During retinal development, a pool of progenitor cells divides to generate daughter cells that eventually differentiate into the seven retinal cell types, including horizontal cells (HCs) and retinal ganglion cells (RGCs). Much about how cells exit the cell cycle and maintain a differentiated state remain unknown. Dysregulation of this process can alter the cellular composition and function of the retina.Thus, by studying this developmental process, we can better understand the mechanisms by which progenitor cells become functional, differentiated cells. Our previous work determined that Maturin (Mturn)is highly conserved in its expression pattern and protein sequence across various vertebrate species. Furthermore, we concluded that it is required for differentiation of primary neurons in Xenopuslaevis. Preliminary work in mice revealed that in the absence of Mturn, extensive folds occur in the retina. I used this model to characterize the expression of Mturnin the mouse retina and ask if Mturn is required for normal mice retinogenesis. By immunostaining retinal sections with various cell type-specific antibodies, I found that Mturn is expressed in differentiating cells and not in proliferating cells. In addition to determining that its expression is maintained in mature HCs, I concluded that Mturn is not required for generating the proper number HCs. Our results from studies on Mturn in both frogs and mice have led us to hypothesize that Mturn may function to maintain HCs in differentiated state and prevent their reentry into the cell cycle. Although preliminary experiments testing this hypothesis were inconclusive, future work should continue to investigate the role of Mturn in retinogenesis.

Reliable, minimally invasive biomarkers that predict the extent of alcoholism-induced CNS damage are currently lacking. This limits the selection of rational interventions and hampers the ability to gauge therapeutic effects. Developing biomarkers that indicate early CNS damage may prove useful in deterring the emergence of alcohol use disorders (AUDs). Extracellular microRNAs (miRNAs) can be informative molecular indicators of neuronal gene expression alterations. They repress large fractions of protein-coding genes and are highly-involved in intercellular signaling between both proximal and distal neurons. This work has focused on (1) examining whether extracellular miRNAs in the serum of individuals diagnosed with AUDs can be used as biomarkers of alcohol-induced brain damage, (2) determining in vivo the ethanol-inducedeffects imparted by miRNAs and their targets in the brain, (3) evaluating their role ininterventions that can reverse behavioral impairment and (4) testing the ability of extracellular miRNAs to transfer ethanol-induced pathologies to ethanol-naive cells. There are five major findings from this work. First, two independent quantification technologies demonstrated comparable differences in miRNA expression levels betweenAUDs and controls and revealed significant correlations between candidate miRNA biomarkers and medical, neuroimaging and drinking parameters. Second, in rats manymiRNAs significantly altered by ethanol in the hippocampus following maternal or postnatal exposure were also changed in the serum. Moreover, postnatal consumption activated cell-cycle pathways in the hippocampus while maternal exposure affected unfolded protein response pathways in adolescent offspring. Third, the lack of social motivation seen following fetal exposure was reversed as a result of social enrichment. Analysis of the integrated data in the amygdala and ventral striatum revealed several functional gene networks whose activation patterns following fetal ethanol exposure were reversed by social enrichment. Fourth, transfer of purified exosomes from ethanol-exposed to ethanol-naive cells conferred many gene expression changes consistent with ethanol exposure. Lastly, examination of all the data revealed consistent changes in miRNAs that independently converged on cell death, cell proliferation and cell cycle regulatory processes, regardless of the species, paradigm and source. The findings in this work illustrate the utility of miRNAs as peripheral biomarkers of AUDs and suggest novel epigenetic mechanisms affected by alcohol.

phageal Cancer-Related Gene 2 (ECRG2) is a novel tumor suppressor which is frequently mutated or downregulated in multiple human cancers. Previous studies have demonstrated that ECRG2 inhibits growth of cancer cells by inducing apoptotic death. However, the molecular basis of its regulation and involvement in DNA damage response remain to be elucidated. The function of tumor suppressor p53 in cellular response to stress conditions, such as DNA damage, has been well-established. In the present study, we report for the first time, that ECRG2 is a novel pro-apoptotic transcriptional target of p53 and ECRG2 expression is induced by DNA damage in a p53-dependent manner. Moreover, we demonstrate that disruption of ECRG2 leads to reduced apoptosis and improved survival following the treatment with DNA damage-inducing anticancer agent despite p53 activation in cancer cells. Significantly, we characterized a natural variant in ECRG2promoter (rs3214447) that is found in the genomes of ~38.5% of world population and showed that ECRG2 promoter with rs3214447 variant is defective in responding to p53 and DNA damage. Thus, ECRG2 is an important executor of p53-mediated apoptosis in response to DNA damage. We also report a novel biological function of ECRG2 and demonstrate that ECRG2 interacts with and stabilizes microtubules. ECRG2 was shown to protect the microtubules against the destabilization induced by cold and nocodazole treatment. In addition, we show that ECRG2 increases acetylation of microtubules, which is associated with more stable microtubules. Importantly, we demonstrate that ECRG2 disruption give rise to increased cell proliferation by elevated activation of Akt. Taken together, our findings ascribe a novel function to ECRG2 in the regulation of microtubule dynamics and cancer cell proliferation. ECRG2-mediated tumor suppressor activities elucidated in this dissertation are clinically significant. Our database analyses reveal that cancer patients with lower ECRG2expression in their tumors had poor prognosis and reduced disease-free survival as compared to their counterparts. These observations suggest that loss of ECRG2 expression and function confers survival advantage to cancer cells. Collectively, this dissertation highlights novel aspects of ECRG2 regulation and function in cancer cell sensitivity to DNA damage-inducing anticancer therapy, microtubule dynamics and cell proliferation.

Gene transcription and protein synthesis are essential molecular processes required for all living organisms. In eukaryotes, messages encoded within DNA are transcribed by three DNA-dependent RNA polymerases (Pols I-III) into ribosomal RNA (rRNA), messenger RNA (mRNA), and transfer RNA (tRNA), respectively. General transcription factors (GTFs) help recruit Pols to their appropriate gene promoters as well as facilitate template opening and transcription start site (TSS) selection. In Saccharomyces cerevisiae, the Pol I pre-initiation complex (PIC) is formed by numerous GTFs that include Upstream Activating Factor (UAF), Core Factor (CF), TATA-binding protein (TBP), and Rrn3. This unique set of GTFs engage ribosomal DNA (rDNA) through interactions with regulatory elements of the promoter known as the Upstream Activating Sequence (UAS) and the Core Element (CE). Here, we resolve the cryo-electron microscopy (cryo-EM) structure of CF bound to the rDNA promoter at 3.8Å near-atomic resolution and determine itsDNA binding preferences in which CF preferentially binds to the GC-minor groove. Briefly, our cryo-EM studies reveal that the CF-DNA interaction is mediated by two CF subunits, Rrn7 and Rrn11. We also found that the path of promoter DNA is relatively straight in the Pol I PIC, which is strikingly different from the bent promoters observed in structures of the Pols II/III PICs. We identified three states of CF engagement with promoter DNA (States 1-3) in which CF acts as a ratchet toforceDNA into the active site of the polymerase that facilitates the melting of the double-stranded DNA template in an ATP-independent manner, another unique feature of the Pol I system. Using in vitroDNA binding assays, we have identified a 12 base pair (bp) region within the CE that is necessary and sufficient for CF binding. We have also demonstrated that the human anticancer compound CX-5461 can inhibit yeast cell growth and blocks yeast CF binding to both yeast and human rDNA promoters in vitro. Furthermore, we show that the human Core Promoter Element (CPE) can functionally replace the yeast CE in a position-dependent manner. Together, these results reveal the unique molecular architecture of the Pol I PIC and suggest a conserved sequence-independent binding mechanism of CF with promoter DNA.

CD11c+ T-bet+ B cells serve crucial roles in both protective immunity and autoimmunity.However, the ontogeny of these cells remains unclear, and strategies to target them in vivo have yet to be identified. Here, we demonstrate that developing CD11c+ T-bet+ B cells received help in the form of IL-21, IFN-γ, and CD40L from a population ofT follicular helper 1(TFH1)cells outside of formal germinal centers (GC). These TFH1cells provided help to developing CD11c+ T-bet+ B cells in two distinct phases: IFN-gwas provided early following infection, and CD40L was provided later. Unlike the TFH1cells, CD11c+ T-bet+ B cells required the GC-associated transcription factor Bcl-6 for their development, but not T-bet. While the CD11c+ B cells that arose in the absence of T-bet appeared nearly identical to their T-bet-competent counterparts,they did not switch to IgG2c. These data support a model where, in the absence of formal GCs, TFH1cells provide GC-like help to developing CD11c+ T-bet+ B cells and while T-bet is not required for the development of these T-bet+ B cells,it is required for appropriate class-switch recombination (CSR). Our work also demonstrates that mature CD11c+ T-bet+ B cells, which arise in both immunity and autoimmunity,wereeliminated following treatment with the adenosine 2a receptor (A2aR) agonist CGS-21680. Depletion of these CD11c+ T-bet+ B cells occurred in a B cell-intrinsic manner and was corelated with improved disease outcome in a mouse model of lupus. Preliminary data indicated that human CD11c+ B cells expressed the A2aR,and these cells were depleted following CGS-21680 treatment in vitro, suggesting that A2aR-agonistadministrationmay also be effective in the treatment of human autoimmune diseaseswhere CD11c+ Bcell play a role. Overall, this work provides novel insight into the development of T-bet+ B cells and identifies the first pharmacological approach to target these cells in vivo.

Folliculin (FLCN) is tumor suppressor protein whose function remains a topic of debate. Germline mutations in FLCN predispose affected individuals to develop Birt-Hogg-Dubé syndrome, which is characterized by facial fibrofolliculomas, pulmonary cysts, spontaneous pneumothorax, and renal cell carcinoma. These kidney tumors exhibit an elevated glycolytic phenotype even in the presence of oxygen, an observation commonly known as the “Warburg effect.” This phenomenon is driven by the activity of lactate dehydrogenase-A (LDHA), the enzyme responsible for the interconversion of pyruvate and lactate in the terminal step of glycolysis. Our work herein shows that FLCN is a specific intracellular inhibitor of LDHA. Biochemical and biophysical analyses mapped the interaction and inhibition of LDHA activity to an unstructured loop in FLCN positioned between the amino and carboxy-terminal domains. Characterization of a minimal 10-amino acid FLCN-derived peptide demonstrated that it was sufficient to both bind and inhibit LDHA activity in vitro. Further, treatment of FLCN-deficient cells with this FLCN-derived peptide is sufficient to suppress glycolysis. Interestingly, evaluated cancer cell lines derived from solid tumors of the lung, breast, prostate, bladder, and colon also demonstrate dysregulation of LDHA activity and dissociation of FLCN-LDHA interaction. Previous work has shown that inhibition of LDHA in cancer cells leads to apoptosis. Accordingly, treatment of these cell lines with the FLCN-peptide results in apoptosis, suggesting a potential therapeutic intervention. Taken together, inhibition of LDHA by the tumor suppressor FLCN provides a mechanistic explanation for the endogenous regulation of glycolysis.

Oligodendroglial progenitor cells (OPCs) are the precursors to the myelinating oligodendrocytes in the central nervous system (CNS). These cells are produced in the ventral neuroepithelium at later stages of cortical development, migrating into the cortex where they contact axons and differentiate, ultimately forming a myelin membrane. During the process of differentiation, OPCs undergo significant morphological changes, extending many processes which will make contact with axons. Once in contact with an axon, the oligodendrocyte process expands and begins to form the myelin membrane which will ensheathe the axon. Reelin is a highly conserved secretory glycoprotein, which has acritical role in directing neuronal migration. Reelin orchestrates the proper cortical layer formation and neuronal organization during brain development. In the absence of Reelin, the cerebral crotex is disorganized, with inverted cortical layers, generating devastating biological effects. Reelin acts through several cellular receptors, activating numerous downstream effectors and complex signaling cascades. If elements of the Reelin signalling pathway are disrupted,similar defects in migration can occur.Oligodendroglial cells, from the early progenitor cells to the mature myelinating cells secrete Reelin, but also express a receptor for Reelin and criticalelements of the intracellular Reelin signaling pathways. It is not known if these cells canrespond to Reelin. In this thesis, we examined the effects of Reelin on oligodendroglial cells, using both in vitroand in vivomethods. We demostrate a potentialrole for Reelin in modulating oligodendrocyte migration, but also identify a novel aspect ofReelin signalling in the biology of oligodendroglia.

Infection with an intracellular pathogen presents the host with the immunological challenge of intracellular access and of clearing infection without excessive damage to host tissues. This challenge was long thought to be addressed primarily by cell-mediated immunity, but is now known to include a significant humoral component. To better understand the B cell-mediated contribution to intracellular pathogen control, we investigated memory and effector B cell responses to the intracellular bacterial pathogen Ehrlichia muris, and SARS-CoV-2 infection. B cells expressing the transcription factor T-bet were identified in both mice and humans, and T-bet played an important role in directing antibody class switch recombination, but not in the generation of memory B cells during E. muris infection. T-bet expression was identified in cells resembling murine B-1 B cells, an innate-like subset of B cells important for early T cell-independent B cell responses. These results suggest that T-bet expression in B-1 B cells may contribute to their participation in the early B cell response to murine intracellular bacterial infection. Following human SARS-CoV-2 infection, we revealed that in addition to the canonical class-switched B cell memory response, the presence of a substantial pool of peripheral blood unswitched IgM+ memory B cells was correlated with reduced symptom duration and enhanced generation of antigen-specific antibody. These IgM+ memory B cells were stable, unlike the contracting plasmablast response. These studies underscore the importance of innate and unswitched B cell subsets to the functional plasticity of the humoral response and contribute to our understanding of correlates of innate protection and adaptive immunity.

Heat Shock Protein- 90 (HSP90) is an essential molecular chaperone. HSP90 relies on its intrinsic ATPase activity as well as interactions with co-chaperone proteins to chaperone its clients. HSP90 is also an extracellular protein, performing both a signaling and chaperoning role. Extracellular client, matrix metalloproteinase-2 (MMP2) relies on HSP90 for its stability. MMP2 mediates extracellular matrix remodeling through its gelatinolytic activity. MMP2 activity is also tightly regulated by its endogenous inhibitor, the Tissue Inhibitor of Metalloproteinase-2 (TIMP2). At present how HSP90 performs its chaperoning role in the extracellular matrix is uncertain. In this thesis, I describe that TIMP2 acts as the first bona fide extracellular co-chaperone of eHSP90, and show that TIMP2 is a stress inducible protein. I describe how TIMP2 directly interacts with HSP90, and how TIMP2 decelerates the HSP90 ATPase cycle. TIMP2 also sensitizes HSP90 to both ATP and N-terminal pharmaceuticals. Overall, TIMP2 acts as both a scaffold and a disruptor of the client/chaperone relationship between MMP2 and HSP90, performing both a HSP90 co-chaperone and MMP2 inhibitor role, non-mutually exclusively. The activatory co-chaperone AHA1 competes with TIMP2 for HSP90 binding. TIMP2 and AHA1 are able to form two independent ternary complexes with MMP2 and HSP90; as a result, the TIMP2 complex is MMP2 proteolytically inactive and the AHA1, active. This competition is further described in vivo where it can be inhibited by both _AHA1 antibodies and TIMP2 AHA1 antibodies and TIMP2 exogenous protein treatments, whilst induced following AHA1 protein and _AHA1 antibodies and TIMP2 TIMP2 antibody treatments. Finally, the role of phos-Y-TIMP2 was examined in relation to its interaction with HSP90. To address this, a novel methodology to purify hTIMP2 from E.Coli without previously necessary refolding strategies in a scale-able manner suitable for therapeutic TIMP2 treatments, was developed. Wild type recombinant human TIMP2 and phospho-mutants Y90E, Y90F, and TE (Y62E, Y90E, Y165E) were purified, were inhibitory towards MMP2, and modulated TIMP2 interaction with HSP90. Taken together, I demonstrate how extracellular HSP90 is regulated by co-chaperones to facilitate the chaperoning of pro-invasive client, MMP2. It further shows ways in which we can manipulate this system to promote an inactive MMP2 protease, a key strategy in cancer therapeutics.