strong population genetic structure across small habitat fragments in the study area, indicating an effect of habitat fragmentation. Further, the gravity models show that elements of topography, microclimate, soil and vegetation types, fire history, and anthropogenic development are important components of functional connectivity in this part of the species' range. Together, my results highlight the importance of natural and anthropogenic landscape factors at- and between-sites, species-specific characteristics, and habitat fragmentation on the patterns of genetic structure and connectivity of P. blainvillii in Southern California.
127. Electrophoretic extraction of adhesion-inhibiting protein
Capelle, Christopher B.
An aggregation inhibitory factor (AIF) has been extracted from mouse ascites teratoma cells that retards adhesion of cultured teratoma cells of the same cell line. Preliminary characterization of AIF on polyacrylamide gels suggests that AIF is a carbohydrate-free protein composed of four subunits. Extraction of AIF from ascites teratoma cells was accomplished without significant loss of viability by a novel technique involving the application of an electric field to large numbers of whole cells suspended in a hypertonic electrode buffer. Assumptions were made regarding the force developed by this field on charged transmembrane proteins in order to measure the potential of this technique for extracting proteins of possible significance in adhesion. In tests of adhesion, AIF consistently inhibited auto-aggregation of cultured teratoma cells after 5, 10, 15, and 30 minutes of incubation with cells. Furthermore, a reduced concentration of AIF resulted in a corresponding decrease in inhibition, suggesting a concentration dependent action. Further work with AIF may reveal if this factor plays a role in the adhesion mechanism of cultured teratoma cells.
128. Improved Plasmid-Based Muscle Gene Therapy
Modarresi, Yasamine
Muscular dystrophies are a group of inherited disorders characterized by progressive muscle weakness, and degeneration. One approach to treatment would be the replacement of the deficient protein via gene therapy. For effective gene therapy, both efficiency of gene delivery and stable expression of the transferred gene are important factors. Our goal was to determine which of the following mammalian expression vectors would be more useful (more stable) for muscle gene therapy; pAcGFP1-C1 and pEPito. We were also interested in switching/substituting both vectors' CMV promoter/enhancer region with the muscle specific promoter, Desmin (DES), to increase their stability for muscle gene therapy. This was accomplished by transfecting C2C12 myotubes with the aforementioned vectors. Both vectors showed relatively continuous GFP expression. Myotubes transfected with pEPito continued to express GFP till day 8. Cells transfected with pACGFP1-C1 also showed continuous GFP expression till day 6. Our results show that both vectors are promising candidates for gene therapy in muscle cells as they maintained stable gene expression of the GFP reporter gene for at least 6 days. Further studies should be done in order to determine the maximum duration that the myotubes would be able to maintain the plasmids and show continuous expression. Future studies could be done to assess stability of these vectors in mice which may lead to future gene therapy trials for muscle disorders and improving gene therapy strategies for other disorders.
129. The Influence of Nutrient and Sediment Loading on Multiple Facets of Coral Functionality in Branching Coral Species
Becker, Danielle
Coral reefs, some of the most diverse ecosystems in the world, face increasing pressures from global and local-scale anthropogenic stressors. Nutrient enrichment and sediment loading could make corals more vulnerable to global climate change by suppressing calcification and reducing their photosynthetic performance. Yet, some nutrient-enriched reef environments have exhibited higher coral growth rates, coral productivity, and coral thermal performance. To properly inform management in coastal coral reef ecosystems, a better understanding of warming and land-based inputs (i.e. sedimentation rates and nutrient loading) on coral reef ecosystems is necessary. This thesis is comprised of two studies that address the ecological and physiological influence of land-based inputs on two branching coral species to thermal stress. The first study tested how a natural nutrient and sedimentation gradient affected multiple facets of coral functionality, including algal endosymbiont and coral host response variables, holobiont metabolic responses, and percent cover of Pocillopora acuta colonies on the north shore fringing reefs in Mo'orea, French Polynesia. Algal endosymbiont % nitrogen content, algal endosymbiont densities, and total chlorophyll a content increased with nutrient input, while algal endosymbiont nitrogen content cell-1 decreased, likely representing competition among algal endosymbionts. Nutrient and sediment loading decreased coral metabolic responses to thermal stress in terms of their thermal performance and metabolic rate processes, and the percent cover of P. acuta colonies decreased by nearly two orders of magnitude along the nutrient gradient. The second study focused on the direct effects of nutrient enrichment on the thermal performance of corals on a fore reef in Mo'orea, French Polynesia. In this study, I measured how chronic nutrient enrichment affected coral physiology, including endosymbiont and coral host response variables, and holobiont metabolic responses of Pocillopora spp. colonies in Mo'orea, French Polynesia. I experimentally enriched corals with nitrogen and phosphorus combined for 15 months on the oligotrophic fore reef of Mo'orea. I first characterized symbiont and coral physiological traits due to enrichment and second used thermal performance curves to quantify the relationship between metabolic rates and temperature for experimentally enriched and control coral colonies. I found that the nutrient-enriched coral's endosymbiont densities, total chlorophyll a content, and tissue biomass were 54 %, 38 %, and 22 % higher, respectively, while algal endosymbiont nitrogen content cell-1 was 74 % lower relative to the control colonies. In addition, thermal performance metrics indicated that the maximal performance rate for gross photosynthesis was 29 % higher in enriched colonies, while the rate of metabolism at a reference temperature (26.8 °C) for gross photosynthesis was 33% higher compared to the control colonies. These findings illustrate that the relationship between land-based inputs, such as nutrient and sediment loading, and coral physiology is dependent on the local environmental context. Together these results help us better understand how local-scale stressors influence coral functionality and thermal performance in varying reef habitats. By understanding the complexities of coral-nutrient dynamics, research-based reef management approaches can be taken, particularly on where to mediate the influx of land-based inputs into coastal coral reef ecosystems.
130. The distribution and physiology Sporosarcina ureae
Pregerson, Bernardine S.
Fifty-one strains of Sporosarcina ureae were isolated from 198 soils by plating diluted soil samples onto a complex agar medium containing 1. 5% filter sterilized urea. S. ureae was found to be widely distributed the United States and in various parts of the world. The primary habitat of this organism appears to be concentrated in certain urban soils closely associated with the activities of man and especially of dogs. However, a variety of soils cannot support growth of S. ureae. This organism was not found in soils obtained from zoos, corrals, stables, grazing lands, deserts, or from cultivated fields. Nor was it found in salt water samples. Sixty-one strains of this organism were tested for their ability to utilize 45 different organic compounds as sources of nitrogen, carbon and energv, or as growth factor requirements. The vast majority of strains required only ammonium salts for a nitrogen source but all of the organisms showed limited ability to metabolize carbon sources. Sugars are not utilized or are utilized very poorly, and glucose could not be attacked by any strain. Acetate (or butyrate) and glutamate are apparently universal substrates. The overall metabolic pattern was one of random variation but there was a clustering of strains according to growth factor requirements. The pattern that emerged was that of gradation in growth factor requirements ranging from organisms which required no growth supplements to those organisms whose nutritional requirements are complex. In general, the ecology, morphology, cultural characteristics, or biochemical reactions revealed no consistent differences that could be correlated with nutritional differences . Nor did this study reveal a constellation of differential characters that would permit recognition of additional species of Sporosarcina. The results did suggest that the leading criterion for species identification and classification may be the nutritional differences shown by this organism.
131. A mathematical model for kelp growth
Anderson, Bruce Nicholas
A deterministic model for growth of species of giant kelp, Macrocystis, of the southern California coast was developed and analyzed using FORTRAN. Factors modifying only light and temperature are required to simulate empirically derived growth curves. The model was used to determine optimum time of the year for new growth which was found to be early June, and optimum time of the year for regeneration of harvested canopy, found to be early July. Critical values for turbidity which will prevent new growth for any given kelp bed depth were determined. At twenty meters depth, reduction of transmitted light to 82% per meter caused by introduction of particles, e.g. sewage effluent, into coastal waters reduces growth time by only 20%, but when transmitted light is below 76% per meter growth time is zero. Possibilities for further development of the model by the quantification of additional variables are discussed.
132. The use of the wire hang test and a novel genotyping method to further determine the M712T mouse model
Qubrosi, Mirey
Hereditary Inclusion Body Myopathy (HIBM) is a disorder caused by a mutation in the GNE gene. Although there are 60 known mutations, this research study focused on the M712T mutation. The mutation causes complications in the production of sialic acid, and produces skeletal muscle wasting. The next step in the development of a cure is to produce an animal model. The purpose of this research study was to use the wire hang test to see if mice heterozygous and homozygous for the disorder developed strength deficits. I also tested a new method of genotyping that is faster and cheaper to perform than testing by restriction analysis. I used an animal model with the M712T mutation that was between 12-16 months of age. I tested their grip strength through the wire hang test. Also, I used an advanced rapid method of genotyping the mouse, which included designing two sets of primers specific for the mice GNE gene to determine which mutation the mice contained. The wire-hang test showed that the wild-type mice (WT) performed much better than both the heterozygote (HT) and mutants (MT). On average as a group, mutants performed the worst (32.62 s), followed by heterozygotes (47.35 s), and then wild-type mice (89.33 s). Moreover, the results show that there is a definite difference between the wild-type and mutant mice; both the mutant and heterozygous mice showed similar levels of strength, which indicates that even heterozygotes for the disorder suffer losses in skeletal muscle. Furthermore, my novel genotyping method also showed that it can reliably determine the genotype of the mice. This method is cheaper and quicker to perform.
133. Cyanide resistant respiration in Bacillus cereus
Vanesian, Michael Ara
Cyanide exerts its toxic effects by inhibiting cellular respiration, specifically by inhibiting cytochrome oxidase. In many bacteria, algae, protozoa and plants cyanide resistance has been demonstrated and is usually associated with an alternate electron transport pathway. A strain of Bacillus cereus was isolated using an elective culture containing cyanide. In 1 mM cyanide, the population growth rate was one-third that of control cells grown in the same medium lacking cyanide. The rate of oxygen consumption was approximately 50% of controls. The addition of 2 mM cyanide to respiring control cells resulted in virtually complete inhibition of oxygen uptake, but only 40% inhibition in cyanide-grown cells. No qualitative or quantitative differences in the cytochromes of the electron transport systems were noted between control and cyanide-grown cells in reduced-minus oxidixed difference spectra. In CO-difference spectra, control cells had one absorption peak at 430 nm. CO difference spectra of cyanide-grown cells lacked this peak, but showed peaks at 500 and 550 nm. The absence of the peak at 430 nm in cyanide-grown cells suggests either that alternate oxidases are produced or that cyanide alters the cytochrome a. (See more in text.)
134. Concanavalin a derivatives and cell agglutination
Knapp, Loren Walter
Agglutination of S-180 ascites tumor cells mediated by Concanavalin A (ConA) and biologically active succinyl and acetyl ConA derivatives was quantitatively measured and compared, using an electronic particle counter assay. ConA causes agglutination of tumor cells in vitro. Compared to ConA, succinyl ConA (SConA) and acetyl ConA (AConA) cause a substantial reduction in agglutination. Agglutination mediated by ConA, SConA and AConA was affected by pretreatment of the tumor cells with cytochalasin B (cyto B) and colchicine. Cyto B causes inhibition of cellular microfilaments while colchicine causes inhibition of microtubules. ConA mediated agglutination of cyto B pretreated tumor cells was greater than ConA mediated agglutination of untreated cells. Agglutination mediated by SConA and AConA contrasted sharply from the ConA profile. SConA and AConA mediated agglutination of cyto B pretreated tumor cells was less than SConA and AConA mediated agglutination of untreated cells. ConA mediated agglutination of tumor cells was not substantially changed by colchicine pretreatment; however, reduced agglutination of colchicine pretreated tumor cells with SConA and AConA was observed. Inhibition of protein synthesis with cycloheximide does not appear to affect agglutination, while reduction in temperature decreases agglutination. This decrease in agglutination may be related to reduced ConA receptor site clustering which is necessary for effective intercellular connections. These results indicate that derivatized ConA may have a reduced binding affinity for ConA receptor sites on the cell surface. This may reduce the clustering of ConA receptor sites and therefore reduce agglutination of these cells. Although microfilaments and microtubules have not been shown as fine structural elements in these cells, the results suggest that ConA receptor sites on the cell surface may be associated with microfilaments and microtubules.
135. Oxidative stress in the cerebellum of the spastic Han-Wistar (sHW) mutant rat, a model of Ataxic Neurodegeneration
Oo, Sanda
Neurodegeneration is the progressive breakdown of neurons resulting in impairment of cognitive functions and motor abilities, and has been attributed to such neurological diseases as Alzheimer's, Parkinson's, Huntington's, amyotrophic lateral sclerosis (ALS), and, important in this study, ataxia. Oxidative and nitrosative stress occurs when the damaging action of the reactive oxygen species (ROS) and reactive nitrogen species (RNS) targets key cellular molecules such as proteins, DNA, and RNA. This phenomenon is usually kept in check by the body's natural antioxidant defense system. However, under certain biomolecular or environmental factors, this defense system can be overwhelmed and shuts down, leading to a degenerative state. Growing data from experimental models and human brain studies suggest that oxidative stress plays an important role in degeneration in neurologic diseases. To determine whether ROS or RNS is occurring in the brains of patients suffering from neurodegenerative diseases, various oxidative stress markers have been utilized, including 8-hydroxydeoxyguanosine (8-OHdG) formed during DNA damage, and nitrotyrosine (NT) a marker of oxidative damage to proteins. My project utilized the mutant spastic Han-Wistar (sHW) rat, an acknowledged model of ataxia. Previous morphological examination of mutant animals indicated progressive losses of cerebellar Purkinje cells correlated with ataxia-like symptoms. Immunohistochemical analyses of expression of 8-OHdG showed increased staining intensity in the Purkinje cells of 55 day old mutant rats compared to littermate controls; while expression of NT was not seen in either mutant or normal siblings. Cerebellar sagittal sections of 50-55 day old mutant and normal sHW rats were stained with 8-OHdG antibody. The sections showed uniform distribution of 8-OHdG in both normal and mutant cerebellums but the mutant sHW rat displayed significantly higher staining compared to normal sHW rats. Specifically, mutant sHW rats showed much greater staining intensity within the Purkinje cells and within the deep cerebellar nuclei. Cerebellar sagittal sections were stained with NT antibody from 50-55 day old mutant and normal sHW rats. The immunohistochemistry staining of NT in the cerebellum showed very light, yet similar staining intensities within both the normal and mutant Purkinje cells. My findings suggest that sHW rat Purkinje cells undergo oxidative stress at the level of DNA only.
136. Bryophytes of the Russian Peak area Siskiyou County, California
McGrew, Karen C.
Russian Peak, at an elevation of 8200 feet, is situ­ated in the Salmon Mountains, a north-south trending range centrally located within the Klamath Region of northwest­ern California. The Salmon Mountains are drained by the Scott River on the east and by the Salmon River on the west; both rivers course northwesterly to discharge into the Klamath River. The Klamath Region is a geologically old and structurally complex series of ranges which grade into the Coast Ranges on the west and into the Cascade Mountains on the northeast. Russian Peak is located in Klamath National Forest, within Siskiyou County, approximately eight miles west­southwest of the town of Callahan. Access to the area is via Forest Service roads and·unimproved roads off of State Highway 3, which runs northward out of Weaverville. Selection of Russian Peak and its environs as a study area was based on several factors: the lack of bryological collections in the area (Personal communica­tion, Norris, 1973); the presence of relict conifer species as described by Sawyer and Thornburgh (1971); and the presence of a diverse, largely circumboreal moss population on the Marble Mountains, located approximately fifteen miles to the northwest (Spjut, 1971). The objec­tive of the following study was to identify bryophyte species within the area and to determine if the findings would lend further support to the concept of a refugium or "central" area, as described by Whittaker(l961), with­in the Klamath Region. Collections were made from around five lakes, includ­ing their headwaters and drainages, during the summers of 1973 and 1974. A total of 786 specimens were collected, representing 152 species. Collections were made at elevations between 5500 feet and 7200 feet in the following drainages: 1)	Sugar Lake and Sugar Creek, August, 1973; 2)	South Sugar Lake, August, 1974 (with D.H. Norris); 3)	Bingham Lake, September, 1974 (with D.H. Norris); 4)	Russian Lake, September, 1973; 5)	Waterdog Lake, Lower Russian Lake and South Russian Creek, September, 1973. Bryophytes were collected along paths, in drainages, and from around the lakes. Effort was made to include special habitats, such as springs, marshes, serpentine outcrops, exposed granite walls, rock crevices, and areas adjacent to perennial snow patches. Each collection was identified, checked with speci­mens from the cryptogamic collection at Humboldt State University, and verified by D. H. Norris. Voucher speci­mens are on deposit at the Humboldt State University Her­barium (HSC). Collection numbers preceded by the prefix "N" are those of D. H. Norris; all others are those of the author.
137. Early secondary succession: a study of clearcut areas in a mixed conifer forest.
Ziser, Brian.
Clearcut logging of numerous small stands of trees over the last fifteen years has provided suitable areas for the study of early secondary plant succession on the western slope of the central Sierra Nevada mountains. Familiarity with early successional patterns in these areas is essential in the development of appropriate silvicultural practices for the maintenance of viable, productive forests, as well as aiding in effective management of wildlife resources. Clearcut logging has been widely practiced and researched in the coastal Redwood forests of Northern California, and in the Douglas Fir dominated forests of Oregon and Washington. However, as indicated by the available literature, no significant research on secondary succession following clearcutting has been conducted in the Sierra Nevada mountains. This project was undertaken to provide information about early regrowth patterns on these clearcut areas.
138. Immatures of Simulium of the Kings River.
Yoshimura, Glenn.
Six species of Simulium larvae found in the Kings River near Piedra, Fresno County, California, are described. The black fly larvae and pupae were reared in the laboratory for taxonomic association of the immature instars, the pupae and the adults. The differential morphology of S. argus Williston, S . aureum Fries, S. canadense Hearle, S. virgatum Coquillett, and S. vittatum Zetterstedt is presented in greater detail than ever before, with the S. arcticum Malloch larva being described for the first time.
139. Nitidulid beetle flight activity correlations with some meteorological factors.
Willson, Ruth.
Nitidulid beetles have been recognized as major pests of figs In California since 1900 (Simmons et al., 1931). Early workers were concerned primarily with Carpophllus hemlpterus (Linnaeus), the dried-fruit beetle, as the principal nitidulid pest (Smith et al., I927). More recent studies have shown four other pestiferous nitidulids: Carpophllus freemanl Dobson, Carpophllus mutllatus Erichson, Urophorus humeralls (Fabrlcius), the pineapple beetle, and Haptoncus luteolus Erichson. Of these species, C. mutllatus was found in 56^ the Calimyrna figs in one sample from Madera County, California (Smilanick and Ehler, 1975)*
140. The effects of grazing on Dipodomys nitratoides exilis in an alkali sink community.
Warner, Donald Remi.
The natural history of the Fresno kangaroo rat, Dipodomys nitratoides exilis, in the San Joaquin Valley's alkali sink habitat was first described by Culbertson (1946). His study included little information on the interspecific interactions with other rodent species or the intraspecific interactions of the Fresno kangaroo rat. Little attention has been given to its ecology since. Instead the emphasis has been placed on the controversy of whether there do or do not exist three distinct subspecies of Dipodomys nitratoides.
141. Partitioning the effects of habitat fragmentation on rodent species richness in Southern California
Johnson, Alex M.
Habitat fragmentation plays a major role in species extinction and the loss of biodiversity around the globe. When fragmentation occurs, the initial loss of overall habitat alone causes species extirpation. However, species that survive this initial loss and persist in the remaining small fragments of habitat continue to experience changes that may lead to their eventual extirpation. Previous research has determined that species richness in habitat fragments is affected by a number of characteristics. These include fragment age, size, and isolation, edge effects, vegetation coverage, habitat heterogeneity, and matrix content. Although most studies focused on one or a few of these characteristics, multiple characteristics work together to affect species richness, showing that the effects of habitat fragmentation are complex. The goal of my study was to partition the complex effects of habitat fragmentation by determining the direct, indirect, and cumulative effects of multiple habitat fragment characteristics on rodent species richness. In 2013, I determined rodent species richness in 25 habitat fragments within a suburban landscape of Thousand Oaks, California. In addition, I measured the following characteristics for each fragment: fragment age, area, isolation, shrub coverage, habitat heterogeneity, perimeter/area ratio, and percent non-urban buffer. Path Analysis was used to test the hypothesized model which described the direct, indirect, and cumulative effect of each habitat fragment characteristic on rodent species richness. Habitat heterogeneity had the greatest direct and total effect on rodent species richness. In addition, fragment area had the greatest indirect effect on rodent species richness through its influence on habitat heterogeneity, suggesting that large fragments containing the greatest diversity of habitats will support the most species. Overall, the path model explained 67% of the variation in rodent species richness among habitat fragments. From a conservation and management standpoint my findings suggest that the most important fragments to protect are the largest and most habitat diverse.
142. Evolution of primordial glial cells and glial genes in invertebrates
Salvo, Mari Ekimyan
Glial cells were first observed in the nervous system of vertebrates as neuronal supportive cells. In recent years they have been credited for not only supporting and sustaining neuronal cells but for playing roles in testis development, neuron migration and the formation of blood brain barriers, to name a few. The glial cells increasing diversity in higher complex nervous systems, has also contributed to the belief that the emergence of glial cells gave rise to the evolution of the central nervous system. Here we examine the possibility of this theory through the study and distinguishing of glial cells in the phylum Cnidaria and the phylum Platyhelminthes, which are considered to possess one of the simplest central nervous systems in metazoans. Through the analysis of Golgi staining, In situ hybridization, transmission electron microscopy (TEM) and immunofluorescence staining we were able to distinguish glial-like cells in the metazoans Nematostella vectensis, Dugesia tigrina, Dugesia dorotocephala and Carybdea species. These glial-like cells appear to have an equivalent relationship with neuronal cells as the glial cells do in the nervous system of vertebrates.
143. Effect of a predator on species diversity : a study under conditions of continuous cultivation
Sweeney, Daniel Stephen
A study was made of the food web formed from a protozcan, Teterhymena pyriformis, two bacteria. Pseudomonas fluorescens and Escherichia coli, and a glucose minimal medium in chemostat culture. An attempt was made to further substantiate the idea that species diversity within a community can be dependent upon predation. The system was divided into simpler parts, first by demonstrating that each bacterial species in pure culture would achieve steady-state growth on the substrate, whereas the protozoa could not. In batch and steady-state growth studies of pure culture of the two bacterial species, P. fluorescens was the more successful. In competition studies between P fluorescens and E. coli in the chemostat. P. fluorescens population level was 100-to 1000-fold greater. Interestingly, E. coli's steady-state population increased approximately 50-fold when P. fluorescens was added. (See more in text.)
144. The influence of the structure of an invasive alga on the distribution of temperate reef fishes
Srednick, Griffin
Algae are key foundation species within temperate subtidal reef systems as their productivity and structure can transform the physical composition of the reef landscape. However, previous assessments of the effects of variation in the algal community on fishes have been mainly focused on changes in the density and identity of algae, and often ignore how the unique structure provided by each alga contributes to such patterns. Recent warming of ocean temperatures has led to decreases in M. pyrifera and increases in the invasive alga Sargassum horneri along the southern California coast. While both species often form mono-specific stands, the physical structure ("architecture") of S. horneri is different from that of M. pyrifera and other native algae with respect to height and shape. The goal of this study was to evaluate the effects of S. horneri on the temperate reef fish community at Santa Catalina Island, California, with emphasis on how unique differences in the physical structure among algae influence the spatial distribution and behavior of fishes. Observational surveys were performed five times across a one-year period at seven sites, to examine distribution patterns of fishes and how they related to differences in the algal community at the scale of sites spread over several km. Additionally, I developed a method to predict volume and surface-area from measurements of height for a number of algae to explore how overall differences in algal structure contribute to variation in fish distribution. In combination, I used surveys of the algal and fish communities to assess the relative importance of algal identity versus size on distribution patterns. The spatial distribution and composition of the fish community varied across the five sampling periods and was partially explained by variation in the density and identity of algae. In particular, I found that S. horneri and M. pyrifera were important predictors of variation in the fish assemblage. However, variation in the fish community was better explained by algal size (i.e., height, volume, or surface area) than by algal density and identity. To better understand the patterns observed at the scale of sites spread over several km, I performed a series of experiments and surveys of individual algae to test the relative importance of algal height versus identity on variation in abundance and behavior of fishes. In three separate experiments, I deployed isolated treatments of algae to examine differences in fish abundance and behavior among various species and size of algae. I found that fish abundance did not vary among algal species but rather among sizes of algae, even when compared across species. In addition, differences in the frequency of feeding and resting behavior were related to size of algae but did not vary among algal species. Field surveys of individual algae showed similar patterns: fish abundance and richness were positively related to algal size. However, differences in the fish community among algal species indicate that certain species (e.g., M. pyrifera) may be more important than others in providing habitat for fishes. These results suggest that the habitat structure provided by algae is important for fishes, but patterns of fish distribution may be more related to size than species of algae. In addition, fishes do not appear to be negatively related to the structure provided by S. horneri, yet a reduction in the tallest form of algal structure on reefs, M. pyrifera, may have important impacts on the distribution and composition of the fish community. The findings of this study highlight the importance of considering how morphological differences among algae influence the fish community.
145. Relationship between intercellular adhesiveness and glutamine synthetase specific activity in synchronized cultures of mouse teratoma cells
Grodin, Martin S.
In cultures of mouse teratoma, cells synchronized with thymidine (3mM) and colcemid (0.5 mcg/ml), cellular adhesiveness displayed an oscillatory pattern with peaks of glutamine synthtase (GS} specific activity occurring lust prior to peaks of adhesiveness. The results demonstrate a correlation between GS specific activity and cellular adhesiveness. Based upon previous work which implies L.-glutamine in intercellular adhesion, it is not unreasonable to speculate that GS specific activity and cellular adhesiveness may be causally related.
146. Avian communities in two urban environment and a natural habitat in Simi Valley, Southern California
Kessler, Margret Elisabeth
Bird communities of two urban habitats of different ages and a natural habitat in Simi Valley, California were compared to determine whether the urban habitats were ecologically equivalent to the original habitat of an area as it existed prior to urbanization. The natural study site was carefully selected so as to closely resemble the original habitat of the urban study areas. The natural habitat supported the highest number of species (31), whereas the older urban area only supported 10 and the younger area only 8 species. Accordingly, species diversity indices were highest for the natural area (H' = 3.3, J' = .96) and much lower for the urban areas (H' = 1.7 and 1.5, J' = .74 and .72). Analysis of population densities showed the highest number of individuals of resident species to be in the older urban area (721/100 ha) closely followed by the natural area with 696/100 ha, and with only 277/100 ha in the younger urban area. The large difference between the two urban study areas was probably due to a difference in age of the developments (birds are still invading the younger, less well vegetated area), and closer spacing of houses (less space and more disturbance to bird activities in the younger area). In general, granivorous and water-dependent birds were the most successful species in invading the urban habitats, whereas insectivorous birds tree cavity nesters, ground-dwellers and birds of prey were generally absent. For those species that are able to adjust to new nesting habits and additional disturbance factors, the urban habitat offers increased food and water and many perching and nesting sites. Apparently urban habitats are capable of supporting high densities of a limited number of species, but invite only a small fraction of the bird species previously occupying the area. They are, therefore, not equivalent to the original habitat present in an area before urbanization.
147. Role if HIF1α in human embryonic stem cell differentiation
Wisidagama, Dona Roonalika
Hypoxia stimulates human embryonic stem cell (hESC) self-renewal. It has been shown that hypoxia inducible factor 2α (HIF2α) up regulates OCT4 (POU5F1) transcription, a pluripotency marker gene. Hypoxia also increases the efficiency of reprogramming of differentiated cells to a pluripotent-like state. Combined, these results suggest that hypoxia, or more specifically HIF, would impair the purposeful differentiation of pluripotent stem cells. Here, we unexpectedly show that HIF expression also promotes hESC differentiation. shRNA knockdown of HIF1α or its binding partner, aryl hydrocarbon receptor nuclear translocator (ARNT), inhibits the early induction of developmental genes GATA2, GATA3, GATA6, HAND1 and HOXB3 during retinoic acid -induced differentiation. shRNA knockdown of HIF1α or ARNT also impaired embryoid body formation, one of the commonlyused in vitro differentiation assays in the human embryonic stem cell world. Conversely, ectopic expression of a stabilized HIF1α mutant, HIF1α (P402A/P564A), in normoxia leads to enhanced developmental genes GATA2, GATA3, GATA6, HAND1 and HOXB3 induction during retinoic acid-induced differentiation. Ectopic expression of HIF1α (P402A/P564A) also increased the chromatin modifier gene JMJD3 induction during retinoic acid induced differentiation in normoxia. Combined, the data show a previously unanticipated role for HIF in promoting early hESC differentiation in hypoxia.
148. Tissue fractionations in reorienting density gradients
Gross, Dennis Michael
In view of their greatly increased sample capacity and optimized internal geometry, zonal rotors have become increasingly popular instruments for quantitative centrifugal fractionations of animal and plant tissues on density gradients. Nearly all zonal rotors presently in use require a rotating- seal assembly which necessitates dynamic loading and unloading of the rotor. As a consequence of certain disadvantages which accompany dynamic unloading and numerous difficulties encountered in the use of a rotating seal, the possibility of employing simpler, seal-less zonal rotors to effect similar quantitative biological separations was investigated. Three seal-less zonal rotors were tested which were loaded either statically or dynamically but were always statically unloaded. The rotors were designed so that the liquid density gradient underwent a stable reorientation between a vertical and radial array during acceleration and deceleration. Rotor performance was evaluated using variety of techniques and the rotors then applied to a series of quantitative tissue fractionations. These included: (l) the separation of whole homogenates of liver into fractions containing nuclei, mitochondria, lysosomes, rnicrosomes and soluble constituents; (2) the separation of discrete populations of liver nuclei on the basis of size differences; (3) the separation of lysosomes and peroxisomes using a replenished gradient technique; (4) the isolation of protein bodies from seeds and (5) the separation of intact and broken chloroplasts from plant leaves. In addition to providing a simpler means for separating cellular components by zonal centrifugation, the reorienting gradient rotors were found to provide a number of interesting and important advantages over zonal rotors with rotating seals.
149. Food-dependent plasticity in C. elegans stress-induced sleep is mediated by TOR-FOXA and TGF-beta signaling
Goetting, Desiree
Plasticity in the drive to sleep represents an important strategy of coordinating and prioritizing competing behavioral drives to offset some of the vulnerabilities associated with sleep, such as the inability to forage. Despite the identification of various neuropeptides and hormones implicated in the dual control of sleep drive and appetite, the mechanism underlying the sleep-suppressing effect of starvation is poorly understood. The nematode C. elegans has recently been identified as a useful model for the dissection of sleep function and evolution, as animals experience a primitive sleep-like state that is triggered by conditions that cause cellular damage. This stress-induced sleep (SIS) appears to be beneficial under certain conditions, as sleepless mutant animals are impaired for survival following noxious heat exposure. Here we show that food deprivation potently suppresses SIS, an effect enhanced at high population density. We present evidence that food deprivation reduces the need to sleep, protecting against the lethality associated with defective SIS. Additionally, we find that SIS is regulated by both TOR and TGF-β nutrient signaling pathways, thus identifying mechanisms coordinating sleep drive with internal and external indicators of food availability.
150. Fanconi Anemia Complementary Group I Gene is Regulated by Sp1 and Elk-1 Transcription Factor Binding SItes
Long, Gavin
Fanconi Anemia Complementary Group I Gene Is Regulated by SP1 and Elk-1 Transcription Factor Binding Sites By Gavin Long Fanconi Anemia Complementary Group I gene (FANCI) plays a role in disorders affecting bone marrow, resulting in anemia, leukopenia, thrombopenia, chromosome instability, and susceptibility to several types of cancer. The FANCI gene produces a protein crucial to the structure and function of a protein complex that repairs double-strand breaks in DNA and interstrand DNA cross-links. The putative promoter region of the human FANCI gene was identified and amplified from genomic DNA. Progressive truncations of the putative promoter were paired with a luciferase reporter vector and assayed for relative gene expression. The greatest relative expression indicates a positive regulatory element(s) likely to drive transcription between the -70bp and +52bp region flanking the Genbank-assigned transcription start site (TSS). Three TSS were experimentally found by 5'RACE thirty or more base pairs downstream of the Genbank-assigned TSS. Computer modeling predicted multiple consensus transcription factor binding sites including Sp1 and ELK-1 within this region, and these were found to be conserved through evolution via multiple sequence alignment of nineteen species. The consensus transcription factor binding sites predicted and evolutionarily conserved were specifically deleted using PCR by exclusion, and relative luciferase activity identified the sequences contributing to the regulation of the FANCI gene. Taken together, these results indicate the FANCI gene is regulated by a dispersed promoter containing two Sp1 sites and an Elk-1 site essential for maximal transcriptional expression. Understanding dysregulation patterns of FANCI can help to develop better treatments and cures for diseases related to Dysregulation of the FANCI gene.
151. Recruitment, growth rates, planktonic larval duration, and behavior of the young-of-the-year of giant sea bass, Stereolepis gigas, off southern California
Benseman, Stephanie
Little is known about the life history of Stereolepis gigas due to the over exploitation of their fishery in the early 1900's, and depressed populations have prevented much research. A completed life history information on an ecologically, and once economically, important species such as the Giant Sea Bass (GSB) is critical for both the continued successful management of that fishery and a baseline for successful scientific studies. Therefore, the goals of this study include 1) determining distribution and general ecology for the YOY of S. gigas populations in the wild, 2) estimating growth rates, based on site aggregations in the wild, and otoliths analysis in the lab, and finally 3) confirming pelagic larval duration and the general temporal scale of their spawning period. One hundred and fifty SCUBA surveys were conducted to census waters with sandy benthos across southern California from 2013-2016. I documented early life ecology, including behaviors such as the "kelping" mimickery, cruising, & resting, as well as their active predator avoidance of burying and their diet of mysids based on field observations & confirmed through gut content analysis. I uncovered a specific distribution pattern spatially along the coast adjacent to underwater canyons, and temporally from July through February. This study was also able to establish a growth rate for the YOY, finally determined their planktonic larval duration around 24 days, and the morphological color changes from black to orange. This study is the first of its kind to examine the YOY of this endangered species making it a key component to their life history, and a baseline for future work on S. gigas and similar long-lived and slow-growing species.
152. Transcriptional regulation of the Slit2 gene core promoter
Saunders, Jacquelyn
The Slit2 gene, located on chromosome 4p15, codes for the production of a membrane bound and secreted glycoprotein. The Slit2 protein has been found to behave as a chemorepellent, allowing for the migration of neural crest cells and inhibiting tumor metastasis. The role of Slit2 in neural development and neoplastic growth and migration has been well characterized in a number of studies, and more recently a possible role in the mediation of the HIV virus has been proposed. However, the genetic mechanisms underlying regulation of the Slit2 gene have not been well studied. Here we identify the core promoter of Slit2 by analysis of its transcriptional activity, the trans-acting factors that contribute to this transcriptional activity, and use bioinformatics to determine the sequence homology for the Slit2 promoter region among a number of species. Data indicate that the core promoter of Slit2 lies within the -420bp promoter fragment in relation to the start site of transcription. Transcriptional analysis suggests the most important region within the Slit2 core promoter lies between -420bp and -305bp and that at least three consensus sequences for transcription factor binding sites within this region contribute to the transcriptional activity of the gene. Bioinformatics analysis of this region shows that it is highly conserved over twenty-one species, and that the DNA sequence follows an expected pattern of divergence through evolution.
153. Extranuclear DNA in Polytomella Parva
Darnall, Karen B.
When DNA was extracted from the phytoflagellate P. parva, and subjected to electrophoresis, a prominent low molecular weight band appeared at a position corresponding to 3.7 kb. The DNA was shown to be associated with mitochondrial membrane fragments and most likely exists as mitochondrial plasmids, common to many plants and fungi. The DNA molecule appears to be linear, and its susceptibility to exonuclease digestion indicates that there are no telomeres present. A Southern blot hybridization was performed with an enzymatic labeling system. The fragment hybridized with itself, but not with DNA in other portions of the gel, suggesting that the sequence of the element is not present in the larger DNA fragments.
154. The role of life-history traits, tradeoffs, and habitat in the rarity of Santa Monica Mountains Dudleya species (Crassulaceae)
Dorsey, Ann
In this study, life-history traits, tradeoffs in those traits, and habitat characteristics of rare and common species were compared in an attempt to explain differences in species distributions. The nine Dudleya species occurring in and around the Santa Monica Mountains were studied. Five are rare narrow endemics with small localized ranges, one is rare with an intermediate range, and three are common with broader ranges. Life-history traits were studied in wild populations and in plants grown from wild collected seeds. Habitat characteristics were recorded in two or three populations of each study species. Differences that could explain why the rare species have smaller distributions than the common species were found in aspects of growth, reproduction, and habitat specialization. In regard to life-history traits and tradeoffs, rare species grew to a smaller size and reproduced earlier than common species. The small body-size of the rare species was correlated with smaller reproductive outputs than the larger-bodied common species. The rare species also tended to have lower seedling survival. Reproductive output and survival affect population size, persistence, and dispersal, all of which affect species distributions. The habitat requirements of the study species differed in terms of co-occurring vegetation, geology, and microclimate, with the rare species being more restricted compared to the common species. To further understand how habitat plays a role in limiting species distributions, the nine species were grown in an inland garden and in a coastal garden. Plants of all species grown in the inland garden were smaller in size than those in the coastal garden. Moreover, the growth disparity between plants in the two gardens was greatest for the rare species. The rare species have a lower tolerance for hot dry conditions compared to the common species. In the Santa Monica Mountains region, the habitat conditions required by the rare species are not as prevalent as those of the common species. Differences in life-histories constrained by tradeoffs affect the prevalence of the species, as well as specialization on rare habitats.
155. Therapeutic response of GBM stem-like cells and the GBM cell of origin
Faal, Tannaz
Glioblastoma Multiforme (GBM) is the most common and aggressive type of adult brain cancer that offers only one to two years of survival and results in the growth of brain tumors. GBM was studied by a group of researchers called The Cancer Genome Atlas (TCGA). This group identified four distinct types of GBM that differ from each other based on unique genetic mutations, rate of tumor growth, and response to treatment. Some cancer cells have stem cell properties such as the ability to give rise to multiple cell types and maintenance of self-renewal, which may advance the rate of tumor growth. It is believed that this cancer stem cell population infers resistance to radiation and Temozolomide (TMZ) chemotherapeutic treatment in GBM. TMZ eradicates cells by alkylating their DNA to cause cell death. Working with primary patient derived GBM stem-like cell cultures, we show that the cultures separate into different response groups that will be used in later studies to classify the cultures into TCGA subgroups. Additionally, we also show that expression of the DNA repair enzyme, MGMT, which removes TMZ induced DNA alkylation, infers GBM culture resistance to TMZ. Though much work has been conducted on the genetic anomalies prevalent and efficacy of treatment of GBM, little is known about the cell of origin in GBM. In fact, the cell of origin for GBM has not yet been identified. The main purpose of the second portion of this research project is to determine the role genetic mutations in the tumor suppressor gene, PTEN, and the oncogenic gene, KRAS, in specific brain cells and their potential to produce GBM tumors in mouse brains. We hypothesize that loss of PTEN and over expression of KRAS in NG2, which is a proteoglycan found on neural progenitor cells, positive mouse cells will result in tumor formation. A CRE-LOX breeding system was used to successfully generate genetic mutations in the mice. We have successfully bred mice that express Pten and contain Kras genetic mutations. We are currently conducting the final genetic cross to determine whether tumors will form from the NG2 positive cells. We have successfully identified groups of our GBM neursophere cultures that differ from each other based on their response to treatment. This data will be used when we begin to classify our cultures into one of the four TCGA GBM subgroups. Additionally, identifying the cell type of origin for GBM will also provide important insight on how to combat this cancer to develop more targeted cancer therapy in the future.
156. Processes affecting the grazing, distribution, behavior, and condition of herbivorous fishes on coral reefs
Tootell, Jesse Scott
Herbivory is a critical process in coral reef systems, which are among the most functionally diverse and productive ecosystems on earth. As coral reefs progressively endure both increasing frequencies and intensities of anthropogenic and natural disturbances, herbivory is arguably more important now than ever. The most critical events in terms of coral reef function are those that result in drastic loss of coral cover, which in turn can lead to phase shift in the benthic community from coral to macroalgal dominance. Herbivores can remove algae, facilitate coral recruitment, and ultimately contribute to the recovery of coral systems. Consequently, a priority of research concerning herbivore-mediated resilience and recovery of coral reefs is to understand the processes that influence herbivory, especially on compromised coral reefs where herbivory may be most important. This research may include herbivore responses to disturbance and benthic shifts, positive feedbacks between herbivores and the environment, and processes or interactions that can reduce herbivory. However, many of these topics are not well understood. To address the relative lack of studies concerning these subjects, this study focused on several aspects of the relationships between herbivorous fishes, reef structure, and algal resources on Moorea, French Polynesia (where all research for this study was performed). The first part of this study evaluated the effects of algal resources on the distribution, behavior and condition of herbivorous fishes among six sites with varied algal resource availability in the back reef environment of Moorea. Here the hypotheses that increased algal resource availability would result in 1) increased biomass of herbivorous fishes, 2) altered behavior (e.g. decreased movement), and 3) increased energy reserves (i.e., lipids) were tested. These were specifically examined using field surveys and algal production estimates, behavioral observations, and collections of adults among all sites respectively. We found that the best predictor of herbivorous fish biomass was algal turf production, where fish biomass increased with increasing algal production. Behavioral observations of the herbivorous parrotfish Chlorurus sordidus (Scaridae) revealed that area traveled increased as algal turf cover decreased. Additionally, the Hepatosomatic Index (HSI) - a proxy for determining excess energy reserves stored as lipids in the liver - showed that energy stores (HSI) were a positive function of algal turf production, and a negative function of herbivorous fish density. The second part of this study addressed the relationship between foraging of herbivorous fishes and structural microhabitats of the back reef habitat. Specifically, it tested three hypotheses, that 1) total foraging intensity would differ among microhabitat types, 2) this pattern would be consistent among sites at larger scales (3-8 km), and 3) differences among microhabitats would be driven by physical structure and not differences in algal turf communities. Foraging was assessed using timed observations of bite rates on algal-turf covered substrata. Foraging rate was compared among five microhabitat types in an observational study; and then differences among microhabitats were evaluated more rigorously with a turf transplant experiment that crossed microhabitat source of turf with microhabitat destination. Vermetid mucous nets were removed to test their effect on foraging. Foraging intensity (bites m-2 min-1) and size-weighted foraging (bites*TL2 m-2 min-1) differed among five microhabitats (high, mid, and low bommies, pavement, and rubble) ranging from ~3-12 bites min-1, suggesting that fishes preferred to graze in certain areas. Algal transplants corroborated preferential grazing, showing similar patterns despite altered origin of turf. In the third and final portion of this study, I examined how foraging of herbivorous fishes around could be altered by negative indirect effects of a vermetid gastropod (Dendropoma maximum). These sessile gastropods extrude a mucous net that is used to indiscriminately collect plankton and settles on the surrounding substrata. Vermetid mucous nets can have deleterious effects on corals, including reductions in the growth and survival of some coral species. To address these potential effects on herbivorous fish grazing, both observational and experimental studies were used. Foraging rates from observations in the previous section of this study were used, with the additional variable of vermetid mucous net coverage as a covariate. Foraging rates were compared among the three microhabitat types commonly inhabited by D. maximum (high, mid, and low bommies). Vermetid mucous nets were also removed in a separate experiment to test their effect on foraging. Foraging rates declined with increasing cover of vermetid mucous nets, ranging from ~2-22 bites m-2 min-1 at 0% coverage to 0-5 bites m-2 min-1 at 100% coverage, but the magnitude of this effect varied among microhabitats. Experimental removal of vermetid mucous nets increased foraging intensity by ~220% relative to when vermetid nets were present. On the whole, the evidence collected from these studies supports the hypothesis that herbivores generate resilience in coral systems, in that herbivores appear to be tightly linked to algal resources in this system. Moreover, the positive relationship between herbivorous fish biomass and algal turf production suggests bottom-up control of herbivores on Moorea. Results from this study support the hypothesis that fundamental changes can occur within populations of herbivorous fishes following shifts in benthic composition that allow them to contribute to coral recovery. Results also elucidated fine scale heterogeneous patterns in herbivory and the production of algae, which could have important implications not only for planning future studies, but also potentially for the resilience of coral reefs.
157. Nitrogenase repression in Klebsiella pneumoniae
Schubert, Wayne Walter
Nitrogenase biosynthesis in a mutant strain of Klebsiella.pneumoniae can be fully repressed by L-glutamine alone. K.pneumoniae SK-25 which has no glutamine synthetase activity or immunologically cross reacting protein, lacks glutamate synthase activity, and has repressed levels of glutamate dehydrogenase cannot directly utilize NH4+, and thus requires L-glutamine as a fixed nitrogen source for growth. When grown in sulfate ion limited chemostat cultures under argon, the presence of excess L-glutamine will fully repress nitrogenase activity. In L-glutamine limited chemostat cultures and in batch cultures, nitrogenase was partly derepressed. These findings indicate that the presence of L-glutamine as a fixed nitrogen source can affect the regulation of nitrogenase by a mechanism which is independent of the adenylylation of glutamine synthetase.
158. Olfactory discrimination between Dipodomys merriami and Dipodomys panamintinus
Mirsky, Edward Neal
A series of experiments was designed to determine the role of the dorsal skin gland in olfactory discrimination and chemical communication in two sympatric species of kangaroo rats, Dipodomys merriami and Dipodomys panamintinus. Experimental animals were tested for responses to odors of the opposite sex of both species. Thin layer chromatographic studies of lipids of the back and of the dorsal skin gland, as well as histological studies of the latter, were performed. Results indicate that, at least under the conditions of these experiments, individuals of D. merriami and D. panamintinus may distinguish between homospecific and heterospecific individuals by chemical cues, although the source of the odor producing chemical is not known. Olfactory stimuli probably play a role in establishment of pair bonds by orienting search behavior of both males and females toward conspecifics. Skin lipids alone, however, are not effective as agents of species discrimination, but may still have a role in chemical communication.
159. Effects of Sugar Alcohols on Sea Urchin Gastrulation in Low Calcium Sea Water
Holmes, Edward
ABSTRACT THE EFFECTS OF SUGAR ALCOHOLS ON SEA URCHIN GASTRULATION IN LOW CALCIUM SEAWATER By Edward Holmes Master of Science in Biology The sea urchin embryo has been designated by the National Institutes of Health as a model system for studying cellular interactions. Over 25 physiological discoveries were made in sea urchins crucial to understanding their function in many taxonomic groups including humans. The sea urchin embryo was used in this study to examine the effects of sugar alcohols on cell- cell interactions during gastrulation. This study examined six different sugar alcohols in 1.5mM low calcium artificial seawater (LCASW) on 24-26 hour Lytechinus pictus sea urchin embryos and 32-34 hour Lytechinus pictus sea urchin embryos. The embryos were incubated in 1.5mM low calcium artificial sea water with and without (controls) 0.1M, 0.05M, 0.01M, 0.005M, and 0.001M adonitol, L-(-) arabitol, dulcitol, D-mannitol, D-sorbitol, and xylitol in 96 well microplates and were fixed at 48-50 hours, observed, and photographed. Three to six separate experiments were conducted for each sugar alcohol concentration at each of the two times. The embryo morphologies observed were: complete archenteron, incomplete archenteron, not invaginated, exogastrulated and dead. Sugar alcohol concentrations for each experiment were tested in 12 replicate wells yielding hundreds of sea urchin embryos in each experiment. Low calcium artificial sea water was utilized in this study because it was found to loosen septate junctions found in the blastula epithelium, accelerating molecular entry into the blastocoel of the embryos without microinjection. An unpaired t-test was performed for each sugar alcohol concentration and morphology compared to the LCASW control. P values less than 0.05 (p<0.05) suggested that the percentages of morphologies in the sugar alcohol concentration were significantly different than in the LCASW control. The sugar alcohols uniformly had little effect on embryo morphologies. The sugar alcohols causing slight effects on archenteron morphology when added at 24-26 hrs: (most effects) adonitol > L-(-) arabitol > xylitol > dulcitol = D- sorbitol > D-mannitol (least effects). The sugar alcohols causing slight effects on archenteron morphology when added at 32-34 hours (most effects) D-mannitol > dulcitol > adonitol = xylitol > D-sorbitol = L-(-) arabitol (least effects). While effects were minimal, this study indicates that very small differences can be identified quantitatively using this assay system.
160. Investigation of formaldehyde effects on electrophoresis banding patterns: are the effects reversible?
Askari, Tina
Formaldehyde has long been a commercially available fixative of common use. Formalin is the term used for an aqueous solution of formaldehyde and has been used over decades as a fixative for preserving tissue integrity. Due to formaldehyde cross-linking of proteins molecular analysis of tissues treated with formalin has not been simple. The mechanisms of formalin fixation and reversal are poorly understood. Previous studies in the laboratory have used formalin to preserve sea urchin embryos for cell adhesion studies. A goal of the research in this laboratory is to learn if formaldehyde fixation allows cell surface components in sea urchin embryo structure to retain function. A first step is this project where I investigate how formaldehyde fixation influences bands on polyacrylamide gels of sea urchin embryo material and the standard protein bovine serum albumin. In about a total of three hundred and thirty seven experiments using numerous protocols this project shows that formaldehyde fixation causes bands to disappear or lighten considerably. Before carefully controlling protein concentrations on gels many experiments were prepared with one or more errors occurring. Finally it was found that attention to solubilization protocols and protein quantity standardization provided reliable results. While the effects of formaldehyde on the appearance of bands could not entirely be reversed, a substantial amount of insight was achieved into the methods that may eventually lead to complete formaldehyde crosslink reversal in these model systems.
161. The isolation and characterization of Simonsiella from dogs and cats
Nyby, Michael David
A survey was conducted in dogs and cats for the presence of simonsiella, a multicellular, filamentous, gliding genus of bacteria. It was found in the mouths of sixty-six of sixty-seven dogs and in eight of nine dogs. The survey which was based on one oral sample from each animal, shows that Simonsiella occurs in nearly all dogs and cats. Nine strains of simonsiella were isolated from dogs and one strain was isolated from a cat using a direct isolation procedure and a serum- supplemented medium. (See more in text.)
162. Characterization of the Promoter Region for the EVI2A Gene
Weinstein, Max
Evolution is driven by natural selection operating on the genes present in a given species. However, it is unclear if the different parts of a gene (coding and non-coding) are under the same types of selection as each other. Determining the similarity of selection between coding and non-coding regions of a gene can be determined using bioinformatics; by analyzing the differences in conserved sites in the coding and non-coding (promoter) regions of the gene. Ecotropic Viral Integration Site 2A was analyzed at both its putative promoter region and its coding DNA sequence to determine its similarity of selection. After identifying the putative promoter region of the EVI2A gene through tracking of conserved regions, the genetic sequences for the EVI2A putative promoter and coding sequences were compared across several different vertebrate species to generate genetic phylogenies, which were then compared against each other and known evolutionary phylogenetic trees. Conserved sequences were identified in both the putative promoter regions and the coding sequences. The motifs conserved in the coding sequence were much more stringently conserved than were the conserved sequences in the putative promoter region, suggesting that the two regions of the EVI2A gene are under different types of selective pressure.
163. An ecophysiological investigation of the effects of macroalgae on juvenile corals and larvae on coral reefs in the Pacific and Caribbean
Widrick, Arien
On macroalgal-dominated reefs, the continuation of coral populations is dependent on successful recruitment and post-settlement success, both of which may be challenged by the presence of macroalgae. This study considers how the environment, particularly macroalgal abundance, contributes to the growth and survival of juvenile corals in two regions in the tropics: the South Pacific and the Caribbean. To determine how juvenile corals and coral larvae are affected by macroalgae in the back reef of Moorea, surveys and manipulative experiments were used to test the hypotheses that proximity to macroalgae with and without contact, or cover of macroalgae, will impact the survival and growth of early life stages of corals. Survival of Pocillopora damicornis larvae did not differ when they were incubated in situ adjacent to coral, macroalgae, or rock. Growth of juvenile colonies of massive Porites spp. and Pocillopora spp. were unaffected by centimeter-scale proximity to macroalgae. Additionally, growth was not affected by cover of macroalgae in 4-m2 plots, or by cages, which protected coral from macroalgal abrasion. Caged corals tended to grow faster, although this was not significant, and I hypothesize that this was related to protection from fish predation on lower cover plots, and from algae on higher cover plots. Macroalgae may not decrease Porites and Pocillopora growth through chemical effects, but factors including macroalgal cover may have indirect effects on the fish community that adversely affect exposed coral colonies. In St. John, US Virgin Islands, 12 sites distributed between White Point and Cabritte were selected and analyzed for benthic cover, herbivore abundance, rugosity, and rock type. Surveys of juvenile corals were conducted to determine whether the abundance and distribution of juvenile colonies within three types of microhabitats were affected by site characteristics, including macroalgae cover, and herbivore abundance. While the characteristics analyzed at the site level explained 75.9% of the variation between sites, these characteristics were not predictive of juvenile microhabitat distribution; ~ 75% of juvenile corals were found on exposed habitats at every site. While juvenile coral distribution among microhabitats was not related to site variation, survival rates within microhabitats may still vary among these sites.
164. The distribution of Cristispira in intertidal bivalve mollusks of the Pacific coast of Southern California
Lasnik, Gerald Louis
Over 1,000 bivalve mollusks, representing eighteen genera and twenty-two species, were collected in four years from twenty geographical sites from Santa Barbara, California to San Diego, California, and their crystalline styles were examined for bacteria. The giant spirochete, cristispira, was widely distributed among bivalves from all types of habitats, open and protected sandy beaches, rocky shores and estuaries, regardless of the season. In any one habitat, certain species never harbored Cristispira. The occurrence of Cristispira was neither correlated with the taxonomy of the bivalves, nor with the types of crystalline styles. The incidence of Cristispira was greater in small than in large specimens of Tivela Stultorum. Most bivalves were maintained successfully in sea water tanks and continued to possess crystalline styles, but certain bivalve species lost Cristispira during captivity. Small Spirochetes were often present in crystalline styles, while rod-shaped bacteria were rarely seen.
165. The spatial and temporal distribution of imp clusters in sea urchin embryos
Ransick, Andrew Joseph
Sea urchin embryos of Strongylocentrotus purpuratus, Lytechinus pictus and Lytechinus variegatus were investigated with respect to appearance, disappearance, and distribution of clusters of larger than average intramembraneous particles and associated cytoplasmic vesicles using thin section and freeze-fracture TEM as well as experimental procedures. This study identified a stable pattern of IMP clusters that becomes established during the eight-cell stage in S. purpuratus embryos. The pattern essentially distinguishes apical surface and lateral surface compartments in blastomere P-face plasma membranes and is spatially the same as the distribution of cytoplasmic (pigment) vesicles which occupy the apical cortices of the blastomeres. Analysis of post 16-cell stages revealed the IMP pattern persists through the early stages of blastulation. Largely due to a process called the vegetal cortical rearrangement, the micromeres are formed without the typical IMP pattern and without the pigment vesicles. Thus, the micromeres have a significantly different plasma membrane organization, and probably, also, a significantly different degree of stability in their cortex at the time of their formation. The implications of these findings are discussed in relation to the development of radial polarity and micromere determination. (See more in text.)
166. A comparison and analysis of the post-release movement of four species of raptors
Cornish, Michael J.
Forty individuals of four species of raptors, Cooper's Hawk (Accipiter cooperii), Red-tailed Hawk (Buteo jamaicensis), American Kestrel (Falco sparverius), and Prairie Falcon (Falco mexicanus) were released after a conditioning period and monitored with the use of radio telemetry. All birds were found to generally increase their activity range over a period of time. Juveniles always moved more than adults. Species that consumed greater proportions of avian prey showed increased differences between juvenile and adult movement suggesting intraspecific predation and consequent adaptive significance in increased juvenile movement. Cooper's Hawks showed the greatest movement followed by American Kestrels and then Prairie Falcons. Red-tailed Hawks showed least movement. In species where the sexes may winter together (Red-tailed Hawk and Cooper's Hawk), both sexes appeared to move the same amount. Both species of Falco had increased male movement. The presence of the radio package had no effect on the birds' activity as long as it remained below 4% of the birds bodyweight. Modified falconry techniques as a method of conditioning appeared to adequately prepare each raptor for survival once released.
167. A temperature sensitive mutant of bacillus subtilis: Analysis of physiological spore defects
Rothman, Joel Eric
A temperature sensitive sporulation mutant of Bacillus subtilis was isolated and its chemical and physical characteristics were investigated. This mutant is able to grow equally well at 30° and 42°C, but is unable to produce a normal healthy spore at the restrictive temperature of 42°C. Properties of the spore such as heat resistance, refractility, and resistance to osmotic shock were normal in the parent strain but were altered in the mutant at the restrictive temperature. Calcium levels were found to be lower in the mutant than in the normal strain. One plausible hypothesis which would explain all of these observations is that the spore cortex , which is the site of the murein–Ca: DPA complex, ma be improperly crosslinked and thus exhibit the altered phenotypic characteristics of the mutant spore.
168. Involvement of hepatocyte membranes in glycogen metabolism
Dowd, Patrick Joseph Jr.
A considerable amount of evidence supports the contention that elements of the endoplasmic reticulum are involved in glycogen metabolism. The present study examined the effects of starvation and glycogen depletion on the membrane elements of rat liver. Membrane elements were isopycnically banded by reorienting gradient zonal centrifugation in sucrose density gradients. Changes in the distribution and density of the various elements were examined during increasing periods of starvation and reflect the structural and physiological changes occurring in the tissue. A remarkable increase in glucose-6- phosphatase activity and protein concentration was observed within a short period of starvation. A membrane fraction corresponding to membrane bound glycogen was found to disappear after 48 hours of starvation. It is proposed that this fraction corresponds to desmo glycogen identified previously. A transitional membrane density stage is reported, and probably represents the intermediate density of rough membranes as ribosomes are removed. Both rough and smooth endoplasmic reticulum increased in density during long-term starvation. This resulted from loss of lipid from both membrane types.
169. The adhesion of sea urchin embryonic cells to polysaccharide beads
Toedter, Gary Paul
Dissociated sea urchin embryonic cells were observed to adhere to Sepharose 4B beads when the cells and beads were rotated together in calcium-and-magnesium-free sea water. When placed in Millipore filtered sea water, the cells would not adhere to the beads. It was shown that both calcium and magnesium ions could separately inhibit the adhesion. Galacturonic and glucuronic acid increased the adhesion, while ether haptens had no effect. The cells would adhere to other composition beads, and could be inhibited by the addition of calcium or magnesium ion. It was also shown that the adhesion to Sepharose 4B was pH dependent. Various mechanisms for explaining the adhesion are discussed. Charge-charge, hydrogen bonding, and van der Waals interactions are possibilities. The cell-bead interaction observed here can serve as a model for demonstrating initial cell-cell interaction, leading to specific cell-cell adhesion.
170. Effects of ecosystem engineering by the giant kangaroo rat on the common side-blotched lizard
Wirth, Camdilla D.
Ecosystem engineers are organisms that control the availability of resources to other species by creating and modifying habitat (Jones et al. 1994). Ecosystem engineers alter habitat in such a way as to reduce physical and biological stresses for other organisms (Crain & Bertness 2006). Burrowing mammals are keystone ecosystem engineers in many communities because burrowing is an engineering activity that can directly and indirectly alter the availability of resources, have effects at multiple spatial and temporal scales, and have a significant role in community organization (Prugh & Brashares 2012). Giant kangaroo rats (Dipodomys ingens), are a federally listed endangered species and keystone ecosystem engineers that modify habitat by building extensive burrowing systems. They are associated with greater density and diversity of vertebrate, invertebrate, and plant populations. However, few studies have addressed the functional mechanisms behind these associations and how ecosystem engineering affects the behavior of associated species. The purpose of this study was to investigate the functional attributes of ecosystem engineering by giant kangaroo rats and quantify their effects on the common side-blotched lizard. Lizard abundance is positively correlated with giant kangaroo rat density in this system, and although an increased presence of arthropods has been suggested as the mechanism, the causative factor has not been determined (Prugh & Brashares 2012). I investigated both landscape and local scale effects of habitat modification by giant kangaroo rats on the density, microhabitat use, and behavior of the common side-blotched lizard in 2012 and 2013. I determined that lizards are associated with greater burrow tunnel density at the landscape scale and are found more often on precincts on a local scale. A translocation experiment revealed that lizards prefer precincts with more burrow tunnels and remain at those precincts longer. I investigated whether giant kangaroo rats facilitate lizard density by three possible mechanisms: by providing refuge from predators, by providing thermal refuge, or by providing increased arthropod prey resources. Additionally, I investigated whether lizards altered their behavioral response to predation risk in the presence of burrow tunnels. I found that lizards use burrow tunnels as refuge from predators and display differences in escape behavior on and off precincts. Precincts also provide thermal refuge where temperature and humidity is more stable than the outside environment. However, at the local scale, there were no differences in arthropod resources. Giant kangaroo rats likely facilitate common side-blotched lizards by a combination of supplying refuge from predators and from extreme temperature and humidity.
171. Separation and characterization of mammalian cells by velocity sedimentation
Doolittle, Mark Henry
Unit gravity (Sta-Put) sedimentation and reorienting gradient zonal centrifugation were used to separate and characterize mammalian cells by velocity sedimentation. The development of both techniques as well as density grad-theory have been reviewed. Zone resolution for both techniques was compared by utilizing a model system of polystyrene beads (of known diameters and densities) in minimum slope gradients of sucrose at unit gravity or predesigned isokinetic gradients of Ficoll in the zonal rotor. Both minimum Slope and isokinetic gradients resulted in constant velocities during sedimentation. Slightly better zone resolution was obtained by unit gravity sedimentation, presumably as a consequence of the mildness of the technique. Nevertheless, both techniques yielded a high level of resolution. Biological application of both techniques was demonstrated by separating and characterizing three sizedensity subpopulations of cells present in the 129/J mouse ascites teratocarcinoma tumor. Buoyant density centrifugation revealed that the tumor is comprised of two distinct density classes of cells with average densities of 1.17 gm/ml and 1.04 gm/ml and average diameters of 15.7 um and 19.9. Unit gravity sedimentation also revealed the presence of independently varying cell densities as well as cell sizes in the tumor. Isolinetic gradients of Ficoll in the reorienting gradient zonal rotor were utilized to probe size variations within each density class. Cells comprising the less dense (1.04 gm/ml) subpopulation of the tumor were shown to have two discrete internal size classes with average diameters of 24 and 9 pm. The denser subpopulation of cells (1.17 gm/ml) appears to be formed by a continuous spectrum of sizes. This work demsonstrates the versatility of both techniques in distinguishing size-density classes in a heterogenous cell population.
172. Freeze fracture analysis of the early sea urchin embryo
Shultz, Samuel Louis
Plasma membrane molecular topography in the early development of the sea urchin embryo, Strongylocentrotus purpuratus was analyzed using freeze-fracture electron microscopy. Membrane topography was analyzed with respect to intramembranous particle densities and size class frequencies in the 2, 4, and 8-cell embryonic stages. Results show similarities in membrane topography between cells of the 2-cell stage, and between cells of the 4-cell stage. Overall membrane particle size class frequencies between the 2 and 4-cell stages are similar, yet the 8-cell embryo is comprised of cells that differ from each other and the previous embryonic stages topographically in membrane particle densities and size class frequencies. Both the 2 and 4-cell embryonic stages contain cells with homogeneous particle distributions, while some cells of the 8-cell stage show membrane particle heterogeneities. Membrane particle clusters appear in some cells of 8-cell embryos, yet no clusters were apparent in membranes of the 2 and 4-cell stages. Results indicate that membrane particle homogeneity is apparent in blastomeres of the 2 and 4-cell stages, yet massive membrane particle insertion and reorganization occurs during division of the 4-cell embryo. The membrane structural changes observed correlate well with the overall pattern of determination in the early development of the sea urchin embryo.
173. Patterns and Mechanisms of Population Diversification in Red-eyed Treefrogs (Agalychnis callidryas)
Jacobs, Leah
A central goal in evolutionary biology is to understand the mechanisms underlying the formation of new species (Ott & Endler, 1989; Coyne & Orr, 1998). Population diversification evolves through ecological and evolutionary processes, which could eventually lead to speciation (Coyne & Orr, 1998; Sobel et al., 2009). These processes include both pre- and postzygotic reproductive isolation. Behavioral isolation is a form of prezygotic isolation, which, along with geographic isolation, can serve to reduce gene flow among populations. Postzygotic reproductive isolation evolves via reduced fitness of outcrossed offspring or chromosomal incompatibilities of the parents (Coyne & Orr, 2004; Giovanni, 2008). Examining the relative strength of multiple forms of reproductive isolation provides insight into the mechanisms of divergence. My research provides valuable insight into mechanisms of population divergence and speciation by focusing on the consequences that phenotypic and genetic diversification can have on both prezygotic and postzygotic reproductive isolation. The first chapter of my thesis examines pre-mating behavioral isolation by testing female mate preference between two divergent populations of red-eyed treefrog. We found that females in each population preferred local mates. Further, females largely chose males in the absence of any male advertisement call, underscoring the importance of non-acoustic cues in mate choice, a known sexual signal for most anurans. This research provides evidence that population divergence likely has consequences for mate recognition and choice. My second chapter examined variation in sperm response to an exogenous hormone among four differentiated populations in Costa Rica. Differences in sperm production in response to the exogenous hormone LHRH (Luteinizing hormone releasing hormone) could indicate that populations have differentiated at a physiological level, which could have consequences for postmating reproductive isolation and eventual lineage divergence. This chapter had two goals. The first was to examine variation among populations in sperm production. We hypothesized that differences in receptivity to hormones or the endocrine system have also evolved among these differentiated populations. To our knowledge, no study has examined sperm response to exogenous hormones across highly differentiated populations within a species. We found that although sperm count did not vary among populations, at least one population showed decreased sperm viability in response to the hormone. From an evolutionary perspective it appears that one population has differentiated in receptiveness to hormone injections. The second goal of this chapter was to develop an Assisted Reproductive Technologies (ART) protocol that could later be used for laboratory breeding, conservation techniques and further studies on reproductive isolation. From a conservation perspective the hormone was effective in producing viable sperm. Thus, we propose that the hormone LHRH is sufficient for conservation protocols for most populations of red-eyed treefrogs. However, for developing an ART protocol for species with highly differentiated populations, we recommend that starting with a baseline dosage efficient at inducing spermiation is adequate but specialized protocols for spermiation, captive breeding or studies on postzygotic reproductive isolation may be needed for species with highly differentiated populations across their range. Combined, the chapters underscore the importance of examining divergence among populations and how population differentiation can lead pre and postmating reproductive isolation and eventual lineage divergence. In addition, the differences among populations can affect conservation and laboratory breeding protocols.
174. Age, growth and genetic diversity of giant sea bass, Stereolepis gigas
Hawk, Holly A.
The giant sea bass, Stereolepis gigas, is a large predatory fish that inhabits the southern California kelp forest community. S. gigas is a critically endangered species, yet little is known about its life history. A more complete knowledge of the life history of this once commercially viable fish is necessary before an effective management strategy can be proposed. Giant sea bass were collected through collaborative efforts with commercial fish landings, scientific gill-netting, and through catch-and-release methods employed by recreational fisherman. A total of 59 samples were used for both mitochondrial DNA and age-and-growth analysis. Genetic analysis of the mitochondrial control region indicated gene flow among all sampling sites, no phylogeographic structure, as well as low haplotype (h) and nucleotide diversity (π). Sagittal otoliths were cross sectioned and analyzed with digital microscopy techniques, resulting in the verification that S. gigas is a long-lived species growing to at least 76 years of age. The calculated von Bertalanffy growth function (lt= 2048.4(1-e -0.041(t+0.839)) for S. gigas was also characteristic of a slow growing, apex predator. Long-lived species that are slow to reach maturity often have a low resilience to over-fishing, therefore it is of paramount importance that we continue to collect essential life history data on this species in order to more effectively protect and manage the S. gigas population.
175. The characterization of obi-1 and obi-3 beetle pheromone insensitive mutants in Pristionchus pacificus nematodes
Aguilar-Portillo, Georgina
Nematodes rely on chemosensation for their survival. Through chemosensation nematodes can avoid harmful chemicals, enter into the dauer alternative developmental stage, find food sources, potential mates, and even insect hosts. Pristionchus pacificus (P. pacificus), isolated from Pasadena, California in 1996 have been found to associate with different beetle hosts around the world. In particular, P. pacificus has shown preference in associating with the oriental beetle found in Japan and northeastern United States and is specifically attracted to the beetle's sex pheromone (Z)-7-tetradecen-2-one (ZTDO) in the laboratory. To determine the genes responsible for insect pheromone sensing, two chemosensory mutants were identified in a genetic screen for lack of attraction towards ZTDO after cGMP treatment, named Ppa-obi-1 and Ppa-obi-3 (oriental beetle pheromone insensitive). Protein expression of a recombinant GST-Ppa-OBI-1 (in E. coli) and recombinant GFP-Ppa-OBI-1 (in C. elegans) have been analyzed using western blots. The second aim of my study is to characterize another mutant allele, Ppa-obi-3 (tu405), which has a short body length and coiling phenotype. In C. elegans, reversal rates are known to affect approach behavior in chemotaxis. I determined Ppa-obi-3's reversal rates by counting the frequency of omega turns on and off food and found that Ppa-obi-3 animals reverses significantly more often than wildtype. Interestingly, C. elegans egl-30 mutants show a similar defective locomotion behavior with a higher reversal rate compared to wildtype. Defects in EGL-30 (Gqα) affects the release of acetylcholine from motor neurons needed for normal muscle movement to occur. I found that the associated behavior is further accentuated in liquid medium. Wildtype PS312 shows a thrashing motion in which the head is restricted to a small angle, whereas Ppa-obi-3 coils into a superimposed circle, and head as a result changes directions continuously. This coiling-swimming phenotype was utilized for positional mapping of Ppa-obi-3 on the genetic linkage map. Homozygotes from Ppa-obi-3 x Japan (RS5195) recombinant inbred lines were rough mapped to a 38cM interval of Chromosome I (out of 6 linkage groups) using simple sequence length polymorphism markers. The long-term goal is to determine if Ppa-obi-3 and Ppa-obi-1 constitute components of an unknown signaling pathway, part of the conserved cGMP-dependent G protein signaling pathway, or TRPV channels lipid signaling pathway (PUFA) involved in sensing insect hosts.
176. Part I, the analysis of spontaneous reversion in a serine auxotroph of Neurospora crassa; Part II, linkage data on a new serine requiring mutant: Ser-5.
Bengtson, Ramona Sundbom
A newly isolated serine auxotroph of Neurosporacrassa, ser (JBM 4-13), exhibited a high rate of reversion in mapping experiments. This study sought to determine whether reversion occurred at a specific frequency , and whether it was time dependent. Individually isolated ascospore cultures of the mutant, as well as subcultures of individual progeny, showed spontaneous reversion frequencies ranging from 1.0 x 10 to 1.0. Other properties of the ser(JBM 4-13) mutant demonstrated by this study include: 1) the apparent meiotic stability of the ser (JBM 4-13) gene, 2) the mitotically unstable and highly revertible state of ser (JBM 4-13) which occurs sometime during vegetative development, 3) the time dependence of the reversion phenomenon, 4) the independence of the reversion phenomenon from conidial population size, and 5) the independence of reversion from cell division or growth. (See more in text.)
177. The effects of water flow and sedimentation in interactions between massive Porites and algal turf
Gowan, Jennifer Caitlyn
Interactions between scleractinian corals and benthic algae can be an important process structuring coral reef communities. The effects of algae on corals and vice versa are not fixed and may be influenced by abiotic factors such as sedimentation, a process often underlying reef degradation. In addition, the rate of sedimentation as well as the effects of trapped sediments may be mediated by water flow, which may influence coral-algal interaction dynamics. The first goal of this study was to test the hypothesis that gradients in sedimentation and flow on the fringing reefs and back reefs of the north shore of Moorea, French Polynesia, correlate with gradients in the frequency of massive Porites-turf interactions, as well as the outcomes of these interactions. In the back reef, Porites-algal turf interaction frequency increased by 73% as sedimentation decreased and flow increased. Along fringing reefs, interaction frequency increased by 97% as sedimentation increased. Results indicated that flow and sedimentation do not interact to influence the frequency or outcome of interactions, but rather exert significant independent effects. The second goal of this research was to test the separate and joint effects of flow and sedimentation on coral-algal interactions in a controlled laboratory setting. Results indicated that despite extensive damage to coral tissue across treatments, Porites growth as measured by the change in dry weight did not differ across treatments. This led to the hypothesis that massive Porites may allocate resources preferentially to the growth of tissues not in direct contact with algal turf, thus accounting for the maintenance of growth rates across treatments. The third goal, therefore, was to test this hypothesis in the field along gradients of flow and sedimentation, and sedimentation alone, for 1 year. Coral growth was quantified as linear extension in three directions: vertical, and horizontal away from and towards the zone of interaction with algal turf. Results indicated that as sedimentation increased, both vertical extension and horizontal extension adjacent to the interacting algal turf decreased by approximately 80%. In contrast, growth of tissue away from the algal turf interaction doubled as sedimentation rate increased. Overall, these data suggest that massive Porites may be able to compensate for the reduction in growth along the zone of interaction by increasing growth rates of tissues away from the interacting turf. The fourth and final goal was to identify a possible mechanism underlying the effects of sediment trapping by algal turf on corals. The hypothesis that spatial variation in sedimentation at the scale of a coral-algal interaction might be an additional important mechanism by which sediments influence coral-algal interactions was tested. For this study, the relative effects of sediment accumulation along interaction boundaries, on the surface of coral and algal tissue not in direct competition, and all possible location combinations was quantified. Results indicate that the accumulation of sediments along the coral-algal interface reduces the area of coral tissue damaged by 56% when sediments also are present on the non-interacting algal turf surface. Knowledge of how fine-scale patterns of sediment deposition may affect the competitive outcome of coral-algal interactions has implications for the study of coral-algal dynamics as well as increasing understanding of the relative importance of sediment trapping by algal turf. Together, these results suggest that flow and sedimentation exert significant, individual, direct and indirect effects on interactions between massive Porites and algal turf.
178. The social ethology of mule deer (Odocoileus hemionus) in Southern California
Koutnik, Debra Lynn
The social behavior and organization of mule deer (Odocoileus hemionus californicus) were studied from 15 Feb 1977 through 12 Feb 1978 in the Chatsworth Reservoir, Los Angeles, California. Results are based on over 733 person-hours of observation. Seasonal changes in social organization were observed and correlated with ecological and behavioral factors. Does were commonly alone during the fawning season (May-June) when they showed high levels of intolerance toward conspecifics. At all other times of the year females associated in doe or mixed groups of 3-30 individuals. Males were frequently observed in buck groups of' 3-5 or in larger mixed groups. During the rut (October-November), bucks usually traveled singly among doe groups. Multi-male breeding associations resulted. Females commonly led walking patterns, whereas males usually occupied the omega position. No specific individual(s) acted as constant group leader, nor was dominance rank correlated with leadership. High ranking males most often led trotting patterns that had resulted from some source of alarm. Dominance hierarchies among males and among females were maintained the year around through continual agonistic interactions. Adult males were always dominant to females. Maternal does rose in social status during the fawning season but previous dominance relationships were re-established after their fawns disappeared. Changes in social rank between males occurred prior to the breeding season and again after antlers were shed. (See more in text.)
179. Radical coupling reactions under neutral conditions and their biological relevance
Sepanian, Ruth
Breast cancer is the second most prominent form of cancer among women in the United States. It is considered to be a multifactorial disorder where estrogens (estradiol and estrone) play a key role in the initiation and progression of the disease. Normally, estrogens are synthesized from testosterone and androstenedione by a Cytochrome P450 19Al enzyme aromatase (steroidogenesis). Its inhibition can effectively reduce the circulating estrogen levels and effectively starve breast cancer cells of the necessary signals. Although there are potent and effective drugs on the market, they cause serious side effects, such as arthralgia, bone pain, bone stiffness, hot flashes, night sweats, and genitourinary diseases. A new generation of aromatase inhibitors should be designed in such a way so that a low toxicity and side effects will become intrinsic properties of these future drugs. The chemistry part of my research is focused on synthesizing novel therapeutics for breast cancer treatment using cobalt-complexed propargyl cations as precursors. These transient species are usually synthesized under acidic conditions (HBF4, BF3, CF3COOH, H2S04), thus severely limiting the substrate base and its applicability to the synthesis of molecular assemblies bearing labile acid-sensitive functional groups (acetal, benzyloxy). In this project, we developed three new organometallic reactions: (1) a high temperature, spontaneous generation of cobalt-complexed propargyl cations (up to 147�C); (2) propargyl methyl ethers interaction with triflic anhydride that, for the first time, allows for generation of prop argyl cations under neutral conditions; and (3) a low temperature (- 50�C) variant of the parent reaction utilizing cobaltocene as an external reducing agent. These new methodologies allowed us to synthesize a variety of topologically diverse, polysubstituted 3,4-diaryl-1,5-alkadiynes m high yields and excellent d,ldiastereoselectivity (up to 99%). Decomplexation with eerie ammonium nitrate afforded metal free d,l-3,4-diaryl-1,5-alkadiynes, the potential aromatase inhibitors. Overall, the generation of cobalt-complexed propargyl cations under neutral conditions substantially expanded the scope of both ionic and radical reactions, allowing for involvement of substrates with acid-sensitive peripheral functionalities. Once these compounds were synthesized, they were tested as potential aromatase inhibitors on a MCF7 breast cancer cell line. These cells were incubated for two days with potential drug candidates, at various concentrations, then cell viability was measured using the MTT assay. Preliminary results indicated that d,l-3,4-di-(4'-methoxyphenyl)-1,5-hexadiyne inhibits cell proliferation with its efficiency being superior to that of aminogluthimide used as a reference.
180. The association of fungi and spiders
Cohen, Randy Wade
Reports of fungi growing on spiders number less than ten. None of the reports had indicated that the spider was alive at the time of the observation of the fungus. No studies have been made on infecting spiders with fungi, although there is extensive knowledge about fungal infections of insects. Collections of spiders from various habitats around Southern California were made to survey the fungi associated with these organisms. These spiders were incubated in the laboratory, in an attempt to allow the associated fungi to infect the spiders. In no case did a fungal infection develop on a living spider. Two methods were used to try to infect spiders with the known insect pathogen, Beauvaria. Spores were dusted on the integument of the spider, and spores were placed on the freshly exposed stump of the coxa, after leg removal. In neither set of experiments did a fungal infection develop. Whole, surface sterilized, living spiders were crushed onto agar plates spread with Beauvaria spores . Zones of fungal inhibition were observed surrounding the crushed spider. These observations led to the conclusion that spiders have a defense mechanism preventing fungal infection.
181. Identification of Legionella pneumophila effector proteins that interact with human endocytic rabs
Thyagarajan, Kavitha
Legionella pneumophila are intracellular bacteria that cause pneumonia by infecting alveolar macrophages. Normally, bacteria internalized by macrophages travel down the endocytic pathway to the lysosome where they are degraded. The endocytic pathway is regulated by Rab GTPases that facilitate movement of vesicles. Legionella escape degradation in the lysosome by secreting effector proteins into host cells. Several effector proteins have been shown to interact with host GTPases ofthe exocytic pathway, Rabl and Arfl. This project has identified for the first time an interaction between a Legionella pneumophila effector protein and a human endocytic Rab protein using a yeast two-hybrid screen. Rab4a, Rab5aQ79L and Rab22a proteins have been used as bait in the 2-hybrid screen. Sequences containing Legionella genes have been sequenced from the positives ofthe yeast two-hybrid screen. Seven positives have been screened for Rab4a; ten for Rab5aQ79L and twelve positives were screened for Rab22a. Two Legionella proteins that bind to Rab4a have been identified. The identified protein, Lpg2229, is annotated as a saframycin MXl synthetase B and Lpg2336 as a peptide chain release factor-1. Four proteins that bound to Rab5aQ79L were identified. These proteins include LegC7, a Legionella effector protein that is thought to affect endocytic transport. Other proteins binding to Rab5aQ79L include a sensory box protein Lpgll68, a transposase Lpg2120 and an unknown protein Lpg2576. Finally, a protein of unknown function, Lpg1644, was found to interact with Rab22a. The data obtained in this thesis may aid in identifying the molecular mechanism by which Legionella avoids destruction in host cells.
182. The masticatory apparatus of four phyllostomatoid bats
Martin, Dennis, Robert
The functional and developmental relationship of the muscles, skull and dentition involved in mastication and feeding in four phyllostomatoid bats were studied. Dry weights of the muscles, weights of the mandibles, and skull measurements were taken. The distribution of dense bone and the development of supportive create suggested major areas of stress arising from the masticatory musculature. Using the variations pf these factors as criteria the four species of bats were classified into three categories of skull type; the breviarcuate, longoarcuate, and pkanoarcuate. The breviarcuate and longoarcuate types of skulls are adaptations to a highly specialized diet as seen in Artibeus and Desmodus. The planoarcuate type is associated with the primative insectivorous diet in Macrotus and with the reduction of the masticatory apparatus due to the increase in importance of another mechanism in Leptonycteris, a necter-eating bat. The mandible and masseteric muscle weights in all four bats suggest a relationship correlated with the lifting function of the muscles compared with the weight of the mandible. The close correlation found between the weight of the pterygoid muscles and the zygomaticomandibular muscle, along with the structure of the jaw aarticulation, suggested lateral motion as an important function of these muscles in Macrotus and Artibeus. In Leptonycteris the zygomaticomandibular muscle are greatly reduced compared with pterygooid muscles. The mechanical efficiency of the mandible as a class III lever was found to be high in Macrotus and Artibeus, corresponding to well-developed masticatory musculature, as opposed to Leptoncteris with low efficiency and reduced musculature. The efficiency of the apparatus in Desmodus, unlike Artibeus and Macrotus, is due to orientation of the temporalis. It is suggested by the articulation, muscle size and orientation, and the dentition that the action of the mandible in Deamodus is significantly flexible in moving anteriorly. Correlated with observations of the biting action of the vampire a possible function is discussed. Hypothetical pathways in the evolution of theses bats from an early primitive insectivorous type are presented.
183. Food habits of Bighorn sheep (Ovis Canadensis Nelsoni) in the San Gabriel mountains
Perry, William Michael
A study was conducted in the San Gabriel Mountains of Southern California during 1979-81 to obtain quantitative data on food habits, diet quality, and preference of bighorn sheep (Ovis canadensis nelsoni). Utilizing a microhistological analysis of fecal pellets, a total of 19 forage taxa were identified. Browse species made up the majority of the diet, both on a monthly and a seasonal basis; the dominant species were Cercocarpus betuloides, Salvia apiana, Eriogonum fasciculatum, Garrya veatchi i, and Prunus ilicifolia. Grasses were eaten year-round, but were taken in significantly greater amounts in the spring. Forbs, the smallest fraction of the diet, were taken when available. Crude protein analysis of the fecal pellets showed both a monthly and seasonal variation with a high of 18.33% in the spring and a low of 10.50% in the fall. Comparing vegetation data with dietary data, the sheep showed a preference for Cercocarpus betuloides in all seasons. Salvia apiana and Eriogonum fasciculatum were preferentially selected during the winter and spring but avoided in the summer and fall. Plants which occurred commonly in the area but were avoided totally included Adenostoma fasciculatum, Eriodictyon trichocalyx, Arctostaphylos sp., and Ceanothus crassifolius.
184. Parents, Predators, or Poop? Testing the Forces Selecting for Aggregation in a Herbivorous Lizard
Rhymer, Rachel
Although physiological, antipredatory, and reproductive benefits have been predicted as important drivers of grouping behaviors in animals, the initial forces selecting for aggregating behavior can be difficult to identify. Grouping often evolves in populations with high densities that occur in challenging environments-conditions that can foster intraspecific interactions and may lead to the evolution of increasingly complex social behaviors, including the formation of kin groups or parental care. Phymaturus extrilidus is a herbivorous and viviparous iguanian lizard that occurs in high densities on rock outcrops in the high-elevation desert (Puna) of San Juan Province, Argentina. This species has been reported to form stable aggregations between adult females and neonates soon after birth, suggestive of maternal care. Parental care is considered rare in squamate reptiles and there have been few tests of the potential benefits or forces selecting for this derived form of grouping behavior. Our goals were to (1) test whether the females associating with neonates were their mothers, and (2) identify the likely evolutionary pressures selecting for aggregating behavior in this species. Such pressures likely include reducing predation risk and/or the need to socialize to transfer vital gut microsymbionts for digesting plant tissues from one generation to the next. I hypothesized that (1) grouping lizards would be commonly observed in the population, (2) groups would be primarily comprised of mothers and their neonates, (3) groups would react more quickly to the threat of predation, and (4) that neonates would acquire and therefore possess microbial communities that are most similar to those of their mothers. I assessed stable grouping rates via focal animal observations over two field seasons (November 2013 and 2014), and although relatively infrequent, groups were nearly always comprised of neonates and adult females. Next, I determined relatedness among 173 individuals via kinship analyses of 10 variable microsatellite loci. Our genetic analyses revealed that only one of 15 stable groups was comprised of related individuals, which did not support our hypothesis that neonates aggregate primarily with their mothers. To assess the role of predation pressure in selecting for grouping behaviors, I conducted predator-approach experiments using models of two confirmed predators (a fox and a hawk). Lizards in groups did not differ from solitary lizards in their responses to predators, rejecting this hypothesis as a potential benefit of grouping. To test whether grouping lizards benefited from exchanging intestinal microbes (Archaea and Bacteria), including taxa presumably required for digesting plant fiber, I characterized these communities by sequencing the 16S rRNA subunit of microbes from lizard fecal samples on the Ilumina MiSeq platform. Using standard workflows (QIIME) I compared these communities among lizard age classes using ANOSIM and PERMANOVA and discovered that lizard spatial organization, not relatedness, was the best predictor of hindgut microbial community composition. When only including a small subset of newborn lizards and their genetic mothers, however, I saw evidence that they are initially acquiring their gut microbes from their mother, but quickly diverge from this primary microbial signature. This finding suggests that lizard acquire their microbial communities over time from common localized source(s) (soil, water, or feces), or that access to resources, such as preferred basking sites, may facilitate the exchange of microbes. Social species exhibit a wide continuum of behaviors, ranging from short-term seasonal interactions to lifelong and complex bonds with mates or kin. I suggest that P. extrilidus has taken the first evolutionary steps along this sociality continuum and, assuming sufficient selective pressure, would be well suited for future studies that seek to understand how more complex social behaviors evolve. More studies are needed on other liolaemids that reportedly exhibit similar grouping behaviors to test whether our findings are generalizable for this clade and to elucidate the broader phylogenetic and ecological diversity of these social interactions among squamate reptiles generally.
185. The effects of ocean acidification, elevated temperature and herbivory on tropical crustose coralline algae
Johnson, Maggie Dorothy
The coral reef ecosystem supports the greatest taxonomic diversity in the ocean and is shaped by a suite of biotic and abiotic factors. One ecological interaction that is important in structuring coral reefs is herbivory. A series of field surveys found that sea urchin grazing on crustose coralline algae was a frequent occurrence on the fringing reefs of Nanwan Bay, southern Taiwan, where more than 50% of grazing scars had been inflicted by sea urchins. Grazing assays conducted in southern Taiwan in August 2009 showed that the closely related sea urchin species Echinothrix diadema and Diadema savignyi had similar grazing effects on the tropical coralline alga Hydrolithon onkodes, where each species grazed similar amounts of coralline algal surface area. Hydrolithon onkodes is an abundant species of tropical coralline algae throughout the Indo-Pacific, and on the northshore of Moorea, French Polynesia H. onkodes accounted for up to 11% of the benthic cover on the shallow backreef. A series of grazing assays conducted in Moorea in January 2010 showed that the sea urchin D. savignyi had a greater effect on the cryptic species of coralline alga Goniolithon improcerum by grazing more surface area than the heavily calcified species H. onkodes. These results suggest that sea urchins are important grazers of tropical coralline algae, and that the extent of thallus calcification may impact susceptibility to grazing. Two environmental factors that are becoming an increasingly important influence on coral reefs are due to the increasing concentration of carbon dioxide in the atmosphere. The effects of elevated carbon dioxide (pCO2) on rates of calcification in coral reef habitats have been explored primarily for scleractinian corals with few studies addressing the impacts on crustose coralline algae. Laboratory experiments conducted in May-June 2010 found that elevated pCO2 significantly decreased calcification rates of the coralline alga H. onkodes by up to 51% and decreased rates of photosynthetic performance by 33%. Experiments conducted in Hawaii in August 2010 showed that elevated pCO2 and warmer seawater temperature combined had a significant interactive effect on the calcification rate of H. onkodes. Calcification was significantly reduced by elevated pCO2, but the effect was non-linear depending on temperature. In addition, elevated pCO2 and warmer temperature also increased the susceptibility of H. onkodes to grazing by sea urchins, where E. diadema grazed 60% more of H. onkodes that had been exposed to elevated pCO2. These results suggest that elevated pCO2 and warming seawater temperature have negative implications for the physiological calcification response in tropical crustose coralline algae, and that this physiological effect may have community-wide effects such as increased susceptibility to grazing by excavating herbivores. Coralline algae serve many important ecological roles in coral reefs, and the abundance of coralline algae, as well as their ecological functions, may be significantly reduced in the future with the progression of ocean acidification and sea surface temperature warming.
186. Activation of puromycin-sensitive aminopeptidase to enhance in the degradation of aggregate prone proteins
Seibert, Charlie
Pathological aggregation of specific proteins underlies many neurological disorders including Tauopathies, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, and spinocerebellar ataxia type-3. Recent research has identified puromycin sensitive aminopeptidase (PSA; NPEPPS) as a potential target in the treatment of tauopathies, as overexpression of PSA reduces total tau levels both in vitro and in vivo, while PSA overexpression does not cause abnormalities in either mouse or fly. PSA may also be involved in the degradation of several other pathologically aggregating proteins. The inhibition of PSA has led to increased aggregation of ataxin-3, huntingtin, SOD1, and -synuclein, which are the major components of the protein aggregates found in spinocerebellar ataxia type-3, Huntington's disease, amyotrophic lateral sclerosis, and Parkinson's disease, respectively. This suggests that PSA may be a key therapeutic target for a number of neurological diseases characterized by protein aggregation beyond tauopathies. In this study, we test the effects of various compounds, previously identified as enhancers of PSA activity, on the degradation of endogenous forms of these aggregate prone proteins at non-toxic concentrations in primary neuronal cultures. We demonstrate that the activation of PSA through the addition of these compounds lowers endogenous huntingtin and SOD1 levels in primary mouse cortical culture, as well as endogenous huntingtin, SOD1, ataxin-3, and -synuclein levels in primary human neural progenitor cells while not causing detectable harm to the cells. This suggests that the compounds tested may be a promising prospect for additional drug development in the treatment of a range of neurodegenerative diseases.
187. Heterologous Expression and Secretion of Aggregatibacter Leukotoxin A by a Tumor-Targeted Salmonella
Vincent, Lena
Despite the advancement of numerous treatment options, conventional cancer therapeutics generally fail to prevent tumor recurrence or successfully treat metastatic cells. A promising alternative involves the use of attenuated Salmonella strains, which preferentially replicate within tumor tissue and suppress tumor growth. Although the tumor-targeted strain VNP20009 localized to some tumors with high efficiency in human clinical trials, they failed to result in eradicating cancer cells or delay tumor growth. To enhance the tumoricidal efficacy of this strain, we engineered the bacteria to express leukotoxin A (LtxA), a pore-forming multigene toxin produced by Aggregatibacter actinomycetemcomitans with natural specificity for β2-integrins found on human leukocytes. Previous studies have shown that the overexpression of this surface antigen on cancer cells is associated with increased metastatic potential, suggesting that LtxA may represent a target for metastatic tumors, which are notoriously resistant to therapy. The LtxA-encoding genes were cloned into the inducible plasmid vector pAra99a and expressed in the tumor-targeted Salmonella strain VNP20009. LtxA expression at the mRNA transcript level was verified by RT-PCR. A codon-optimized LtxA sequence was generated and tested for its ability to enhance expression in the VNP20009 background. To improve the secretion of the LtxA protein into the culture supernatant, the wild-type LtxCA was co-expressed with the E. coli hemolysin-α (HlyA) secretion complex, HlyBD. C-terminal modifications were generated to facilitate interaction of the LtxA protein with the HlyBD complex. Protein expression of the wildtype and modified LtxA constructs generated in this study could not be consistently determined by immunoblot analyses, although Columbia agar hemolysis assays suggested that active LtxA protein was functionally expressed and secreted by the wild type LtxCABD construct and an LtxCA with a modified C-terminus together with the E. coli HlyBD expressed in trans. In preliminary experiments, the human monocytic cell line THP-1 was sensitive to wildtype LtxCABD and to the C-terminal fusion constructs when co-expressed with HlyBD by VNP20009.
188. Determining the In-Vitro Transcriptional Regulon of SigG in Nostoc punctiforme
Lee, Jamie
Nostoc punctiforme is a cyanobacterial species that is capable of adapting its morphology into several different states of which the spore-like akinetes are the least understood. A time-course array previously revealed that Npun_F4153, a putative ECF sigma-encoding gene, was significantly upregulated upon akinete induction along with Npun_F4154, the cognate anti-sigma factor to Npun_F4153. Based on sequence alignments, Npun_F4153 was initially designated as putative RpoE-like protein, SigG, with subsequent experiments having since supported this identification. In this thesis, bioinformatics analysis suggests that Npun_F4154, designated SapG, shares homology with RseA-like sequences in organisms both from its own phylum and others. A knockout mutant strain of sigG, previously thought to not show any difference in phenotype from a WT strain, was discovered to exhibit a more buoyant cell pellet compared to WT N. punctiforme in log-phase vegetative cells. Furthermore, nitrogen deprivation in an attempt to induce heterocysts resulted in a massive rate of hormogonia formation in sigG mutant relative to WT. Attempts to complement the mutant were unsuccessful. Notably, sigG mutant showed increased susceptibility to lysozyme stress in both vegetative and akinete-induced strains, while akinete-induced mutant showed increased susceptibility to cold stress relative to akinete-induced WT cells. Once these phenotypic findings were established, N. punctiforme RNA polymerase core enzyme, as well as hexahistidine-tagged SigG protein were expressed and purified. Selected promoter templates based on previous in-vivo regulon results were also amplified, of which one was shown to be a direct target of SigG under heterocyst-inducing conditions. Encouraging preliminary results using these purified products with selected promoter templates and optimized in-vitro reaction conditions culminated in a runoff-transcription microarray-analysis (ROMA) using sheared, N. punctiforme genomic DNA templates. The in-vitro regulon generated from this experiment was in turn analyzed to develop a consensus sequence logo and for in-silico SigG regulon determination. Finally, regulons from all three approaches were compared to determine the most likely gene promoter targets of N. punctiforme SigG.
189. Limnological features of a culturally eutrophic pond in Southern California
Johnson, Mark Alan
A study of a culturally eutrophic pond including structural features and their dynamics was carried out. Data analysis from measured physical, chemical, and biological parameters collected over a 15 week period from a 15 million gallon pond receiving secondary treated wastewater indicated a distinct and eutrophic character. Physical and chemical measurements revealed a marked degree of stratification and correlation for dissolved oxygen, temperature, and pH. Low Secchi disk transparencies and high concentrations of nitrogen and phosphorous are primary evidence of cultural enrichment. Evidence indicates that carbon may be acting as a limiting nutrient for the pond's phytoplankton. Phytoplankton densities and distributions varied widely. Genera common to eutrophic systems were dominant-blue-green algae of the genus Oscillatoria and green algae such as Ankistrodesmus sp. and Sphaerocystis sp. . Populations of net phytoplankton were not dependent or influenced by the measured nutrients - nitrogen and phosphorous. Photosynthetic activity clearly influences the chemical equilibrium of the pond. (See more in text.)
190. Identification of acid proteinase activity in the ascites fluid of mouse teratocarcinoma
Behringer, Richard
A mouse teratocarcinoma in our laboratory recently converted from a slow growing multicellular form (embryoid body) to an apparantly more malignant fast growing single cell form. Associated with this conversion was an accumulation of ascites fluid in which acid proteinase activity was found to be present. Inhibitor studies allowed identification of this proteinase activity as the carboxyl peptidase Cathepsin D. Mouse Cathepsin D has a pH optimum of 3.0 and is inactive at pHs higher than 5.0. Titrations of Cathepsin D by the specific inhibitor Pepstatin A yielded estimations for the amount of Cathepsin D present in the ascites fluid. These studies set the ground work for investigations into the possible role of proteinases in malignant transformation.
191. Vocal repertoire and female choice in an Ecuadorian treefrog, Dendropsophus carnifex
Oliva, Mark V.
Frogs predominantly communicate acoustically, using advertisement calls to mediate both inter- and intrasexual interactions. In many frogs, these signals comprise a single component; however, in others they are composed of distinct multiple components. Male executioner treefrogs, Dendropsophus carnifex, exhibit a multicomponent advertisement call composed of an introductory screech note followed by two or more secondary click notes. However, the vocal repertoire of this species, including spectral and temporal properties, has not been fully characterized. Therefore, the purpose of my study was to identify and characterize the different call types used by male D. carnifex, and to determine the effect of the multicomponent advertisement call on both female call recognition and choice. I conducted field recordings of calling males, and carried out three 3-choice female phonotaxis experiments. I tested (1) female phonotaxis to each call component and to a complete call, (2) female choice for differences in the number of click notes, and (3) female choice for differences in call rate. Based on previous literature on multicomponent advertisement calls, I hypothesized that females would recognize the click notes of the advertisement call as the mating signal, and that females would choose calls with more notes and at faster rates. The results demonstrate that both notes, presented separately or together as a complete call, evoked similar female phonotactic responses, suggesting that either note was sufficient to elicit a mate-recognition response. Additionally, I found that females preferred calls with greater numbers of added click notes and faster call rates. Because females preferred stimuli with greater call output, males calling with more click notes or at faster rates should experience a greater mating advantage. As calling is energetically expensive and possibly indicates male quality, selecting mates based on these call parameters may provide females with indirect benefits, such as good genes. Determining female phonotaxis towards multicomponent calls and call preferences is important in understanding the evolution of signal complexity in light of sexual selection.
192. Genetic diversity, population structure and connectivity of Millepora alcicornis (Hydrozoa: Anthomedusae: Milleporidae) in the Florida Reef Tract
Jacinto, Diana M.
Coral reefs are experiencing global declines due to changing environmental conditions triggered by climate change and anthropogenic effects impacting important reef-building organisms and their inhabitants. Millepores are calcareous hydrocorals found on shallow reefs worldwide, however little information is known about their genetic diversity and population biology. The present study sought to determine the population structure and genetic diversity of Millepora alcicornis, a branching fire coral, in reefs found in the Florida Reef Tract (FRT) and population connectivity was inferred. Five microsatellite markers were used to detect genetic differentiation between 12 sampling sites from reefs from the middle Keys and Miami within the FRT. A single panmictic population of M. alcicornis in the FRT (K=1; FST=0.001) was found with moderate levels of genetic diversity (Ho=0.426, SE=0.023; Na=6.0, SE=0.763) inferring high connectivity and gene flow among reefs in the FRT. High connectivity of M. alcicornis in the FRT along with moderate levels of genetic diversity is a hopeful indication that M. alcicornis will be better able to acclimate to changing environmental conditions.
193. Scanning electron microscope study of chemically induced surface modulations of sarcoma 180 cells
Jamgotchian, Nora
Sarcoma 180 ascites tumor cells were perturbed with the chemical agents cytochalasin B, manganese, the local anesthetics procaine and tetracaine, adrenalin, and serotonin. These agents induced topographical modulations at the cell surface which were examined with the scanning electron microscope. Cytochalasin B treatment resulted in large surface blebs and fewer surface microvilli than S-180 control cells. Treatment with the local anesthetics showed shorter, stubby microvilli with a few blebs on the cell surface, while in the manganese treated cells, microvilli expanded to form broad wedges or ruffles covering the entire cell surface. By contrast, adrenalin had little apparent effect with the exception of slightly shorter microvilli. Surface microvilli quadrupled in length in serotonin treated cells. In the combined reagents experiments, manganese had the strongest overall influence by illiciting its characteristic response of expanded microvilli while seemingly masking the influence of the other reagents.
194. Divers' preferences and proposals for underwater park management at Carmel Bay, California.
Stewart, Katherine.
The California State Park system, hereafter referred to as CalDPR, has sought for five years to include Carmel Bay, California, in its park system as an underwater park. Carmel Bay is approximately 15 km (5 mi ) in area, located between Pescadero Point, 36°33'40"N by 121°56130"W, and Carmel Point (Point Lobos) at 35°33'40"N by 121°55'42"W, immediately south of the Monterey Peninsula (Fig. 1). It is about 125 km (80 mi.) south of the city of San Francisco, and is a popular weekend trip for visitors from the Bay Area and inland cities such as Sacramento and Fresno. Since the advent of recreational diving in the 1950's, divers have found that the subtidal scenery here is equally as beautiful as the world-famous beauties of its shoreline.
195. The association of sediment size and natural enrichment with littoral foraminifera, Año Nuevo Point, California.
Shumaker, Evelyn.
An important aspect of any group of organisms from both ecologic and evolutionary points of view is the part played by that group in channeling the flow of energy within the community in which it lives. Foraminifera, marine protozoans with calcium carbonate or sand grain (agglutinated) tests, are recorded as feeding chiefly upon bacteria, small diatoms, and nannoplankton in a wide variety of marine environments (Myers, 1943; Bradshaw, 1961; Lee et al., 1966; Muller and Lee, 1969; Murray, 1963). Myers (1943) found the diet of foraminifera includes soft-bodied flagellated organisms such as gametes of larger algae as well as filamentous algae, minute eggs, spores, and cysts. Their food items are usually below 25 y in size (Lipps and Valentine, 1970). Predators upon foraminifera range from highly specialized microcarnivores that feed largely on foraminifera to less selective ones that include foraminifera in a mixed diet and to generalized feeders that ingest foraminifera along with much other material (Lacaze-Duthiers, 1856; Mare, 1942; Graham, 1955; Morton, 1958; Lipps and Valentine, 1970). The present study considers the effect of organic enrichment on the abundance and diversity of intertidal benthic foraminifera. This requires an understanding of regional differences in their habitat and local variations of rnicroenvironments within that habitat. Sediment is an important microhabitat feature and must be included in a complete study of foraminifera! assemblages.
196. Feeding habits of silversides (Family Atherinidae) in Elkhorn Slough, Monterey Bay, California.
Ruagh, Amer Ali.
The primary purpose of this study was to-investigate the feeding habits of silversides (Family Atherinidae) in Elkhorn Slough, Monterey Bay, California, since little detailed work on the feeding habits of silversides from California has been published.
197. Are non-native invasive species more phenotypically plastic than co-occurring native species?
Messineo, Charlotte
It is essential to understand the conditions that enable invasive plant species to be one of the greatest threats to biodiversity. Phenotypic plasticity can be an important contributor to invasiveness, but evidence in the literature is not consistent. Few studies have examined whether invasive species with higher capacities for plasticity than native species contribute to invaders possessing fitness advantages over natives. Furthermore, environmental factors including elevated soil nitrogen levels due to deposition from air pollution have been found to facilitate invasion success. A common garden experiment was performed to compare plasticities in performance and fitness related traits of two non-native invasive species (Hirschfeldia incana and Erodium cicutarium) and six co-occurring native California species (Eschscholzia californica, Camissoniopsis bistorta, Amsinckia intermedia, Calandrinia ciliata, Plantago erecta, and Lupinus microcarpus) in a range of soil nitrogen conditions (control 0 g/L, low 0.4 g/L, medium 1.4 g/L, and high 2.3 g/L; n=30 for each species in each nitrogen treatment). Both invaders had greater phenotypic plasticity than the six native species. However, all of the native species, except L. microcarpus, were also relatively plastic. Interestingly, H. incana and E. cicutarium demonstrated greater plasticity in traits associated with plant size. Excluding L. microcarpus, all species exhibited high capacities for plasticity in the number of flowers produced in the nitrogen treatment gradient. My results indicate that the invasiveness of H. incana and E. cicutarium is related to a high capacity for phenotypic plasticity found in performance traits that lead to increases in plant size, and that this gives them an ecological advantage over co-occurring plant species. However, this edge may be short-lived. A great decline of survival in the medium and high nitrogen treatments, among a majority of the species, suggests that even species with a plastic advantage will experience reductions in survival when nitrogen levels become too high. With nitrogen levels continuing to increase, this could have a detrimental effect on plant communities with changes in plant compositions including declines in species richness. Even the newly established competitive species will decline in numbers if the nitrogen levels become so high that they are unable to survive in such extreme conditions.
198. Isolation of acid protease from murine teratocarcinoma ascites fluid
Steiner, Robert Clayton
An acid protease that appears responsible for causing cell-cell disaggregation in a mouse ascites teratocarcinoma was partially purified and characterized. The protease was purified from the mouse ascites fluid by affinity chromatography on Pepstatin A agarose beads. The purified material yielded one broad band on silver stained native polyacrylamide slab gels and two bands on silver stained SDS, 8-mercaptoethanol, slab gels, with approximate molecular weights of 66,000 and 20,000 Daltons. The affinity purification method yielded a 46 fold purification of the protease from the ascites fluid. These studies represent a first approach to purification and characterization of an enzyme that appears to be involved in altering the adhesiveness of a specific mouse ascites tumor.
199. The occurrence of Metabacterium polyspora in guinea pig feces and a microscopic study of its endospores
New, Roger Alvin
Fecal material from 39 guinea pigs was collected and microscopically surveyed for the presence of Metabacterium polyspora, a large, Gram-positive bacterium uniquely forming many endospores. It was found in the feces of 9 of 11 adult female guinea pigs, 5 of 14 adult males, and 3 of 14 infants. The abundance was high in pregnant sows near the time of delivery. The persistence in newborn infants depended on continued contact with feces containing Metabacterium. Mechanical enrichments of M. polyspora were prepared from fecal suspensions by filtration to remove coarse plant debris followed by centrifugation to eliminate smaller bacteria. M. polyspora sporangia measured from 10 - 25 pm in length by 5 pm in width and enveloped 2 - 8 endospores. Individual endospores measured 8 pm by 2 Jlm. Scanning electron micrographs of sporangia displayed a relatively smooth cell surface. In some cases the outer envelope was folded and snugly contoured the endospores. Subpolar appendages were commonly present at one end of the sporangia; they measured about 60 nm in width and up to 720 nm in length. The spores were brightly refractile in phasecontrast illumination and retained Wirtz-Conklin's spore stain. They could not be resolved in Sacks and Alderton's aqueous polymer two-phase system. Germination depended on nutrients, was activated by ethanol, and was decreased by heating at 80°C for 30 minutes. M. polyspora failed to grow on a variety of media under aerobic and anaerobic conditions. The common occurrence of M. polyspora within the indigenous microflora of guinea pigs and current knowledge justifies that Metabacterium Chatton and Perard 1913 be described and be placed as a genus of uncertain affiliation with the endospore-forming rods and cocci in future editions of Bergey's Manual of Determinative Bacteriology.
200. Fatty acid content of membranes of Phytophthora cinnamomi in cholesterol supplemented media
Ballard, Richard Grant
Phytophthora cinnamomi Rands, a root pathogen of worldwide importance, is a member of a family of fungi that lacks the ability to synthesize sterols. Sterols are considered important as hormones, membrane structure stabilizers, and steroid precursors. The effect of cholesterol on growth, asexual reproduction, total fatty acid content and fatty acid content of polar lipids of vegetative and asexually reproducing cultures was studied. Cholesterol supplementation was shown to enhance growth and sporulation. Levels of total fatty acids, myristic acid (14:0) and linoleic acid (18:2) were shown to increase while palmitic acid (16:0) decreased with increasing amounts of cholesterol supplementation. No recognizable trends were observed in the fatty acid content of the polar lipid fraction of vegetative or sporulating mycelia, though definite rearrangements occurred with increasing levels of cholesterol supplementation. Sporulation response also increased as concentration of cholesterol supplementation increased.