• ALTERATIONS IN CELLULAR GLUTAMATE TRANSPORT DO NOT CONTRIBUTE TONEURONAL CELL DEATHIN A MIXED CORTICAL CELL CULTURE MODEL OF HYPOGLYCEMIA

      Hewett, Sandra; Thorn, Trista (2013)
      Severe hypoglycemia is associated with neurological deficits thatwhen left untreated can lead to frank neuronal cell death. Despite longstanding evidence in both in vitro andin vivomodels that hypoglycemic neuronalcell death is mediated by glutamateexcitotoxicity, the cellularand molecular mechanisms involved remain incompletelydefined. Toward this end, werecently reported that glutamate efflux from astrocytes via the anionic cystine/glutamate antiporter, system xc-, contributed to glucose-deprivation (GD) induced neuronal cell death in vitro. However,the precise mechanism by which system xc-activity links to glutamate-mediated injury has yet to be determined. Thus, the overall purpose of this thesis was toinvestigate whetherchanges insystem xc-expression in our astrocyte and mixed cortical cell cultures and/or alterations in glutamate handling in a mixed cortical culture modelfollowing glucose deprivationoccur(s). Toward the former, no change in the expression of mRNA (GD up to 4 h) or protein(GD up to 8 h) ofxCT, the functional light chain of system xc-, in either astrocyte or mixed cortical cell cultureswas demonstrated via quantitative RT-PCR or western blot analysis, respectively. Further, aglycemic neuronal injury, induced by 6 or 8 h of glucose deprivation, was not prevented by the addition of either actinomycin D (10 μg/mL) or cycloheximide (1 μg/mL), demonstrating no requirement for transcription or translation, respectively. Toward the latter, alterations in classical glutamate re-uptake transporter function also did not appear to be altered. Media containing added glutamate taken from control astrocytes or astrocytes deprived of glucose (6 h) was equally toxic to pure neuronal cultures, demonstrating no alterations in glutamate removal between control and glucose-deprived cells. However, neurons in mixed cortical cell cultures deprived of glucose showed increased neuronal cell death over those maintained in glucose-containing medium when exposed directly to equimolar concentrations of either glutamate or NMDA.Similarly, this increased neuronal death in glucose deprived mixed cortical cultures was shown across several different time points using constant concentrations of either glutamate or NMDA. Lastly, we show that neurons in our mixed cortical cultures are fully protected from excitotoxic cell death when system xc-and NMDA receptor inhibitors are added up to two hours following the initiation of glucose deprivation. Overall, our data reveal that neither enhanced system xc-expressionnor impaired glutamate uptake could account for the neuronal cell death induced by glucose deprivation, but that energy deprived neurons appear simply more susceptible to excitotoxic insults. Therefore, physiological levels of glutamate releasedfrom astrocyte system xc-maybe sufficient to mediate neuronal cell death under aglycemic conditions.
    • Alternative splicing dysregulation in mental disorders

      Glatt, Stephen; Cohen, Ori S (2014)
      The brain's ability to adapt ultimately depends on the efficiency with which neuronal connections are made, destroyed, or manipulated. This connectivity is largely controlled by synaptic plasticity, which creates, strengthens, or weakens signals that are necessary for appropriate functioning of the organism. This constant rewiring allows an organism to learn, mature, and cope with the ever-changing environment. However, this rewiring is dependent on the ability to make new proteins, which highlights the importance of transcription, translation, and post-translational modification in the process of synaptic plasticity. Among these cellular functions, transcription plays a key role in providing the necessary variability that is required to regulate neurodevelopment and cognitive behaviors. During transcription, alternative splicing regulates the contents of transcriptomic elements by cutting and stitching the transcribed pre-mRNA and adjusting the configuration of the mature mRNA(s) to meet the necessary cellular requirements. Therefore, it is conceivable that alternative splicing abnormalities can result in inappropriate adjustment of the transcriptome and result in pathological adaptation. In this dissertation, I review the evidence of dysfunctional gene splicing in neuropsychiatric disorders. Then I evaluate the extent of alternative splicing in an animal model for social interaction. This model utilizes valproic acid exposure at a critical developmental period to illicit significant and long-lasting changes in social interaction behavior. Next, I explore the abundance and types of alternative-splicing dysregulationin postmortembrain tissue samples from schizophrenia patients as compared to non-psychotic comparison subjects. Finally, I describe the mechanisms by which a schizophrenia-associated polymorphism in a strong candidate gene (DRD2, which encodes the D2 dopamine receptor) disrupts alternative splicing and leads to inappropriate transcription that is associated with cognitive dysfunction. Collectively, these results reinforce the notion that consideration of genetic variants that dysregulate particular mRNA isoforms and understanding the biological consequence of expressing such isoforms is a crucial step in our efforts to understand human behavior and to develop therapeutic interventions for mental disorders.
    • Analysis of cdGAP in Extracellular Matrix Rigidity Sensing and Cell Migration

      Turner, Chris; Wormer, Duncan (2014)
      CdGAP is a Rac1/Cdc42 specific GTPase activating protein that localizes to cell–matrix adhesions through an interaction with the adhesion scaffold α-parvin/actopaxin to regulate lamellipodia formation and cell spreading. In chapter 2 of this thesis, using a combination of siRNA-mediated silencing and over expression, I show that cdGAP negatively regulates directed and random migration by controlling adhesion maturation and dynamics through the regulation of both adhesion assembly and disassembly. Interestingly, cdGAP was also localized to adhesions formed in three-dimensional matrix environments and cdGAP depletion promoted cancer cell migration and invasion through 3D matrices. Cell migration in 3D CDMs more closely approximates the topography of in vitroconnective tissues, suggesting that cdGAP likely plays an important regulatory role in cell migration in vivo. Other aspects of the extracellular matrix also influence cell migration. Specifically, motile cells are capable of sensing the stiffness of the surrounding extracellular matrix through integrin-mediated focal adhesions and migrate towards regions of higher rigidity in a process known as durotaxis. Durotaxis plays an important role in normal development and disease progression, including tumor invasion and metastasis. However, the signaling mechanisms underlying focal adhesion-mediated rigidity sensing and durotaxis are poorly understood. In chapter three of this thesis, I utilizefibronectin-coated polydimethoxysiloxane gelsto manipulate substrate compliance, and show that cdGAP is necessary for U2OS osteosarcoma cells to coordinate cell shape changes and migration as a function of extracellular matrix stiffness. CdGAP regulated rigidity-dependent motility by controlling membrane protrusion and adhesion dynamics, as well as by modulating Rac1 activity. I also found that CdGAP was necessary for U2OS cell durotaxis. Taken together, these data identify cdGAP as an important component of an integrin-mediated signaling pathway that senses and responds to mechanical cues in the extracellular matrix in order to coordinate directed cell motility.These findings highlight the importance of GAP proteins in the regulation of Rho family GTPases andprovide insight into how GAPs co-ordinate the cell migration machinery.
    • ANALYSIS OF INOSITOL 1,4,5-TRISPHOSPHATE RECEPTOR-ERLIN1/2 COMPLEX-RNF170 AXIS MUTATIONS THAT RESULT IN NEURODEGENERATIVE DISEASE

      Wojcikiewicz, Richard; WRIGHT, FORREST (2017)
      Inositol 1,4,5-trisphosphate receptors (IP3Rs) are endoplasmic reticulum (ER) proteins that assemble into tetrameric IP3- and Ca2+-gated Ca2+ channels. Activation of IP3Rs begins with stimulation of cell surface receptors that elevate cytosolic IP3 levels. IP3, with its co-agonist Ca2+, binds to IP3Rs and causes a conformational change that results in the opening of the channel aperture, allowing for Ca2+ ions to flow from stores within the ER lumen to the cytosol and thereby promoting a number of Ca2+-dependent cellular events, including secretion, neurotransmitter release and cell division. Intriguingly, it appears that the same conformational change that IP3Rs undergo during activation makes them a target for degradation by the ubiquitin-proteasome pathway. This processing allows the cell to fine-tune its internal Ca2+ responses to extracellular stimuli. In the Wojcikiewicz lab, it was discovered that processing of activated IP3Rs is mediated by the Erlin1/2 complex, a large (~2MDa) complex composed of the proteins Erlin1 and Erlin2. Constitutively-associated with the Erlin1/2 complex is the E3 ubiquitin ligase RNF170. Thus, we employed TALEN and CRISPR/Cas9-mediated gene editing technologies to abrogate expression of these three proteins to define their roles in this process. Remarkably, analysis of cells lacking RNF170 showed that it is required for all ubiquitination of activated IP3Rs. Investigation into the roles of Erlin1 and Erlin2 uncovered that Erlin2 is the “dominant partner” in the Erlin1/2 complex, mediating complex interaction with activated IP3Rs and bringing RNF170 into place to allow for ubiquitination to proceed. Mutations to RNF170 (R199C) and Erlin2 (T65I) have been identified as causative for progressive neurodegenerative diseases. Investigation of the R199C mutation on IP3R processing by RNF170 uncovered that while the mutation did not affect normal RNF170 function, it destabilized the RNF170 protein, resulting in a significantly reduced cellular complement of RNF170 and inhibition of IP3R degradation. Analysis of the Erlin2 T65I mutation showed that the effect of the mutation on Erlin1/2 complex function was two-fold. First, Erlin2 T65I interaction with activated IP3Rs was completely blocked, thereby inhibiting recruitment of RNF170 and subsequent ubiquitination and processing. Second, normal binding of the Erlin1/2 complex to phosphoinositol-3-phosphate (PI(3)P) – the significance and function of which remains undefined – was drastically inhibited. Examination of complex assembly and stability by SDS-PAGE and Native PAGE showed no destabilization of individual Erlin2 proteins nor of overall Erlin1/2 complex assembly. These data demonstrate that proper and tight control of IP3R levels in the cell are critical to overall cellular homeostasis, as disruptive mutations to requisite mediators of IP3R processing - the Erlin1/2 complex and RNF170 – result in the development of progressive neurodegenerative disease.
    • ANALYSIS OF TEMPERATURE SENSITIVE CYK-1 MUTATIONS EFFECT ON MUSCLE DEVELOPMENT ON CAENORHABDITIS ELEGAN SLARVAE

      Pruyne, David; Laszlo, Arianna (2017)
      Sarcomeres are the most basic unit of muscle cells. Formins are actin regulatory proteins that are important for actin filament polymerization and nucleation, and might be responsible for the actin filamentassembly in sarcomeres. In Caenorhabditis elegans, two formins (CYK-1 and FHOD-1) were found in the body wall muscle (BWM), specifically in the Z-line of sarcomeres. Previously, BWM were analyzed in null cyk-1(ok2300) mutant worms, derived from heterozygous parents, and fhod-1(tm2363) mutantworms, andwere found to have smaller muscles than wild-type worms. Yet, there was still functional CYK-1 present in the worms due to inherited maternal CYK-1. To eliminate this potential source of CYK-1, cyk-1(or596ts) temperature sensitive mutant worms were used to allow for all CYK-1 to be non-functional at 26°C. The focus of this study was to understand the importance of cyk-1on muscle development in C. eleganslarvae. Wild-type, fhod-1(tm2363), cyk-1(or596ts), and fhod-1(tm2363);cyk-1(or596ts) double mutant wormsat L1 larval stage were observed for worm shape and muscle abnormalities. Abnormal worm shapes were observed in fhod-1, cyk-1, and more commonly in fhod-1;cyk-1mutant worms at permissive and restrictive temperatures. Abnormal muscle was observed in both permissive and restrictive temperatures forfhod-1, cyk-1, and fhod-1;cyk-1mutant worms that had abnormal worm shapes, while all worms that had normal body shape usually had normal muscle at both temperatures. Worms were alsotested to determine long-termeffects of cyk-1and fhod-1mutations on muscle development. Worms were held at permissive or restrictive temperatures for various times. Fhod-1and cyk-1mutant worms showed reduced muscle size compared to wild-type, while fhod-1;cyk-1mutant worms displayed a more severelyreduced muscle size. Next, cyk-1orfhod-1were tested to see whether they can rescue muscle cell size after mosaic expressionin a cyk-1(-) or fhod-1(-) mutant worm background. Fhod-1(+) muscle cells showeda significant increase in muscle cell size compared to fhod-1(-) cells, while cyk-1(+) muscle cells showeda non-significant increase in muscle size compared to cyk-1(+/-) cells. This shows cell-autonomousexpressionoffhod-1influencesmuscle growth, while cyk-1expression in another organ might influence muscle development.
    • ANTIVIRAL ACTIVITY AND MECHANISM OF ACTION OF A NOVEL URACIL ANALOG FOR VARICELLA-ZOSTER VIRUS

      Moffat, Jennifer; DE, CHANDRAV (2015)
      The alphaherpesvirus varicella-zoster virus (VZV) is widespread in humans. VZV causes primary and recurrent diseases that are preventable with live vaccines and are treatable with antiviral drugs. New antiviral drugs for varicella-zoster virus (VZV) with increased potency are needed, especially to prevent post-herpetic neuralgia. The purpose of this project was to evaluate β-L-1-[5-(E-2-Bromovinyl)-2-(hydroxymethyl)-1,3-dioxolan-4- yl)] uracil (L-BHDU) and 5′-O-valyl-L-BHDU for efficacy, safety, resistance, and mechanism of action in three models of VZV replication: primary human foreskin fibroblasts (HFFs), skin organ culture (SOC) and in SCID-Hu mice with skin xenografts. We found that L-BHDU and valyl-L-BHDU were safe and effective against VZV in culture and in a mouse model. Herpes simplex virus Type 1 was also sensitive to LBHDU in cultured cells. The mechanism of action of L-BHDU and its effect on drugdrug interactions were not known. Given its similar structure to brivudine (BVdU), we addressed whether L-BHDU, like BVdU, inhibits 5-fluorouracil (5-FU) metabolism. LBHDU did not interfere with 5FU metabolism, indicating that L-BHDU is a safer drug than BVdU. However, L-BHDU antagonized the activity of acyclovir (ACV), BVdU and foscarnet (PFA) in cultured cells, which was due to competition for phosphorylation by VZV thymidine kinase (TK). The mechanism of action of L-BHDU was studied by evaluating its activity against related α-herpesviruses and by analyzing resistant VZV viii strains. VZV strains resistant to L-BHDU (L-BHDUR ) were cross-resistant to ACV and BVdU but not to PFA and cidofovir (CDV). Whole genome sequencing of L-BHDUR strains identified mutations in ATP-binding (G22R) and nucleoside binding (R130Q) domains of VZV TK. The purified L-BHDUR TKs were enzymatically inactive and failed to phosphorylate the drug. In wild type VZV- infected cells, L-BHDU was converted to L-BHDU mono- and diphosphate forms; cells infected with L-BHDUR virus did not phosphorylate the drug. We also investigated whether addition of nucleosides reversed LBHDU inhibition of VZV in dividing and quiescent HFFs. Excess thymidine and uridine, but not purines, in proportion to L-BHDU restored VZV replication only in dividing cells, suggesting that the active form of L-BHDU interfered with pyrimidine biosynthesis. Like other herpesviruses, VZV infection induced thymidine triphosphate (dTTP) in confluent cells while L-BHDU treatment decreased the dTTP pool. Some herpesviruses raise dNTP pools by inducing cellular enzymes. However, VZV infection did not increase cellular thymidylate synthase (TS) expression to facilitate viral replication. Furthermore, the active form of L-BHDU did not interfere with cellular metabolism, suggesting a viral target. Further studies are required to identify the target(s) of L-BHDU active form(s).
    • Autism spectrum disorder traits in SLC9A9 knock-out mice

      Faraone, Stephen; Yang, Lina (2014)
      utism spectrum disorders (ASDs) are a group of neurodevelopmental disorders which begin in childhood and persist into adulthood. They cause lifelong impairments and are associated with substantial burdens to patients, families and society. Genetic studies have implicated the sodium/proton exchanger (NHE) nine gene, SLC9A9, to ASDs and attention-deficit/hyperactivity disorder (ADHD). SLC9A9 encodes, NHE9, a membrane protein of the late recycling endosomes. The recycling endosome plays an important role in synapse development and plasticity by regulating the trafficking of membrane neurotransmitter receptors and transporters. Here we tested the hypothesis thatSLC9A9 knock-out (KO) mice would show ADHD-like and ASD-like traits. Ultrasonic vocalization recording showed that SLC9A9 KO mice emitted fewer calls and had shorter call durations, which suggest communication impairment. SLC9A9 KO mice lacked a preference for social novelty, but did not show deficits in social approach; SLC9A9 KO mice spent more time self-grooming, an indicator for restricted and repetitive behavior. We did not observe hyperactivity or other behavior impairments which are commonly comorbid with ASDs in human, such as anxiety-like behavior. Our study is the first animal behavior study that links SLC9A9 to ASDs. By eliminating NHE9 activity, it provides strong evidence that lack of SLC9A9 leads to ASD-like behaviors in mice and provides the field with a new mouse model of ASDs.
    • Biological importance of TIMP-2 phosphorylation on MMP-2 activity

      Bourboulia, Dimitra; Bullard, Renee (2016)
      Matrix metalloproteinases (MMPs) are proteolytic enzymes that are secreted from the cell and play an important role in embryonic development and tissue remodeling. In cancer, MMPs are hyperactive, promoting degradation of the ex-tracellular matrix. Enhancement of MMP proteolytic activity allows tumor cells to migrate and invade surrounding tissues, increasing the chance of metastasis. Tissue inhibitor of metalloproteinases (TIMPs) are also known to act extracellu-larly, and are the endogenous inhibitors of MMPs. To inhibit the protease activi-ty of MMPs, the N-terminus of the TIMP protein binds to the catalytic domain of MMP at a ratio of 1:1. Studies from our lab have found that TIMP-2 is phosphor-ylated on three tyrosine residues, and this phosphorylation increases the inter-action with MMP-2. This is the first time that phosphorylation of TIMP-2 has been reported. Fascinatingly, the proto-oncogene tyrosine kinase c-Src was found to phosphorylate TIMP-2. This is significant in that c-Src has not yet been shown to act extracellularly, and there are no details within the current lit-erature describing how this protein may function outside of the cell. In this the-sis, we usedmammalian cells as a model to decipher whether TIMP-2 phosphor-ylation wasable to occur extracellularly,as well as the effect that phosphoryla-tion of TIMP-2 hadon its functionto both inhibit/activate MMP-2. We found that(1) c-Src is able to phosphorylate TIMP-2 extracellularly in conditioned me-vidia; and (2) phosphorylation of TIMP-2 enhances its function of inhibiting MMP-2 proteolytic activity, as well as assisting in the activation of pro-MMP-2. Our results suggest the presence of anovel mechanismin whichphosphoryla-tion of TIMP-2is able to regulate the extracellular environment through en-hanced interaction with MMP-2. The information gained from this research couldlead to development of novel therapies that use phosphorylated TIMP-2 as a means of decreasing cellular migration and invasion with the overall goal of preventing metastasis.
    • BRAIN SPECIFIC NEURAL EXTRACELLULAR MATRIX EXPRESSION AND MODIFICATIONS IN NEUROLOGICAL DISEASE AND DISORDERS

      Matthews, Rick; Dwyer, Chrissa (2013)
      The central nervous system (CNS) is extraordinarily complex in both structure and function. The neural extracellular matrix (ECM) is one of the key classes ofmolecules that regulates thedevelopment of the CNS and maintains its structure and function in the adult.Thereby understanding the function of the neural ECMis key to understanding the CNS. The neural ECM is composed of several nervous-system specificproteins, which are hypothesized to uniquely contribute to the defining physiological functions of the CNS. However,work in this area has been hindered by the highly complex molecular properties of the neural ECM, which stem from alterations in expressionand modifications (resulting from glycosylation and proteolytic cleavage) of its constituents. Further defining mechanisms that alter the expression and modifications of neural ECM constituents are critical to fully understanding its complex array of functions. Often in neuropathologies, the neural ECM undergoes dynamic changes providing a valuable tool to further understand its function andthe opportunity to explore its contribution to disease pathology and utility as a therapeutic target. The work presented herein investigates the role of altered expression of the nervous-system specific ECM constituent, Brain Enriched Hyaluronan Binding (BEHAB)/ brevican(B/b), in glioma,and altered glycosylation of the nervous system enriched ECM constituent, RPTPζ/phosphacan, in O-mannosylrelated congenital muscular dystrophies (CMDs). Our work suggests that increased expression of B/b in the glioma tumor microenvironment (TEM) contributes to the pathological progression of these tumors, and reducing its expression is a valuable therapeutic strategy. Additionally, our work evaluates the transcriptional regulatory mechanisms leading to increases inB/b expression in glioma and highlights the potential value of these mechanisms as therapeutic targets. Our work also identifies the absence of O-mannosyl linked carbohydrates on RPTPζ/phosphacan in the brains of CMD models and suggests that altered glycosylation of RPTPζ/phosphacan may have a role in the neuropathologies underlying these disorders. Overall this work provides valuable insight intothe molecular complexities of the neural ECM stemming from changes in the expression and glycosylation of its constituents and furthers our understanding of its function in the normal CNS and in neuropathologies.
    • Bundling of cytoskeletal actin by the formin FMNL1 contributes to celladhesion and migration

      Blystone, Scott; Miller, Eric (2018)
      Metastasis is one of the leading causes of death in the world, affecting thousands every year. This is especially true of breast cancer, which can often result in the formation of secondary metastatic sites in the lung, liver, and bone marrow. There are many aspects to metastasis and an innumerable amount of molecular, biochemical, and cellular interactions contribute to its pathology. The ability of primary tumor cells to disseminate from the primary tumor, degrade the basement membrane, invade through the ECM, and eventually intravasate across the endothelial cell lining of the circulatory system or lymphatics requires a plethora of proteins, all working together in concert to achieve this. Nowhere in the cell is this more apparent than the actin cytoskeleton.Locomotion of cells requires several alterations in the actin cytoskeleton component of the cellular machinery. Generally speaking, cells must be able to polarize, form protrusions, adhere to the substratum, translocate, and then retract their tail, repeating this process as they continue to navigate to their destination. While there are many underlying aspects to this activity, spatiotemporal rearrangements of the actin cytoskeleton are key to the successful cellular motility. The mechanics behind dynamic actin cytoskeletal modifications are varied and complex, demonstrating the requirement for a variety of actin-associated, regulatory proteins.A crucial family of proteins involved in this process is the formin family of proteins. Formins are a relatively “new” group of actin modifiers which possess the unique ability to modify and generate linear actin filaments. While the members of this protein family all share some of the same actin modifying processes, many of these proteins also have functions exclusive to themselves. As a result, research into this field has blossomed and several novel features of different formins have been identified. Furthermore, alternative splice isoforms of several formins are often expressed in a variety of cell types, with specific functions attributed to each.The formin FMNL1 was originally identified in cells of a myeloid lineage and for many years was mostly thought to be involved in leukocyte adhesion and migration. Indeed, our lab has characterized many of the functions of this protein in both human and murine macrophages. However, as a result of the work in this dissertation, we have generated sufficient evidence suggesting that FMNL1 not only plays a role in breast cancer migration, but also exhibits functions unique to a specific alternative splice isoform of this protein.Our work on FMNL1 has pushed the field of study into this protein family in new directions. Herein, we have demonstrated that all three alternative splice isoforms of FMNL1 are expressed in a variety of cell types and the FMNL1ɣalternative splice isoform distinguishes itself from these isoforms via its ability to bundle linear actin filaments. Additionally, our data indicates that this is accomplished independently of the trademark FH2 domain, often thought to be the essential component of all formins. More specifically, we have identified a unique amino acid sequence in the C-terminal region of this isoform that most likely regulates this function. As a result, we have not only identified a potential therapeutic target for the treatment of metastasis via inhibition of cellular locomotion, but also pushed the field of formin research into a novel direction by providing insight which may foster new hypotheses and challenge classical theories regarding the relationship between formins and actin.
    • CD11C+ T-BET+ B CELLS IN INFECTION AND AUTOIMMUNITY

      Winslow, Gary; Levack, Russell (2020)
      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.
    • Characterization of Hic-5 in Cancer Associated Fibroblasts: A Role in Extracellular Matrix Deposition and Remodeling

      Turner, Christopher; Goreczny, Gregory (2017)
      Hic-5 (TGFβ1i1) is a focal adhesion scaffold protein that has previously been implicated in many cancer-related processes. However, the contribution of Hic-5 during tumor progression has never been evaluated, in vivo. In Chapter 2 of this thesis, I crossed our Hic-5 knockout mouse with the MMTV-PyMT breast tumor mouse model to assess the role of Hic-5 in breast tumorigenesis. Tumors from the Hic-5 -/-;PyMT mouse exhibited an increased latency and reduced tumor growth. Immunohistochemical analysis of the Hic-5 -/-;PyMT tumors revealed that the tumor cells were less proliferative. However isolated tumor cells exhibit no difference in growth rate. Surprisingly, Hic-5 expression was restricted to the tumor stroma. Further analysis showed that Hic-5 regulates Cancer Associated Fibroblast (CAF) contractility and differentiation which resulted in a reduced ability to deposit and reorganize the extracellular matrix (ECM) in two-and three-dimensions. Furthermore, Hic-5 dependent ECM remodeling supported the ability of tumor cells to metastasize and colonize the lungs.The molecular mechanisms by which CAFs mediate ECM remodeling remains incompletely understood. In Chapter 3 of this thesis, I show that Hic-5 is required to generate fibrillar adhesions, which are specialized structures that are critical for the assembly of fibronectin fibers. Hic-5 was found to promote fibrillar adhesion formation through a newly characterized interaction with tensin1, a scaffold protein that binds to β1 integrin and actin. Furthermore, this interaction was mediated by Src-dependent phosphorylation of Hic-5 in two and three-dimensional matrix environments to prevent β1 integrin internalization and subsequent degradation in the lysosome. This work highlights the importance of the focal adhesion protein, Hic-5 during breast tumorigenesis and provides insight into the molecular machinery driving CAF-mediated ECM remodeling.
    • Characterizing the Role of the Epsilon Subunit in Regulation of the Escherichia coli ATP Synthase.

      Duncan, Thomas; Shah, Naman (2015)
      The F-type ATP synthase is a rotary nanomotor central to cellular energy metabolism in almost all living organisms. In bacteria, the enzyme also plays a role in nutrient uptake and pH regulation underlining its importance. All ATP synthases can be inhibited by ADP, whereas in bacteria, the enzyme is alsoautoinhibitedbyits ε subunit. The inhibition involves a drastic conformationa l change of the C-terminal domain of the ε subunit (εCTD)thatblockscatalytic turnover. Thisregulation by ε is believed to play an important role in maintaining viability of the cell. Recent development in the field of antibiotics has validated ATP synthase as a drug target against pathogenic bacteria. Thus, there is a renewed interest in studying the role of the ε subunit in regulation of the enzyme and exploiting it to develop antimicrobials that can kill pathogenic bacteria. The present work describes advances in our understanding of the regulatory interactions of εCTD in E. coli ATP synthase.In the first approach, we used an optical binding assay to understand the transitions of εCTD between its active and inhibitory conformations.Using different ligands we revealedthe relationship between ADP inhibition and ε inhibition. In the second novel approach, the terminal five amino acids of εCTD were deleted to observe the effects on in vivo and in vitro functions of ATP synthase. The results obtained from these studies advance our understanding of εinhibition inbacteria and also provide a noveltarget within bacterial ATP synthase to obtain antibacterial drugs.
    • The Chemosensory-­Related Consequences of Fetal Ethanol or Fetal Nicotine Exposure: Their Contribution to Postnatal Nicotine Acceptance

      Youngentob, Steven; MANTELLA, NICOLE (2015)
      Human studies demonstrate a predictive association between gestational exposure to alcohol or nicotine and the probabilityoflater nicotine dependence.The flavor qualitiesof both drugsare known to influencetheir earlyacceptance and they share the perceptual attributesof an aversive odor, bitter taste and oral irritation.This dissertationexamined whether there are chemosensory-­‐related consequences offetal: (1) alcohol exposurethat contribute toenhanced nicotine acceptance; or (2)nicotine exposure that also enhances acceptance. The study rationale was drivenby overlappingliteraturesrelated to: (1) the relationship between gestational exposurewith chemosensory stimuli and their postnatal acceptance; (2) lessons learned from prenatal alcohol exposure and its postnatal consequences; and (3) perceptual commonalities between the flavor of alcohol and nicotine.Alcohol studies: rats were alcohol-­‐exposed during gestationvia the dams’ liquid diet. Control damsreceived ad libaccessto an iso-­‐caloric, iso-­‐nutritive diet. Nicotine studies: dams’ were implanted with a mini-­‐osmotic pump containing nicotine.Control animals received either vehicle only or no pump. Behaviorally, we found that fetal alcohol exposed adolescent rats showed anenhanced nicotine odor
    • Connexin43 and immunity : macrophage phagocytosis, cardiac calcinosis and autoimmune myocarditis

      Steven Taffet; Aaron Glass (2013)
      Connexin43 (Cx43) is a gap junction protein best known for coupling the cytoplasms of cardiac myocytes and allowing the efficient conduction of action potentials throughout the heart. In addition to the heart, Cx43 is also highly expressed in many immune cells and it has been attributed numerous roles in immunity. One such reported role was in macrophage phagocytosis. The first chapter in this dissertation explored the phagocytic activity of cultured and primary murine macrophages from wild type (WT) and Cx43-deleted (Cx43-/-) macrophages. No difference in phagocytic uptake was observed between the two groups using a series of target particles, indicating that Cx43 is dispensable for phagocytosis in macrophages. Given the spectrum of immune functions in which Cx43 has been ascribed a role, we set out to characterize its effect on a model of autoimmune myocarditis (EAM). Using the area of cardiac inflammatory infiltrate as a correlate of disease severity, we observed the progression of the disease to be independent of Cx43 status utilizing WT and Cx43-heterozygous (Cx43+/-) animals as well as radiation chimeric mice reconstituted with cells from donor WT, Cx43+/- and Cx43-/- mice. Although the severity of EAM did not measurably change when induced in animals with differing levels of Cx43 expression, substantial changes to ventricular Cx43 were noted in diseased hearts. Large foci were observed that completely lacked Cx43 immunofluorescence signal. Areas surrounding these foci exhibited disrupted Cx43 patterns such as internalization and lateralization. Similar alterations to Cx43 were also observed in the BALB/cByJ strain of laboratory mice that develop a spontaneous myocarditic disease. To investigate the electrophysiological ramifications of EAM, especially in the context of Cx43+/- mice, ECGs were recorded from animals over the course of EAM. Significant changes to the QRS interval were noted, including prolongation that was only observed in Cx43+/- animals.
    • Deciphering a hippocampus to hypothalamus feeding circuit via the septal nucleus.

      Yang, Yunlei; Sweeney, Patrick (2017)
      The neural circuits controlling feeding are concentrated in the hypothalamus and hindbrain. These circuits primarily control homeostatic feeding behavior, which can be broadly defined as increasing feeding in response to hunger or decreasing feeding in response to satiety. However, non-homeostatic factors, such as the emotional state of an animal, can also profoundly affect feeding behavior. Therefore, the current thesis project sought to determine how primary emotion centers in the brain influence the known homeostatic feeding circuitry in the hypothalamus. In particular, given that ventral hippocampus (vHPC) and septum are involved in emotional processes, influence feeding behavior, and are anatomically connected to hypothalamic feeding circuitry, this dissertation aimed to determine the cell-types in vHPC and septum that control feeding and to functionally connect these cell-types to the primary feeding circuitry located in the hypothalamus. To accomplish these central aims, chemogenetic and optogenetic approaches were utilized to selectively manipulate neural activity within distinct ventral hippocampal and septal cell types and neural circuits. These approaches were complemented by traditional anterograde and retrograde tracing techniques and chemo/optogenetic circuit mapping approaches to define the neural circuits responsible for vHPC and septal control of feeding behavior. We find that chemogenetic activation of ventral hippocampal glutamate neurons reduces feeding, while inhibition facilitates feeding. We further dissect a functional neural circuit pathway from ventral hippocampus to lateral septum that is sufficient to suppress feeding behavior. Within the septum, both chemo/optogenetic activation of septal GABAergic neurons reduces feeding, while inhibition of these neurons increases food intake. Utilizing optogenetic circuit manipulation approaches, we demonstrate that septal GABAergic neurons reduce feeding, at least in part, by projecting to hyperphagia-inducing GABAergic neurons located within the lateral hypothalamus. Taken together, our findings expand upon the known roles for ventral hippocampus and septum in energy homeostasis by providing the specific cell-types and neural circuits governing vHPC and septal control of feeding behavior. Given the role for ventral hippocampus and septum in emotional processes and energy homeostasis, we propose that the described vHPC and septal circuits represent promising neural circuits for investigating interactions between feeding, emotional state, and motivated behavior.
    • DECIPHERINGGLUTAMATERGIC NEUROTRANSMITTER SPECIFICATION IN THE ZEBRAFISH SPINAL CORD

      Lewis, Kate; Hilinski, William (2016)
      The correct specification of neurotransmitter phenotypes is crucial for properly functioning neuronal circuitry. Neurons specify their neurotransmitter phenotypes via transcription factors that they express as they differentiate. Often, transcription factors that specify neurotransmitter phenotypes are expressed in multiple populations with the same neurotransmitter phenotype. Since, the transcription factors that specify this characteristic are not yet known for all spinal cord glutamatergic populations, we identified additional transcription factors expressed at relatively higher levels in glutamatergic neurons compared to inhibitory neurons. We have functionally tested three of these (Lmx1bb, Skor1a and Skor1b) to determine if they are required for correct spinal, glutamatergic phenotypes. We demonstrate that Lmx1b likely maintains a subset of glutamatergicphenotypes in the spinal cord. In lmx1bb mutant embryos, the number of cells that initially express glutamatergic markers are unchanged but become reduced at 36 h and to a greater degree at 48 h. In contrast, we observe no changes in the total number of dI5 or V0v neurons, which express lmx1bb,nor do we detect elevated levels of apoptosis between 36 h and 48 h in lmx1bb mutants. Lastly, we show that at least some of the cells that lose their glutamatergic neurotransmitter phenotype are likely to be V0v cells.Additionally, we demonstrate that skor1a and skor1b are expressed predominantly by glutamatergic spinal interneurons, many of which are V0v neurons. When skor1a and skor1b are knocked-down, we observe a significant reduction in the number of glutamatergic neurons and no change in the number of inhibitory neurons suggesting that these genesmay be required to specify the glutamatergic neurotransmitter phenotype of a subset of spinal neurons. In parallel studies, it was shown that evx1 and evx2, genes expressed exclusively by V0v spinal neurons, are required to specify the V0v glutamatergic phenotype. Interestingly, we show that lmx1bb, skor1a and skor1b require evx1 and evx2 for their expression. In summary, these results suggest that skor1, skor1b and lmx1bb may function downstream of evx1 and evx2 to specify and/ormaintain the glutamatergic neurotransmitter phenotype ofat least a subset of V0v neurons.