<p> Many reptiles display temperature-dependent sex determination (TSD), in which the primary sex is determined by incubation temperatures rather than sex chromosomes. However, temperature is not the only factor that play critical roles in sex determination in the species with TSD. Previous studies in the snapping turtle, a species with TSD, showed that dihydrotestosterone (DHT) induces ovary development at temperatures that normally produce males or mixed sex ratios. In addition, the feminizing effect of DHT was found to be associated with increased expression of the ovary-determining gene <i>Foxl2</i>, suggesting a potential androgen-<i>Foxl2</i> regulatory mechanism. This dissertation aims to clarify the molecular mechanisms underlying TSD in several aspects. First, determine the role of androgen in TSD; second, identify novel thermosensitive genes involved in TSD and lastly, reconstruct gene regulatory networks underlying sex determination. </p><p> To test the hypothetical androgen-<i>Foxl2</i> interaction, I cloned the proximal promoter (1.6 kb) and coding sequence for snapping turtle <i> Foxl2</i> (t<i>Foxl2</i>) in frame with mCherry, a red fluorescent protein. The t<i>Foxl2</i>-mCherry fusion plasmid or mCherry plasmid were stably transfected into mouse KK1 granulosa cells. Although expression of t<i>Foxl2</i>-mCherry was not affected by androgen treatment in KK1 cells, androgen inhibited expression of the endogenous mouse Foxl2 gene, suggesting the androgen-Foxl2 interaction does exist but it differs between species. We also found t<i>Foxl2</i>-mCherry potentiated low dose DHT effects on aromatase expression, which has not been reported in any other studies. </p><p> To identify novel sex-determining genes in TSD, I first <i>de novo </i> assembled and annotated the transcriptome of the snapping turtle using next-generation sequencing (NGS) and then performed RNA-seq analyses on the newly assembled reference transcriptome. With the differential gene expression analyses, I identified 293 thermosensitive genes. Among these genes, I find AEBP2, JARID2, and KDM6B of particular interest because these genes could influence expression of many other genes via epigenetic modifications. </p><p> To further investigate the molecular mechanisms underlying sex determination, I reconstructed gene regulatory networks using an entropy based network reconstructing algorithm&mdash;ARACNE with public microarray experiments in mouse gonads. The subsequent hub gene analyses revealed the basic molecular pathways underlying gonadal development and the master regulator analyses identified 110 candidate sex-determining genes including both known sex-determining genes and novel candidate genes. </p><p> My findings demonstrate that androgens can influence expression of key ovarian genes but further studies are needed to understand the androgen signaling in TSD. Furthermore, my study provides a first description of the snapping turtle transcriptome and the effects of temperature on transcriptome-wide patterns of gene expression during the TSP. In addition, hub genes and master regulators identified for mammalian gonad determination will guide the direction of future studies in the field of sex determination. However, additional studies are needed to validate the computational findings.</p><p>

The effect of mechano-chemically bound polypropylene modifiers on the mechanical performance and thermal-oxidative stability of polypropylene composites has been studied. The mechanical performance of unmodified polypropylene containing silane coupled glass and Rockwool (mineral) fibre was poor by comparison with a similar commercially produced glass reinforced composite; this was attributed to poor fibre-matrix adhesion. Mechano-chemical binding with unsaturated additives was obtained in the presence of a free radical initiator (di-cumyl peroxide). This process was inhibited by stabilisers present in commercial grades of polypropylene composites by chemical bond formation between the chemically bound modifier and the silane coupling agent on the fibre surface, resulting in a dramatic improvement in the mechanical properties, dimensional stability and retention of mechanical performance after immersion in fluids typically found in under-bonnet environments.A feature unique to some of these modifiers was their ability not only to enhance the mechanical properties of polypropylene composites to levels substantially in excess of currently available commercial materials, but their ability to act as effective thermal-oxidative polypropylene stabilisers. The mode of action was shown to be a chain-breaking mechanism and as a result of the high binding levels achieved during melt processing, these modifiers were able to efficiently stabilise polypropylene in the most severe volatilising and solvent-extracting environments, thus giving much better protection to the polymer than currently available commercially stabilised grades of polypropylene.

<p> Dietary fat provides essential nutrients, contributes to energy intake and regulates blood lipid levels. These functions are important to health; however, when dysregulated they contribute to dyslipidemia and increase risk for development of obesity, diabetes and cardiovascular disease. </p><p> Dietary fat absorption is efficiently mediated by the small intestine. The digested products of dietary fat in the gut lumen are taken up by enterocytes, the absorptive cells of the intestine, where they are re-synthesized to triacylglycerol (TAG). The resulting TAG is packaged onto chylomicrons (CMs) for secretion into the circulation, contributing to postprandial blood lipid levels. When levels of dietary fat are high, TAG is also packaged into cytoplasmic lipid droplets (CLDs) for temporary storage within enterocytes. The CLDs increase and then decrease overtime in response to fat consumption, indicating that the stored TAG is mobilized for secretion or to the other fates at later time points. The intestinal metabolism of CMs and CLDs together regulate the rate and the amount of TAG secreted into the circulation. The objective of this dissertation work is to explore the mechanisms through which the TAG is partitioned or mobilized to certain metabolic fates in enterocytes. </p><p> First, we investigated the effect of endurance exercise on genes of intestinal lipid metabolism using Otsuka Long-Evans Tokushima Fatty (OLETF), an obese and diabetic rat model. We found that exercise training in these animals resulted in parallel upregulations of genes involved in TAG anabolic and catabolic processes and promoted mitochondrial biosynthesis in enterocytes compared to sedentary rats. We proposed that these changes lead to a more efficient fatty acid oxidation in the intestine and a consequent reduction of intestinal TAG secretion in this model. Overall, this work highlights that endurance exercise training programs intestinal lipid metabolism, contributing to the beneficial effect of endurance exercise on improving obesity and metabolic disease. </p><p> Nest, we investigated the differential roles of acyl CoA: diacylglycerol acyltransferase 1 (Dgat1) and Dgat2, in regulating dietary fat absorption. Mice with intestine-specific overexpression of Dgat1 (<i>Dgat1</i>^Int) or Dgat2 (<i>Dgat2</i>^Int), or lack of Dgat1 (<i>Dgat1</i>^{&ndash;/&ndash;}) were previously reported to have different intestinal phenotypes in response to fat consumption and altered susceptibilities to obesity and hepatic steatosis; the underlying mechanism(s) is unknown. By conducting an ultrastructural analysis on enterocytes from these Dgat mouse models in response to fat consumption, we found that Dgat1 and Dgat2 altered intracellular TAG distribution for CM and CLD synthesis. Based on the observations in the study, Dgat1 is proposed to preferentially synthesize TAG for the subcellular pool that promotes CM expansion in the ER lumen and thus limits TAG storage in CLDs. In addition, Dgat2 is proposed to preferentially synthesize TAG for the subcellular pool that determines the number of CMs generated in the ER lumen and for storage in CLDs. In this study, we provide the mechanism of how intestinal Dgat1 and Dgat2 exert regulatory effects on postprandial blood lipid levels and whole-body physiology. Overall, this work demonstrates non-redundant cellular roles of Dgat1 and Dgat2 in dietary fat absorption. </p><p> Lastly, we investigated the regulation of lipophagy, where CLDs are targeted by autophagy and catabolized in acidic lysosomes, in enterocytes of <i> Dgat1</i>^{&ndash;/&ndash;} mice. We found an increased number of autophagic vesicle (AV) and abnormal TAG accumulation within AVs in enterocytes of <i>Dgat1</i>^{&ndash;/&ndash;} compared to WT mice, suggesting an impaired AV turnover and an inefficient lipophagy by Dgat1 deficiency. In addition, we identified that this impaired lipophagy process was due to a lysosome dysfunction, as indicated by the decreased mRNA levels of genes involved in lysosome acidification and higher lysosome pH in enterocytes of <i>Dgat1</i>^{&ndash;/&ndash;} compared to WT mice. Furthermore, we found alterations in cellular lipid composition and levels of reactive oxygen species (ROS) in enterocytes of <i>Dgat1 </i>^{&ndash;/&ndash;} compared to WT mice. These changes may possibly contribute to the lysosome dysfunction seen in <i>Dgat1 </i>^{&ndash;/&ndash;} mice. Based on the results in the study, we propose that the lysosome dysfunction limits lipid supply from the storage pool for secretion, resulting in a greater intestinal TAG storage and a reduced rate of intestinal TAG secretion seen in <i>Dgat1</i>^{&ndash;/&ndash;} mice. Together, this study highlights that lysosome function plays a critical role in lipophagy and that lipophagy may serve as a potential target for treating postprandial hyperlipidemia and its related diseases. </p><p> The findings presented in this dissertation expand the current knowledge of regulation of dietary fat absorption. The proposed models generated from these studies provide novel therapeutic strategies for managing postprandial blood lipid levels and preventing obesity and its related diseases.</p><p>

<p> Understanding enzyme catalysis is one of the major goals in biology. Ribonuclease A (RNase A) is a key system to understanding protein structure and function provides an attractive system to investigate the catalytic role of active site interactions. Crystal structures show a lysine residue (Lys41) situated in the RNase A active site, and mutagenesis studies suggest this residue is important for catalysis. To evaluate the catalytic importance of the Lys41-phosphate interaction, double mutant cycle analysis was used. Individual mutation of lysine to arginine (K41R) and substitution of a phosphate oxygen with sulfur led to &sim;350 and &sim;100-fold decrease in <i> k_cat/K</i>_M, respectively. However, in the K41R background, substitution of the same oxygen with sulfur decreased activity by a similar amount (within 2-fold) as it did with the wild-type enzyme. This result provides evidence that functional interaction between Lys41 and the phosphate backbone of RNA substrates may not be solely limited between the two groups.</p><p>

<p> Large intracranial arteries are unique compared to conduit vessels in the extracranial vasculature. They differ in resistance, blood flow regulation, reactivity to vasostimuli, and the accumulation of atherosclerotic plaque formation. Large cerebral vessels are atheroprotective, while conduits are atheroprone. Central to the observed differences are the vascular smooth muscle cells (VSMCs). Unique transcriptional activity in cerebral VSMCs may be responsible for the atheroprotective nature of the intracranial vasculature. Kr&uuml;ppel-Like Factor (KLF) 4 helps promote cellular de-differentiation, while KLF5 facilitates proliferation. Working together, the two KLFs may facilitate the early onset of atherosclerosis in the aorta, relative to the cerebral vasculature. The current study aimed to address whether or not those KLFs were partially responsible for the observed differences in the two types of arteries. Laser microdissection was used to isolate VSMCs from the aorta and basilar artery to measure KLF abundance using rt-qPCR.</p><p>

<p> <i>Aedes aegypti</i> mosquitoes can be found globally in tropical and subtropical urban areas and spread Zika, Dengue fever, yellow fever, and Chikungunya viruses. Current vector control methods are limited and nonspecific. The female <i>Ae. aegypti</i> mosquito uses blood meal proteins to obtain nutrients required for oogenesis; inhibition of the midgut trypsin-like serine proteases responsible for blood meal digestion may provide a novel method of vector control. <i>Ae. aegypti</i> blood meal digestion is complex and the role of uncharacterized serine proteases in blood digestion is unclear; specifically, a group of trypsin-like serine proteases (AaSPII&ndash;V) is expressed at constant levels before and following <i>Ae. aegypti</i> blood meal acquisition. This research focuses on the <i>in vitro</i> biochemical study of two specific <i>Ae. aegypti</i> trypsin-like serine proteases (AaSPII and AaSPIV) in order to gain further understanding of their role in blood meal digestion. The approach involved the successful cloning and bacterial expression of these soluble, recombinant proteases. Results from attempts to purify these proteases were unsuccessful but indicative of potential autocatalytic and autodigestive behavior. Future studies will focus on obtaining purified recombinant proteases for further study. The study of AaSPII and AaSPIV, as well as other midgut <i>Ae. aegypti</i> proteases, will aid in understanding the overall role proteases play in blood meal digestion and may eventually allow for the development of mosquito-specific enzyme inhibitors.</p><p>

<p> Evolutionary pressure to protect against phage-induced lysis and rampant horizontal gene transfer has created a wide repertoire of defensive pathways in bacteria. CRISPR-Cas (<u>c</u>lustered <u>r</u>egularly <u> i</u>nterspaced <u>s</u>hort <u>p</u>alindromic <u> r</u>epeats, <u>C</u>RISPR-<u>a</u>ssociated) systems are adaptive immune pathways that use RNA-guided nucleases to direct cleavage of invading nucleic acids. The programmable nature of these enzymes has enabled a revolution for DNA-targeting applications including gene editing, transcriptional control, and genomic imaging. In addition to DNA-targeting enzymes, specific subtypes of CRISPR-Cas systems recognize and degrade single stranded RNA (ssRNA) substrates. Repurposing these ssRNA-targeting enzymes into biotechnological tools is currently limited by a lack of mechanistic information. In this work, we address this issue by redirecting a well-studied DNA-targeting CRISPR nuclease, Cas9, to ssRNA targets and investigating the enzymatic mechanisms of a novel ssRNA-targeting CRISPR nuclease, Cas13a (formerly C2c2). </p><p> Typically, Cas9 ignores ssRNA while searching for dsDNA targets due to ssRNA&rsquo;s inherent single-stranded structure and lack of a <u> p</u>rotospacer <u>a</u>djacent <u>m</u>otif (PAM). We redirected Cas9 to bind and recognize ssRNA targets through addition of a third component, a target-complementary DNA oligonucleotide or PAMmer, that provides a DNA:RNA hybrid PAM. Using primary microRNAs as a model system, we provide proof-of-concept evidence that Cas9:PAMmer complexes can mediate the isolation and subsequent mass spectrometry analysis of protein complexes bound to specific RNAs. </p><p> The complexity of redirecting Cas9 to ssRNA substrates motivated us to investigate CRISPR proteins that natively target RNA. We focused on Cas13a, a predicted ribonuclease from Type VI CRISPR-Cas systems. We discovered that Cas13a possesses two distinct catalytic activities, one for site-specific cleavage of its CRISPR RNA (crRNA) and the second for nonspecific ssRNA degradation activated by target binding. These insights allowed us to establish a revised model for ssRNA-targeting by Type VI CRISPR-Cas systems. Through biochemical characterization of the entire Cas13a protein family, we revealed hidden diversity in substrate preferences and defined orthogonal enzyme subfamilies. These diverse Cas13a homologs can be harnessed in parallel for detection of distinct RNA species within complex mixtures for both bacterial immunity and diagnostic applications. Together, this work presents two novel biotechnological applications of CRISPR-Cas nucleases for RNA isolation and RNA detection.&not;&not;</p><p>

<p> Sanfilippo syndrome type B (Mucopolysaccharidosis type IIIB; MPS IIIB) is a lysosomal storage disorder affecting primarily the brain and is characterized by profound intellectual disability, dementia, and a lifespan of about twenty years. The cause is a mutation in the gene encoding &alpha;&ndash;<i> N</i>-acetylglucosaminidase (<i>NAGLU</i>), a lysosomal enzyme, leading to the deficiency of NAGLU and accumulation of heparan sulfate. I am investigating a stem cell gene therapy approach in a <i>Naglu-/-</i> mouse model. I think that iNSCs overexpressing NAGLU can engraft and reduce neural pathology in the mouse model. Here I report that NAGLU overexpressed in neural stem cells derived from induced pluripotent stem cells (iNSCs) is capable of being taken up by deficient cells. I used flow cytometry and Lysotracker to demonstrate that NAGLU can reduce deficient cells&rsquo; lysosomal volume <i> in vitro</i>, suggesting that NAGLU treatment has a biological effect. iNSCs overexpressing <i>NAGLU</i> were injected into the brains of 1 day old <i>Naglu-/-</i> mice. iNSCs were detected 10 weeks after injection. Brain sections possessed NAGLU activity greater than or equal to heterozygous controls, activity was detected distal to injection sites, and transplanted animals showed reduction in LAMP1, GFAP, and CD68. The results suggest that engineered iNSCs could be used to deliver enzyme and treat MPS IIIB.</p>

<p> There are approximately 230,000 new diagnoses of prostate cancer every year in the U.S., making prostate cancer the most diagnosed cancer in men. It is responsible for approximately 30,000 deaths per year, with only lung cancer taking more lives. An important distinction must be made in men with prostate cancer. The majority of men with prostate cancer have a relatively indolent form of the disease, meaning high survival rates (100% survival 5 years after diagnosis) and no invasion of the tumor to other organs. However, approximately 4% of men are diagnosed with an aggressive form of the disease, and for these men, the survival rate is a mere 30% after 5 years. And for many patients, it is clinically difficult to differentiate between the indolent and the aggressive forms of the disease. Therefore, it is imperative to develop new genetic models of prostate cancer, and the mouse is an excellent model organism in which to do so. In 2009, mice were used to discover a new type of stem cell, called a castration-resistant Nkx3.1-?expressing cell in the luminal cell population of the prostate. We have used a mouse model targeting these cells to study the roles of two tumor suppressors, adenomatous polyposis coli (Apc) and Smad4. Apc down-regulates the Wnt signaling pathway, which is a carcinogenic pathway in the prostates of humans and mice. Deletion of Apc in mice causes an increase of Wnt signaling and prostate cells to proliferate but not invade, which represents a relatively indolent, precancerous phenotype. Smad4 is a transcription factor that controls the signaling of two pathways: transforming growth factor &beta; and bone morphogenetic protein signaling. Deletion of Smad4 causes these pathways to shut off. When Apc and Smad4 are deleted simultaneously, mice develop aggressive, invasive prostate cancer. This work suggests that these two tumor suppressors &ndash; and the pathways they control &ndash; are important regulators of prostate cancer, could allow for clinicians to differentiate between indolent and aggressive disease, and should be targeted therapeutically in prostate cancer patients.</p><p>

<p> The word "cancer" rarely stands alone, usually prefaced with its anatomical location: lung cancer, prostate cancer, brain cancer. With the advancement of high-throughput omics approaches, specific oncogenic events are reorganizing the landscape of cancer classification, at once creating commonalities between cancers arising in diverse anatomical locations and dividing organ-centric classifications of cancer into a multitude of subtypes. The term "precision medicine" postulates that these new, data-driven groupings based on molecular characterization are the key to making rational therapeutic choices. </p><p> The majority of this dissertation addresses the disconnect between extensive molecular characterization and poor cancer therapy outcomes for patients with glioblastoma multiforme (GBM). Despite clear evidence that hyperproduction of the ligand for PDGFR (platelet-derived growth factor receptor &alpha;) is sufficient to generate GBM of the proneural subtype, anti-PDGFR&alpha; therapeutics have proven disappointing in clinical trials. Cell adaptation contributes to therapeutic escape. In GBM, proneural tumor cells adopt transcriptional profiles of the mesenchymal subtype. The interconversion between the proneural and mesenchymal transcriptional classes within a tumor population presents both a challenge and an opportunity for therapeutic approaches. The proneural subtype has a proliferation phenotype and presents druggable targets such as PDGFR&alpha;. The mesenchymal subtype presents an invasive phenotype, but the targets are more challenging to drug. The typical screening for combination therapies that synergize to induce cell death is not as advantageous here, where the disease management is expected to include cytostatic drugs that act on two different aspects of the phenotype: proneurally mediated proliferation and mesenchymally mediated invasion. This work examines the applicability of a combination approach against a proneural target, PDGFR&alpha;, and mesenchymal targets in the STAT3 (signal transducer and activator of transcription 3) pathway, in the context of a proneural model of GBM.</p><p> The work is concluded with collection of work applying precision medicine in other disease contexts, most notably canine lymphoma.</p><p>