<p> <b>Statement:</b> A global <i>Rbp1</i> knock out (<i> Rbp1-/-</i>) mouse model was used to correlate direct retinoid measurements with vitamin A metabolizing and atRA biosynthesizing enzyme activities, Crbp function and tissue microenvironment for the first time. </p><p> <b>Methods:</b> atRA was quantified by LC-MRM³ and ROL/RE/RAL was quantified by HPLC-UV. Enzyme activities were measured from enzymes present in subcellular fractions isolated from WT and <i>Rbp1-/- </i> tissues. Mouse CrbpI and CrbpIII were purified from transformed <i> Escherichia coli</i> for functional comparative studies. Tissue were formalin fixed for histological examination. Relative gene expression was analyzed using quantitative PCR. </p><p> <b>Results:</b> Reduced atRA was consistently quantified in extrahepatic tissues with elevated ROL/RE. Relative gene expression showed altered expression in retinoid pathway proteins and atRA loss preceded expression changes in some cases. Tissue microenvironments also consistently showed a loss of structure and organization along with accumulation of extracellular matrix and hyperplasia without apparent disease. Functional studies showed that CrbpIII binds retinol with less affinity than CrbpI and does not function equivalently to CrbpI in regulation of atRA biosynthesis. Also, metabolizing enzymes had altered activities in the <i>Rbp1-/-</i> tissues with reduced atRA biosynthesis. </p><p> <b>Conclusions:</b> Loss of CrbpI results in altered regulation of enzyme activity and atRA homeostasis cannot be maintained by other Crbp homologs in extrahepatic tissues. Dysfunctional atRA biosynthesis due to loss of CrbpI results in altered tissue microenvironment characteristic of dietary vitamin A deficiency and precancerous dysfunction associated with cancers that are observed to have silenced CrbpI.</p>

<p> In both mice and humans, males and females exhibit differences in behavior and response to neurological and psychological diseases that are linked to the cortex and hippocampus. The perinatal exposure of males to testosterone secreted by the testes creates alterations in neural structures and behaviors that can persist throughout their lives; however, the molecular mechanisms that underlie the actions of sex steroids to produce these lasting changes are still unclear. Given that regulation of gene expression is a primary mechanism whereby sex steroids exert changes to an organism, I sought to identify genes expressed at different levels between the sexes in the cortex and hippocampus and to determine the effect of testosterone on expression of these genes. Using gene expression microarrays and RT-qPCR, I identified genes that are differentially expressed between the sexes in the neonatal mouse cortex and hippocampus; however, whether perinatal testosterone is regulating these differences remains unclear.</p>

<p> The vacuole in <i>Saccharomyces cerevisiae</i> serves as a model for the mammalian lysosome. In a genome wide screen for mutants with severe growth <u>h</u>ypersensitivity to <u>h</u>ygromycin B, our lab identified 14 <i>HHY</i> genes. Each of the <i> hhy</i> mutants is defective in vacuolar trafficking and/or function and also sensitive to rapamycin and caffeine, suggesting a compromised target of rapamycin (TOR) kinase pathway. My research divides the <i>hhy</i> mutants into two groups based on quantitative growth analyses in the presence of hygromycin B. (1) a super affected group (<i>s-hhy's</i>) and (2) a dose-dependent group (<i>d-hhy's</i>). The <i>s-HHY</i> genes include <i>CHC1, DRS2, SAC1, VPS1, VPS34, VPS45, VPS52,</i> and <i>VPS54</i>. Evaluation of the known functions of <i> s-HHY</i> gene products reveals vesicular trafficking function at the trans-Golgi and late endosome interface to be a common factor. In yeast, the TORC1 complex localizes to the vacuole. Because a compromised TORC1 complex signaling is suggested in <i>hhy</i> mutant strains due to their caffeine or rapamycin sensitivity, I hypothesized that compromised TORC1 signaling in <i>HHY's</i> may be due to defects in the vacuolar localization of Tor1 kinase. To assess Tor1 kinase localization, we utilized a strain expressing endogenously tagged Tor1-GFP and assayed localization to the vacuolar membrane in each of the s-hhy mutants using confocal microscopy. In wild-type cells, Tor1-GFP co-localizes with the vacuolar membrane marker FM4-64 while s-hhy deletion strains fail to localize Tor1-GFP to the vacuolar membrane when treated with hygromycin B. Our results implicate that Tor1p is transported to the vacuole membrane via the late endosome (CPY pathway) and not the ALP (Vam3) pathway. Additionally, <i>s-hhy</i> mutants are unable to recover growth after a 4-hour treatment with hygromycin B, similar to EG0 mutants, which fail to exit from G0 after treatment with the TORC1 inhibiting drug rapamycin. Based on our data, we propose a model in which the <i>s-HHY </i> gene functions in vesicular trafficking at the trans-Golgi/late endosome interface are involved in recruitment and subsequent transport of Tor1p to the vacuolar membrane, and that interface is hypersensitive to hygromycin B. We also propose that Tor1 kinase localization at the vacuole is essential for its cell cycle regulatory function.</p>

<p>The success of Drosophila as a system for genetic analysis is closely linked to its amenability to genetic manipulation. Part 1 of the dissertation elucidates a novel scheme for long-range targeted manipulation of genes. We integrated an 80-kb genomic fragment at its endogenous locus, utilizing a targeted attP attachment site for the phiC31 integrase. We achieved single-copy reduction of the resulting region duplication by inducing recombinational DNA repair. We showed that this two-step scheme of integration and reduction is efficient and useful for delivering modifications. We established a vector configuration that facilitates the recovery of modifications. The integrating genomic fragment allowed for delivery of a new attachment site at 70 kb from the existing <i>attP</i> into a new locus, making it susceptible to targeted mutagenesis. We extrapolate that with this scheme, only 1 200 lines bearing att-sites throughout the genome would suffice to render all Drosophila genes amenable to targeted mutagenesis. Excitingly, this method should be readily applicable to other systems. </p><p> In Part 2 of the dissertation, I explored the question of telomere organization in Drosophila. Telomeres demarcate the ends of linear chromosomes to distinguish them from broken ends. In yeast, they cluster at the periphery of the nucleus establishing a compartment of silent chromatin. To bring insight into telomere organization in a higher organism, we followed EGFPlabeled Drosophila telomeric protein HOAP <i>in vivo</i> and found that the 16 telomeres cluster into 4-6 foci per nucleus in somatic tissues. Interestingly, HOAP signal intensity in the clusters doubles in interphase, potentially due to loading of HOAP to newly replicated telomeres. We tested several predictions about rules governing clustering. First, by inspecting mutant embryos that develop as haploids, we found that clustering is not mediated by associations between homologs. Second, by demonstrating clustering capability for a telomere of novel sequence, we eliminated DNA sequence homology and identity as important factors. Third, by marking both ends of a chromosome, we ruled out predominance of intra-chromosomal interactions. We propose that clustering is indiscriminate of sequence and is likely maintained by a yet undetermined factor. </p>

<p> We have used the <i>C. elegans</i> primordial gonad to understand how stem cells assemble into a niche during development. The <i>C. elegans </i> primordial gonad contains two somatic gonad precursor cells (SGPs) and two primordial germ cells (PGCs). The primordial gonad assembles during embryogenesis when PGCs and SGPs come together adjacent the intestine. </p><p> As a first step in understanding niche assembly, we investigated how PGCs move to the site where the primordial gonad forms. PGCs and somatic cells move into the interior during gastrulation. Because somatic cells require transcription to ingress whereas PGCs are transcriptionally quiescent, we hypothesized that somatic cells might push or pull the PGCs into the embryo. We used videomicroscopy to identify cells that contact the PGCs, and used laser killing to determine if the contacting cells are required for PGC ingression. The PGCs are surrounding by adjacent mesodermal cells and internal endodermal cells. We found that the only contacting cells necessary for PGC ingression were the endodermal cells, which ingress into the embryo an hour before the PGCs. Killing or altering the fate of the endodermal cells prevented PGC ingression but not ingression of other somatic cells. Using fluorescent membrane markers and live imaging, we showed that PGCs and endodermal cells maintain contact throughout gastrulation, and that endodermal cells move dorsally as PGCs ingress form the ventral surface. PGCs express high levels of E-cadherin/HMR-1, and knocking down E-cadherin/HMR-1 caused PGCs to detach from endodermal cells and remain on the surface of the embryo. Finally, we show that the enrichment of HMR-1 protein in the PGCs is not due to transcriptional upregulation, but is instead due to an increase in protein expression mediated by the hmr-1 3' UTR. We propose that PGCs upregulate E-cadherin/HMR-1 to maintain tight adhesion with endodermal cells, which pull the PGCs into the embryo and position them at the site of primordial gonad assembly. Our results highlight the importance of germ cell - gut interactions during development and of E-cadherin-mediated adhesion in niche formation.</p>

<p> Human genes are split into regions that code for protein, exons, and regions that don't, introns. Upon transcription, the removal of these intervening introns is necessary if a usable mRNA molecule is to be translated. The process of intron removal and subsequent ligation of exons is called splicing and is carried out by a large complex called the spliceosome. This process is driven by sequence elements within the pre-mRNA itself and is the major contributor of diversity to the human transcriptome. Due to the ubiquitous nature of alternative splicing in almost every multi-exon gene, the regulation pathways of exon inclusion are a subject of wide study. </p><p> The different lengths of introns and exons as well as location of splice sites in a pre-mRNA molecule have been shown to have differing affects on the spliceosomes ability to recognize them. Using <i>in vitro</i> splicing and complex formation assays in parallel with cell transfection experiments, we determined that the distance between two splice sites across the intron or across the exon are strong predictors of splice site usage. Additionally, we found that two splice sites interact differently when placed at different lengths apart. Our findings suggest a mechanism for observed selection of specific intron/exon architectures. </p><p> Splice site recognition is also influenced by the presence of protein binding sequence elements in the pre-mRNA that alter spliceosomal recruitment. Previously, these proteins and sequence elements had been rigidly classified into splice enhancing or inhibiting categories. We show that this rigid classification is incorrect. We found that the location of these elements relative to the splice site determines their enhancing or silencing effect. That is, an enhancing element found upstream of a splice site imposes a silencing effect when relocated downstream of the splice site (and vice versa). </p><p> Spliceosomal proteins are conserved from yeast to humans. The sequence elements used in pre-mRNA sequences have been evolving over time but under pressure from multiple cellular processes, including splicing. To observe the effect of splicing on evolution, we took advantage of the synonymous mutation positions that are under the least amount of selective pressure from the genetic code. We mutated these positions and found that some caused a large decrease in exon inclusion. When we analyzed the comparative alignment data, we found that these specific nucleotide mutations were selected against across species in order to maintain exon inclusion. SNP analysis showed that this pattern of selection was broadly observable at synonymous positions throughout the human genome.</p>

<p> The redistribution of nitrogen from old leaves to young leaves during senescence is crucial for plant survival. The <i>Arabidopsis thaliana </i> chloroplast residential protease S33Ab (At5g11650) was predicted to contribute to the degradation of chloroplast stromal proteins during senescence by bioinformatics tools. Rubisco degradation did not co-segregate with the T-DNA insertion mutant <i>s33Ab.</i> The location of T-DNA insertions into <i>S33Ac</i> (At1g18360) and <i>S33Ad</i> (At1g73480) were confirmed by sequencing. Total leaf proteins from <i>Ab, Ac, Ad </i> single, <i>Ab/Ac, Ab/Ad, Ac/Ad</i> double and <i> Ab/Ac/Ad</i> triple mutants were isolated from senescing leaves, subjected to immunoblot analysis to quantify Rubisco large subunit (LSU), glutamine synthase 2 (GS2), Rubisco activase (RCA), and light harvesting complex protein for photosystem II, b (Lhcb1) and compared to wild type. There is no significant difference among all the genotypes. This genetic analysis shows that S33Ab and its two closely related homologs, S33Ac and S33Ad do not individually contribute or work additively in chloroplast stromal protein degradation.</p>

<p> Between 8% and 10% of patients diagnosed with common variable immunodeficiency (CVID) have known mutations in the gene coding for transmembrane activator and calcium-modulating ligand interactor (TACI). Family members of CVID patients carrying the same mutations, but lacking clinical symptoms of CVID, have left the role of TACI in hypogammaglobulinemia unclear. There are two described isoforms of TACI observed at the mRNA and protein levels in humans, a long and a short form, identical save for the exclusion of the second cysteine-rich domain in the short form. The aim of this study was to determine which isoforms contain mutations in a subset of patients and their healthy relatives all known to carry at least one heterozygous mutation in TACI at the genomic level. All patients and healthy relatives carried TACI mutations within their long isoforms. However, in the cohort examined, mutations in the short isoform were only present in CVID patients and one healthy relative. These results may implicate the short isoform of TACI in CVID progression. Our lab has observed that the short isoform induces plasma cell differentiation more efficiently than the long, so this data also supports a more active role for the short isoform in B cell immunology. Mutation localization within additional CVID patients and mutation carrying relatives must be studied to determine if this trend is still significant.</p>

<p> The ascomycete fungus <i>Magnaporthe oryzae,</i> causative agent of rice blast disease, poses a threat to global food security, destroying enough rice to feed 60 million people each year. Characterization of the host-pathogen interaction between rice and <i>M. oryzae</i> is critical, as better understanding of the system may lead to better disease control strategies. The sequenced genome and repertoire of molecular tools available have made <i> M. oryzae</i> an ideal model system for understanding general plant-pathogen interactions as well. </p><p> The objective of this dissertation was to characterize the <i>M. oryzae</i> homologs of <i>Histoplasma capsulatum RYP</i> (<i><u> R</u>equired for <u>Y</u>east <u>P</u>hase </i>) genes that are required for transition to the parasitic phase. <i> H. capsulatum</i> is a human pathogen that undergoes a dimorphic switch from filamentous to yeast cell growth at 37&deg;C, the host body temperature. Four<i>H. capsulatum RYP</i> genes were identified in a forward genetic screen to identify genes required for entry into the yeast phase. <i> RYP1</i> is a member of the Gti1_Pac2 family, which contains previously characterized regulators of dimorphic switching. <i>RYP2</i> and <i> RYP3</i> are homologs of <i>vosA</i> and <i>velB,</i> members of the Velvet family, best characterized in <i>Aspergillus nidulans, </i> where they coordinate morphological differentiation with secondary metabolism. <i>RYP4</i> is a zinc binuclear cluster protein, a main class in the zinc finger transcription factor family. Deletion of the <i> M. oryzae RYP1</i> homolog, <i>RIG1</i> (<i><u> R</u>equired for <u>I</u>nfectious <u>G</u>rowth </i>), resulted in a non-pathogenic mutant on susceptible rice cultivars, even upon removal of the host penetration barrier. <i>&Delta;rig1</i> was blocked in the transition to infectious hyphal growth, similar to <i> H. capsulatum ryp1,</i> which could not transition to the yeast phase. Deletion mutants of <i>M. oryzae RYP2, RYP3,</i> and <i>RYP4 </i> homologs were similar to the wild type in somatic growth and pathogenicity indicating that although <i>RIG1</i> is a pathogenicity factor conserved in plant and animal pathogens, such conservation does not apply to all of the <i>RYP</i> pathogenicity genes identified in <i>H. capsulatum. </i> </p><p> <i>&Delta;rig1</i> is the first <i>M. oryzae</i> mutant known to be blocked in production of primary infection hyphae. Overall, the study suggests limited parallels exist in phase transition of fungal pathogens of plants and animals.</p>

<p> Coordinated movement of cardiac valves controls unidirectional flow of the blood with every heart beat. Cardiac valves are composed of thin, pliable leaflets that withstand compressive tension, fluid shear stress, and bending stress as blood flows through them. The structure and the mechanical properties of the valves render them durable during the lifetime of human beings. However, changes in hemodynamic environment, inflammatory responses, and congenital valvular defects can all cause valves to undergo irreversible structural changes, one of which is calcific aortic stenosis (CAS). CAS affects 2-3% of the population over 65 years old in the western world, and the only effective treatment is valve replacement surgery. CAS is characterized by tissue stiffening and the formation of calcified nodules, the development of which is associated with abnormal differentiation of resident fibroblasts known as valvular interstitial cells (VICs). Upon tissue injury, VICs are activated to myofibroblasts which deposit excessive collagen and stiffen the matrix. Understanding how the pathogenic phenotype of VICs is regulated by cues from the matrix may lead to new therapeutic treatments for CAS. In this thesis, I examined how matrix elasticity and TGF-&beta;1 regulate VIC phenotypes. First, I characterized the VIC population from porcine aortic valves and showed that this population is relatively homogeneous. When I cultured these primary cells on different substrates, I found that poly(ethylene glycol) hydrogels mimicked the native valve matrix better than tissue culture polystyrene plates with respect to preserving the quiescent fibroblast phenotype. At the level of signaling, I demonstrated that this is mediated through an elasticity-regulated PI3K/AKT pathway. Additionally, I showed that reduced matrix rigidity redirected activated valvular myofibroblasts into dormant fibroblasts without inducing significant apoptosis. Finally, I examined the effect of TGF-&beta;1 on VIC gene expression over time with microarray-based gene expression profiling and found that TGF-&beta;1 up-regulated cell-cell contact proteins (e.g., OB-cadherin, N-cadherin) in order to regulate valvular myofibroblast activation. Collectively, my thesis work revealed novel mechanosensing mechanisms employed by VICs to respond to matrix elasticity and explored the complex interactions among multiple extracellular cues, including matrix elasticity, TGF-&beta;1 and cell-cell adhesion, to direct the cellular fate of VICs.</p>