Because Drosophila do not grow after initiation of metamorphosis, their final body size is determined by larval growth rate and duration of the larval growth phase. Drosophila metamorphosis is triggered by the steroid hormone ecdysone, which is produced in the prothoracic gland (PG). Ecdysone synthesis requires expression of the "Halloween" genes, which encode ecdysone biosynthetic enzymes. Growth rate is regulated by Insulin-like peptides, which are released from the insulin-producing cells (IPCs). Genetic ablation of the IPCs decreases growth rate and delays onset of metamorphosis, suggesting that ecdysone synthesis is induced by insulin signaling. Inhibiting PI3 Kinase (PI3K), the major effector of insulin signaling, in the PG similarly delays metamorphosis as a consequence of decreased ecdysone synthesis and decreased Halloween gene expression. In contrast, activating PI3K in the PG advances the onset of metamorphosis and increases Halloween gene expression. Here I report that increased insulin signaling, accomplished inhibiting the protein kinase A pathway in the IPCs increases insulin signaling and increases growth rate but also advances the onset of metamorphosis by increasing expression of at least one Halloween gene. Ecdysone synthesis is promoted by a second peptide hormone, PTTH, which activates Halloween gene expression via the Torso receptor followed by Ras and Raf in the PG. Null mutations in the transcription factor broad (br ) prevent torso transcription and thus prevent Halloween gene expression and metamorphosis. Here I identify Br as the mechanistic link between PI3K activity and Halloween gene expression. I found that PI3K activity is required for br expression by inhibiting the downstream kinase GSK-3. I provide evidence that three nuclear hormone receptors, βFTZ-F1, HR3 and E75, link GSK-3 activity with br expression: RNAi-mediated βFTZ-F1 or HR3 knockdown, or E75A overexpression, in the PG prevents br expression. I also found that ectopic Torso pathway activation, accomplished by expressing the constitutively active Rafgof , restores Halloween gene transcription to larvae lacking br or βFTZ-F1 , suggesting that these larvae fail to express Halloween genes because they fail to transcribe torso . These studies identify a potential molecular mechanism linking growth rate with competence to respond to the PITH metamorphic signal and thus initiate metamorphosis.

<p>Traditional <italic>Drosophila</italic> hearts screens have focused on early patterning and development, and adult heart phenotypes have only recently been pursued due to difficulty in accurately measuring cardiac function in adult <italic>Drosophila</italic>. For my dissertation I performed a screen in <italic>Drosophila</italic> using optical coherence tomography (OCT) to phenotype cardiac function in awake, adult <italic>Drosophila</italic>, in order to discover novel disease-causing and disease-modifying genes for heart failure. I initiated a screen of X chromosome deficiency stocks for mutants displaying abnormal cardiac function in the adult, and I identified two mutant strains from the X chromosome with the phenotype of dilated cardiomyopathy. These deficiencies of 125kb and 92kb each correspond to 10 and 16 deleted genes in each, respectively. Interestingly, the candidate genes did not include any sarcomeric proteins, nor any proteins previously implicated in heart function. Utilizing genetic tools including customized deletions, RNAi constructs, and transgenic rescues, I identified the causative gene in each deficiency. I show that cardiomyopathic genes can be identified in adult <italic>Drosophila</italic> using genetics and noninvasive phenotyping methodologies.</p> / Dissertation

<p>Non-alcoholic fatty liver disease (NAFLD) is now the most common liver disorder in Western countries. NAFLD is characterized by hepatic steatosis, or an intracellular accumulation of lipids in hepatocytes. An excess of intracellular lipids can cause toxicity, leading to hepatocyte cell death and progression of NAFLD to Non-alcoholic steatohepatitis (NASH). NASH is very severe and is additionally characterized by inflammation and fibrosis, leading to liver failure. In order to develop appropriate strategies to prevent the progression from NAFLD to NASH, it is essential to define the molecular mechanisms leading to hepatocyte cell death that promote this transition.</p><p>Since caspase-2 has been reported to respond to different types of intracellular metabolic stress to promote apoptosis, we investigated whether this enzyme promotes lipid-induced apoptosis, or lipoapoptosis, in tissue culture cells. We discovered that the enzymatic activity of caspase-2 is activated by saturated free fatty acids, and that expression of caspase-2 is necessary for lipoapoptosis in multiple hepatocyte cell lines. We also explored the role of caspase-2 in lipoapoptosis in vivo using a mouse model of NASH and patient biopsies. We found caspase-2 expression increases in the liver of both patients with NASH and in mice fed a methionine choline-deficient (MCD) diet, a model for NASH. In addition, we fed WT and caspase-2 knockout mice the MCD diet and observed a decrease in both apoptotic and fibrotic markers in caspase-2 knockout mice, indicating caspase-2 promotes lipoapoptosis in vivo. Finally, we investigated the molecular mechanism of lipotoxicity-induced caspase-2 activation, and identified a novel caspase-2 binding protein, TRAF3. TRAF3 knockdown was also able to reduce lipoapoptosis in cultured hepatocytes. Reducing the binding between these two proteins decreased the pro-apoptotic activity of caspase-2. In conclusion, we identified caspase-2 as a key protein in the regulation of lipoapoptosis both in vitro and in vivo, and discovered a novel regulator of caspase-2 activity. This work furthers our knowledge on the mechanisms leading to NAFLD progression, and may inform future studies in the development of treatment options to limit this progression.</p> / Dissertation

<p> Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the third leading cause of cancer death in the United States. Twin cohort studies indicate that inherited susceptibility accounts for approximately 35% of all CRC cases, but only 5-6% of CRC cases can be attributed to known functional mutations. We were the first to identify a germline mutation in Transforming Growth Factor Beta Receptor 1 (<i>TGFBR1</i>) that is also somatically acquired in tumors, a 9 bp in frame deletion within exon 1 (rs11466445), which results in a receptor with decreased TGF-&beta; signaling properties. The observed association between this hypomorphic variant and cancer risk led us to hypothesize that constitutively decreased TGF-&beta; signaling may contribute to the development of CRC. </p><p> In this dissertation, we developed a novel mouse model of <i>Tgfbr1 </i> haploinsufficiency (<i>Tgfbr1</i>^+/&minus;) and found that <i>Tgfbr1</i>^+/&minus; mice were twice as likely as <i>Tgfbr1</i>^+/+ mice to develop CRC. We subsequently identified two human haplotypes associated with constitutively decreased <i>TGFBR1</i> expression and CRC risk and found that decreased <i> TGFBR1</i> expression is strongly associated with three SNPs: rs7034462, rs11466445 and rs11568785. Further examination of <i>TGFBR1</i> haplotype tagging SNPs suggests that the <i>TGFBR1</i> rs7034462-TT is a novel moderate penetrance risk genotype, which has high penetrance among African Americans, the ethnic group with the highest risk for CRC. Our results provide strong support for the novel notion that rs7034462-TT is a potentially clinically relevant CRC susceptibility genotype that may identify individuals at high risk of dying from CRC.</p>

<p> Mitosis ensures the proper segregation of chromosomes into daughter cells, which is accomplished by the mitotic spindle. During fission yeast mitosis, chromosomes establish bi-orientation as the bipolar spindle assembles, meaning that sister kinetochores become attached to microtubules whose growth was initiated by the two sister poles. This process includes mechanisms that correct erroneous attachments made by the kinetochores during the attachment process. This thesis presents a 3D physical model of spindle assembly in a Brownian dynamics-kinetic Monte Carlo simulation framework and a realistic description of the physics of microtubule, kinetochore, and chromosome dynamics, in order to interrogate the dynamics and mechanisms of chromosome bi-orientation and error correction. We have added chromosomes to our previous physical model of spindle assembly, which included microtubules, a spherical nuclear envelope, motor proteins, crosslinking proteins, and spindle pole bodies (centrosomes). In this work, we have explored the mechanical properties of kinetochores and their interactions with microtubules that achieve amphitelic spindle attachments at high frequency. A minimal physical model yields simulations that generate chromosome attachment errors, but resolves them, much as normal chromosomes do.</p><p>

<p> The yeast vacuole is a dynamic organelle that is functionally analogous to the mammalian lysosome and serves as a model for the study of membrane fusion and fission. Mechanisms of membrane fission and fusion dynamics have been well conserved from yeast to humans. However, the regulatory mechanisms that govern cellular fission and fusion dynamics remain poorly understood. Our lab has previously established that Env7 is a conserved yeast palmitoylated protein kinase that localizes to the yeast vacuole and negatively regulates vacuole membrane fusion during budding and hyperosmotic stress. Phosphorylation of Env7 is dependent on another vacuolar membrane kinase, Yck3, and is essential to Env7 stability and negative regulation of vacuolar membrane fusion. In this study, we aim to further our understanding of the role Env7 plays at the vacuole by 1) characterizing the phosphorylation of Env7 as a function of cell cycle using cell cycle arrest and synchronization techniques, and 2) generating functional biochemically tagged Yck3 to be used in interaction and phosphorylation assays with Env7. Cell cycle arrest and synchronization techniques have not previously been established in our lab. Here, we report reliable protocols of inducing cell cycle arrest using &alpha;-factor mating pheromone and Hydroxyurea. Results show that Env7 is hyperphosphorylated when cell cycle is arrested at G₁ phase using &alpha;-factor mating pheromone. In both cell cycle arrest approaches, vacuoles show significant increase in fragmentation, and Env7 remains localized to the membrane of fragmented vacuoles. In cell culture synchronized with &alpha;-factor, Env7 shows an increase in phosphorylation between S-phase and G₂, with decreased phosphorylation in M and G₁. We were successful in engineering biochemically tagged Yck3 and established that the expressed 6XHis-Yck3 is functional and able to restore phosphorylation of Env7 <i>in vivo</i>. We also established that overexpressed 6XHis-Yck3 localized correctly to the vacuolar membrane. These tools will be used in future studies on interactions and regulation of membrane fusion.</p><p>

<p> My research using the model plant <i>Arabidopsis thaliana </i> is focused on unraveling the signal transduction pathways involved in elicitor-mediated plant defense, particularly defense pathways involved in resistance to fungal pathogens. I have isolated a group of related genes, the <i>ATL</i> family, which appear to play a direct role in defense against fungal pathogens. Previous research has shown that ATL proteins can be induced by chitin and they are involved in basal resistance to the fungal pathogens. </p><p> ATL9, an <i>Arabidopsis</i> RING zinc finger protein, is an E3 ubiquitin ligase that can be induced by chitin and is involved in basal resistance to the fungal pathogen, <i>Golovinomyces cichoracearum</i> (<i>G. cichoracearum</i>). In order to understand the expression and regulation of ATL9, I studied the expression pattern of <i>ATL9</i> and the functions of its different protein domains. Using a p^ATL9:<i> GUS</i> transgenic <i>Arabidopsis</i> line I found that ATL9 is expressed in different tissues in <i>Arabidopsis</i> at various developmental stages and that GUS activity was induced rapidly upon wounding. Previous research in our lab also showed that ATL9 is a short-lived protein within plant cells and it is degraded via the ubiquitin-proteasome pathway. Protein prediction software indicated that ATL9 contains two transmembrane domains (TM), a RING zinc-finger domain, and a PEST domain. Data from confocal microscopy and western analysis indicate that both the PEST domain and the RING domain have effects on ATL9 degradation. To study the importance of these domains in ATL9's function, I constructed a series of deletion mutants and generated transgenic <i>Arabidopsis</i> plants. As expected, transgenic <i> Arabidopsis</i> containing the deletion constructs showed that both the RING domain and the TM domains are important to its resistance phenotype against <i> G. cichoracearum</i>. Interestingly, the PEST domain was also shown to be significant for the resistance to fungal pathogens. Additionally, I discovered that ATL9 can bind the defense related proteins FBS1, PCC1, and PDF1.2 directly and degrade them via the proteasome. Finally, I propose a hypothesized mechanism is proposed describing the function of ATL9 and its possible interaction with other proteins in enhancing the plant defense response.</p><p>

<p> Over half of all pregnancies worldwide occur in malaria endemic regions. Placental malaria, a serious condition caused by the malaria parasite <i> Plasmodium falciparum</i>, occurs when malaria-infected red blood cells adhere to the tissue of the placenta, with potentially devastating consequences for both mother and infant. Placental malaria infections are responsible for approximately 30% of preventable low birth weight newborns, 20% of stillbirths, and 200,000 infant deaths per year in Africa alone. Placental malaria infection is mediated by VAR2CSA, a <i>P. falciparum</i> protein that is expressed by the parasite only when in a pregnant woman, and translationally repressed outside of pregnancy. However, the mechanisms by which this repression and expression occur or, indeed, how the parasite senses when its host is pregnant are unknown. Elucidation of the genetic determinants of this specific translational regulation could provide insight for therapeutic development for placental infection. Additionally, further study of overall translation and its pharmacologic inhibition under &ldquo;normal&rdquo; circumstances may help identify novel therapies for malaria in general.</p><p> Utilizing a novel <i>in vitro</i> translation system derived from <i>P. falciparum</i> cultures, I have shown that synthesis of VAR2CSA is repressed under normal conditions, and that multiple elements in the 5&rsquo; untranslated region of the <i>var2csa</i> gene contribute to this repression. Further, this repression occurs only in <i>P. falciparum </i>, and not mammalian <i>in vitro</i> translation systems, indicating a <i>P. falciparum</i>-specific mechanism of inhibition. Importantly, I have found that circulating factors present in maternal serum during the first and second trimesters of pregnancy relieve repression of VAR2CSA translation, identifying two placental enzymes as candidate factors. Both enzymes serve to alter the pH of the microenvironment and, in fact, increasing pH in the <i>P. falciparum</i> <i>in vitro </i> translation system mimics the increase in VAR2CSA production induced by pregnant serum. Separately, I have utilized this <i>in vitro</i> translation system to identify inhibitors of translation among clinically approved antimalarial drugs and found that none utilize this mechanism of action. Importantly, this disproved the recent assertion that mefloquine inhibits translation, while also underscoring the therapeutic potential for targeting the translational apparatus as a novel and orthogonal mechanism of action. </p><p>

abstract: Malignant brain tumors are devastating despite aggressive treatments such as surgical resection, chemotherapy and radiation therapy. The average life expectancy of patients with newly diagnosed glioblastoma is approximately 15 months. One novel therapeutic strategy involves using a ketogenic diet (KD) which increases circulating ketones and reduces circulating glucose. While the preclinical work has shown that the KD increases survival, enhances radiation and alters several pathways in malignant gliomas, its impact on the anti-tumor immune response has yet to be examined. This dissertation demonstrates that mice fed the KD had increased tumor-reactive innate and adaptive immune responses, including increased cytokine production and cytolysis via tumor-reactive CD8+ T cells. Additionally, we saw that mice maintained on the KD had increased CD4 infiltration, while T regulatory cell numbers stayed consistent. Lastly, mice fed the KD had a significant reduction in immune inhibitory receptor expression as well as decreased inhibitory ligand expression on glioma cells, namely programmed death receptor -1 (PD-1) and its ligand programmed death receptor ligand -1 (PD-L1). Further, it is demonstrated that the ketone body beta-hydroxybutyrate (BHB) reduces expression of PD-L1 on glioma cells in vitro suggesting it may be responsible in part for immune-related changes elicited by the KD. Finally this dissertation also shows that the KD increases the expression of microRNAs predicted to target PD-L1 suggesting a potential mechanism to explain the ability of the KD to modulate immune inhibitory checkpoint pathways. Taken together these studies shed important light on the mechanisms underlying the KD and provide additional support for its use an adjuvant therapy for malignant glioma. / Dissertation/Thesis / Doctoral Dissertation Biology 2018

<p> Although castration-resistant prostate cancers no longer respond to anti-androgen therapies, the androgen receptor (AR) is still required to promote tumor survival. However, the signaling pathways downstream of AR that promote this survival are not well known. We recently identified an AR-dependent survival pathway whereby AR induction of integrin &alpha;6&beta;1 and adhesion to laminin activates NF-&kappa;B/RelA signaling and Bcl-xL. This pathway acts in parallel with the PI3K/Akt pathway in Pten-null tumor cells such that combined inhibition of both PI3K and integrin &alpha;6&beta;1 is required to effectively kill tumor cells adherent to laminin. However, PTEN-null castration-resistant tumors were not effectively killed by this combination. I discovered that BNIP3, a hypoxia-induced BH3-only, pro-mitophagic Bcl-2 family member, is induced by androgen in castration-resistant cells through integrin &alpha;6&beta;1 and HIF1&alpha;. Furthermore, castration-resistant cells adherent to laminin were much more efficient at inducing autophagy in response to androgen. Androgen blocked the ability of the PI3K inhibitor PX866 to kill castration-resistant tumors, but this was reversed by loss of BNIP3. Although BNIP3 was dispensable for androgen-induced autophagy, its mitophagy function was required for BNIP3 to promote resistance to PX866. Thus, enhanced hypoxia signaling in cooperation with AR/&alpha;6&beta;1/HIF1&alpha; signaling on laminin in castration-resistant cells drives the expression of BNIP3 and enhances autophagy, both of which contribute to PX866 resistance through induction of mitophagy.</p><p>