Primary cilia are sensory organelles that are important for initiating cell division in the brain, especially through sonic hedgehog (Shh) signaling. Several lines of evidence suggest that the mitogenic effect of Shh requires primary cilia. Proliferation initiated by Shh signaling plays key roles in brain development, in neurogenesis in the adult hippocampus, and in the generation of glial cells in response to cortical injury. In spite of the likely involvement of cilia in these events, little is known about their characteristics. Centrosomes, which are associated with primary cilia, also have multiple influences on the cell cycle, and they are important in assembling microtubules for the maintenance of the cell’s cytoskeleton and cilia. The cilia of terminally differentiated neurons have been previously examined with respect to length, incidence, and receptors present. However, almost nothing is known about primary cilia in stem cells, progenitors, or differentiated glial cells. Moreover, it is not known how the properties of cilia and centrosomes may vary with cell cycle or proliferative potential, in brain or other tissues. This dissertation focuses first on neurogenesis in the hippocampal subgranular zone (SGZ). The SGZ is one of the few brain regions in mammals that gives rise to a substantial …

The goal of this project was to reduce processing time for forensic DNA testing without incurring significant added costs and/or the need for new instrumentation, while still generating high quality profiles. This was accomplished by: 1) extraction normalization using the ChargeSwitch® Forensic DNA Purification Kit such that a small range of DNA concentrations was consistently obtained, eliminating the need for sample quantification and dilution; 2) developing fast PCR protocols for STR primer sets using shorter amplification methods, low volume reactions and non-fast thermal cyclers; and 3) developing a quicker 3130xl Genetic Analyzer detection method using an alternative polymer/array length combination. Extraction normalization was achieved through a reduction in bead quantity, thereby forcing an increase in bead binding efficiency. Four products (AmpliTaq Gold® Fast PCR Master Mix, KAPA2G™ Fast Multiplex PCR Kit, SpeedSTAR™ HS DNA Polymerase and Type-it Microsatellite PCR Kit) were evaluated for low volume (3μl) fast PCR on a 384-well Veriti® thermal cycler with the Identifiler primer set. KAPA2G™ was selected for 3μl fast PCR protocols using PowerPlex 16 HS and Identifiler Plus primer sets (42-51min), as well as 5μl and 6μl Identifiler fast reactions on a 9700 thermal cycler (51-60min). Alternative detection (POP-6™/22cm) achieved 24-28min run times, but …

The purpose of this project was to further elucidate the effects post-exercise alcohol ingestion. This project had many novel aspects including using a resistance exercise (RE) only exercise design and the inclusion of women. To our knowledge, we are the first to investigate the effect of post-RE alcohol ingestion in women. In the first chapter of this project, information on the prevalence of alcohol use and the importance of skeletal muscle as a dynamic and metabolic tissue was provided. In chapter two, the effects of post-RE alcohol ingestion in men and women are detailed. The major findings of this study was that although RE elicited similar mTORC1 signaling both in men and in women, alcohol ingestion appeared to only attenuate RE-induced phosphorylation of the mTORC1 signaling pathway in men. The third chapter focused on examining the effects of post-RE alcohol ingestion on acute testosterone bioavailability. The primary findings of this study was that alcohol substantially elevated serum total and free testosterone concentrations during recovery from a bout of resistance exercise. The fourth chapter detailed factors that contribute to bone density in men. The major findings of this study was that young adult male long-distance runners who participated in resistance training …

Absence of functional phenylalanine hydroxylase results in phenylketonuria (PKU). Viable treatments remain few, expensive and secondary conditions such as osteopenia occur in most PKU patients. Objective 1: Given the recently described roles of gut microbes to aid host digestion, an orally administered genetically engineered probiotic as the delivery vehicle for enzyme replacement therapy was created. The engineered probiotic, pHENOMMenal, produced phenylalanine ammonia lyase with significant production of trans-cinnamate (phenylalanine cleavage product) in vitro and resulted in a reduction of 515 μM in blood phenylalanine when fed to PKU animals for 14 days (from 2307µM ± 264µM to 1792µM ± 261µM, n = 6, P < 0.05). The control probiotic produced no change in blood phenylalanine. Thus, pHENOMMenal treatment in PKU mice demonstrated engineered microbes could compensate for a metabolic deficiency of the host. Objective 2: Evaluate the PAHenu2 mouse model of PKU for a genetic discrepancy causing ocular enlargement and delayed development observed only after the PAHenu2 mutation was crossed to the C57BL/6J mouse. When compared to healthy littermates, ELISA indicated a consistent but insignificant decrease in plasma IGF-1 and an increase in ocular IGF-1 in PKU animals. SNP screening demonstrated a differential inheritance of IGF-1 alleles in healthy and …

An organisms’ diet represents an exogenous influence that often yields colossal effects on long-term health and disease risk. The overconsumption of dietary sugars for example, has contributed to significant increases in obesity and type-2 diabetes; health issues that are costly both economically and in terms of human life. Individuals who are obese or are type-2 diabetic often have compromised oxygen delivery and an increased vulnerability to oxygen-deprivation related complications, such as ischemic strokes, peripheral arterial disease and myocardial infarction. Thus, it is of interest to identify the molecular changes glucose supplementation or hyperglycemia can induce, which ultimately compromise oxygen deprivation responses. By utilizing the Caenorhabditis elegans genetic model system, which is anoxia tolerant, I determined that a glucose-supplemented diet negatively impacts responses to anoxia and that the insulin-like signaling pathway, through fatty acid and ceramide biosynthesis and antioxidant activity, modulates anoxia survival. Additionally, a glucose-supplemented diet induces lipid accumulation. Use of RNA-sequencing analysis to compare gene expression responses in animals fed either a standard or glucose-supplemented diet revealed that glucose impacts the expression of genes involved with multiple cellular processes including lipid and carbohydrate metabolism, stress responses, cell division, and extracellular functions. Several of the genes we identified are homologous …

Natural flow regimes are critical for sustaining biodiversity and river integrity. Floods and droughts form an important component of river systems and control population sizes and species diversity across space and time. Modification of flow regimes, including disruption of the timing, magnitude and duration of flooding, is a global problem, and many new impoundments are planned for large river-floodplain ecosystems in the tropics. Flow modifications may cause dramatic non-linear responses in population sizes and have lasting effects through time, but such topics are poorly investigated over multi-year scales, especially in highly diverse tropical ecosystems. Using a long-term dataset from the Upper Paraná River floodplain, Brazil, I tested for threshold and legacy effects of fish assemblages to flood attributes, such as timing, magnitude, duration, rate of change and variation. Specifically, I hypothesized that long duration, high magnitude floods would elicit threshold responses in long-distance migratory fish species and these responses result in significant legacy effects detectable over multiple years. Consistent positive threshold responses to increasing flood duration and magnitude were detected for many species and not significantly correlated with reproductive guilds. Legacy effects were prevalent (i.e. identified for more than 90% of species) and including flood attributes from previous years increased …

This study determined the effect of hypoxia on air-breathing onset and physiological and morphological characters in larvae of the air breathing fishes Trichopodus trichopterus and Betta splendens. Larvae were exposed intermittently (12/12 h daily) to 20, 17, and 14 kPa of PO2 from 1 to 40 days post-fertilization. Survival, onset of air breathing, wet body mass, O2, Pcrit were measured every 5 dpf. Hypoxia advanced by 4 days, and delayed by 9 days, the onset of air breathing in Betta and Trichopodus, respectively. Hypoxia increased larval body length, wet mass, and labyrinth organ respiratory surface of Betta, but did not affect these factors in Trichopodus. Hypoxic exposure increased O2 by 50-100% at each day throughout larval development in Betta, but had no effect on larval Trichopodus. Hypoxia decreased Pcrit in Betta by 37%, but increased Pcrit in Trichopodus by 70%. Larval Betta reared in hypoxia showed a modified heart rate:opercular rate ratio (3:1 to 2:1), but these changes did not occur in Trichopodus. Compared to Betta, the blood of Trichopodus had a higher P50 and much smaller Bohr and Root effects. These interspecific differences are likely due to ecophysiological differences: Betta is a non- obligatory air-breather after 36 dpf with …

Austrofundulus limnaeus is a species of annual killifish which inhabits ephemeral ponds in South America. The species is able to survive seasonally desiccating ponds due to their ability to produce robust embryos. The embryos of this species are capable of entering a developmental arrest, termed diapause II, which precedes the onset of drought. While in this arrested state embryos exhibit the greatest tolerance to anoxia of any characterized vertebrate at 25ºC. Furthermore, when raised at 30ºC, embryos escape the entrance to diapause II and go on to develop directly. Currently, little is known about the molecular mechanisms which induce and maintain this developmentally arrested state. In this study I have developed methods to analyze changes in histone modifications in the context of diapause II. Histone modifications were chosen due to their extreme conservation and well characterized role as modulators of gene expression in other systems. Results utilizing adapted immunobased assays show significant changes in the global amount of H3S10P, H3K27me and H3K4me, as the embryos progress from early embryogenesis through the exit of diapause. Additionally, it is revealed that there exists a degree of phenotypic plasticity with regards to the entrance into diapause II which is modulated by the environment …

In this project, the effects of dietary polyphenols on exercise-induced muscle damage and vascular health are examined. Dietary polyphenols exert well-known anti-inflammatory effects; however, how these effects are realized with respect to vascular health and EIMD is relatively unknown. I begin by reviewing the available literature surrounding the impact of three dietary polyphenols (curcumin, catechins, and quercetin) on inflammation associated with EIMD. It is well established that their primary means of anti-inflammation is through alterations of NF-κB and AP-1 transcription activities. Given this, their inclusion into training strategies seems reasonable. Consistent evidence is presented making a case for the anti-inflammatory effects of dietary polyphenols following EIMD. I follow this review up by completing an in-depth study on the consumption of curcumin prior to EIMD. I found curcumin (1000 mg/day) can reduce subjective soreness and decrease inflammation compared to placebo controls. To further understand the effects of dietary polyphenols on health, I investigate the effects of a four-week supplementation period of cocoa (catechins) on vascular. I concluded that atherogenic risk in obese women is reduced after consumption of cocoa. In addition to these experimental projects, I developed two novel methods that can be used to investigate vascular health (EMP concentration) and …

The accumulation of industrial contaminants in the natural environments have rapidly become a serious threat for human and animal life. Fortunately, there are microorganisms capable of degrading or transforming environmental contaminants. The present dissertation work aimed to understand the genomic basis of microbial degradation and resistance. The focus was the genomic study of the following bacteria: a) Pseudomonas fluorescens NCIMB 11764, a unique bacterium with specific enzymes that allow cyanide adaptation features. Potential cyanide degradation mechanisms found in this strain included nit1C cluster, and CNO complex. Potential cyanide tolerance genes found included cyanide insensitive oxidases, nitric oxide producing gene, and iron metabolism genes. b) Cupriavidus sp. strain SK-3 and strain SK-4. The genome of both bacteria presented the bph operon for polychlorinated biphenyl (PCB) degradation, but we found differences in the sequences of the genes. Those differences might indicate their preferences for different PCB substrates. c) Arsenic resistant bacterial communities observed in the Atacama Desert. Specific bacteria were found to thrive depending on the arsenic concentration. Examples were Bacteroidetes and Spirochaetes phyla whose proportions increased in the river with high arsenic concentrations. Also, DNA repair and replication metabolic functions seem to be necessary for resistance to arsenic contaminated environments. Our …