• 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.
    • STUDIES ON PHOTORECEPTOR GENE EXPRESSION

      Knox, Barry; ZHUO, XINMING (2013)
      Rod photoreceptors are a group of specialized retinal neurons that convert light into a neuronal signal in low light condition. The phototransduction function of rodsrequires expression of a group of rod genes. The homeostasis of these genes is primarily regulated by a photoreceptor regulatory network, which contains two major retina-specific transcription factors, neural retina leucine zipper (Nrl) and cone-rod homeobox(Crx). Nrl and Crx synergistically activate the expression of several phototransduction genes, most notably rhodopsin. Base on the studies done in cell culture, the synergy is theconsequence of interaction of Nrl and Crx. However, the interaction of Nrl and Crx has not been studied in live rods. The goal of thesis is to develop methods that can be used for studying Nrl and Crx interaction in live rods.In order to study Nrl and Crx in live rods without altering cell fate and inducing cell degeneration, I developed a novel inducible system, G3U, which can regulate gene expression in live rods. In this chapter, we investigated the characters of G3U by using rhodopsin-mCherry as a reporter and monitoring the induction response in transgenic Xenopus. The results of live rod imaging suggest that the inducible system has negligible background expression before induction and significant fold increase of expression after induction. Moreover, the induction response of G3U is reproducible in rods. These findings suggest that G3U system is a good candidate for expressing Nrl and Crx temporally in rods. In the second part of my thesis, I developed a flow cytometry based FRET method, FC-FRET, to study Nrl and Crx interaction in live cells. This method allows non-invasively analysis of protein-protein interaction in large population of live cells in a very short time. Furthermore, researchers will be able to analyze the concentration effect of FRET conveniently. In this study, I investigated the orientation of Nrl-Nrl, Crx-Crx and Nrl-Crx interactions. Our studies revealed that the Nrl-Nrl homodimer has a head-to-head and tail-to-tai l conformation. Both the Crx-Crx and the Nrl-Crx dimers have a head-to-head conformation in interacting complex. I also performed structure-function studies on both Nrl and Crx and classified the role of their different domains in the interactions. In addition to the known Nrl basic leucine zipper domain (b-ZIP) and Crx homeobox domain (HD), we found that the Nrl extended homology domain (EHD) plays an important role in the Nrl-Crx interaction. Two methods presented in this thesis are my major achievement during my graduate studies. The G3U inducible can be used to generate transgenic animals carrying inducible Nrl and Crx in rods. These transgenic animals allow researchers to study the interaction of Nrl and Crx in live rods by FC-FRET assay.
    • 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.
    • 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.
    • Specific mutations in the α and ß subunits of the Kluyveromyces lactis F1-ATPase enhance ATP hydrolysis in the absence of the central γ-rotor

      Xin Jie Chen; Thuy La (2013)
      In eukaryotic cells, the mitochondria are vital organelles which are required for cell viability. Mitochondrial stresses such as oxidative stress, loss of membrane potential or loss of mitochondrial DNA are considered extreme and are associated with many neurodegenerative diseases and aging. The mitochondrial FoF1-synthase, where the majority of cellular ATP is synthesized, is composed of one inner membrane bound Fo domain and a water soluble F1 domain in the mitochondrial matrix. F1 contains the hexameric α3β3core and the centrally located γ subunit. The γ subunit is believed to play a key role in inducing conformational changes while rotating within the α3β3 core during ATP hydrolysis/synthesis. Previous studies have shown that the α3β3 core alone from the Thermophilic bacterium PS3 has a detectable hydrolyzing activity. In recent years, evidence of the rotary catalysis of Thermophilic Bacillus sp. PS3 F1-ATPase without its rotor - subunit γ - was shown using high-speed atomic force microscopy[1]. Moreover, previous study undertaken in our lab had utilized a unique genetic screen that allowed the identification of two specific mutations in the α and β subunits in the aerobic yeast Kluyveromyces lactis that stimulate ATP hydrolysis by the mitochondrial F1-ATPase in the absence of γ. This allows cells to survive upon the loss of mitochondrial DNA. In current work, we confirmed that the αF446I and βG419D mutations on the DELSEED loop are sufficient to allow ρ0 cells to survive in the absence of γ. Biochemical experiments showed that the γ -less F1-ATPase can be assembled to actively hydrolyze iv ATP in vivo, but this activity becomes extremely labile in vitro. These studies give insights into the catalytic mechanism of the α3β3 subcomplex and help to better understand the evolutionary origin of the mitochondrial F1-ATPase.
    • DISSECTING THE ROLE OF YEAST RIM8 IN THEINTERNALIZATION OF THE PLASMA MEMBRANE PROTON PUMPPMA1

      Kane, Patricia; Shoniwa, Makandiwana (2013)
      The vacuolar A-TPase (V-ATPase) is a proton pump that is found ubiquitiously throughout the cells. It uses the hydrolysis of ATP to transport protons across membranes, thereby maintaining homeostatic pH. pH control in the cells of an organism is vital, a disturbance in cellular pH may be lethal. The maintenance of homeostatic pH within the cell appears to be a result of the interplay between V-ATPasesandproton exporters. In yeast and plants, the major proton exporter isthe plasma membrane proton exporter, Pma1. Pma1 is the transporter that is primarily involved in themaintenance of cytosolic pH. In cells in which the function of V-ATPase has been compromised (vma mutants) Pma1 is partially mislocalized.It is known thatmembrane transporterslacking the PY motifare endocytosed via the action of an Arrestin Related Trafficking (ART) protein, which translocates an E3 ligase into close proximity with the transporter, so as to allow for the ubiquitination of the transporter. Rim 8 is the ART protein (adaptor) that has been linked to the endocytosis of Pma1, along with E3 Ubiquitin ligase Rsp5. It is of interest to this project that Rim8 is well studied in its role as an adaptor in the alkaline ambient pH pathway. We thus propose that there may be crosstalk between the ambient pH pathway and the pathway that leads to the internalization of Pma1. Therefore, in this body of work we seek to find other players that may be involved in the Pma1 pathway, as well as to elucidate theareas of interaction between Rim8 and Pma1. Ourfinal goal isbringing a better understanding ofthe pathway that leads to the endocytosis of Pma1. To answer the question posed in this work we monitored the growth phenotype and the localization of Pma1 indouble mutants lacking both V-ATPase subunits and key players in the ambient pH pathway. In addition, we looked to see which cytosolically exposed terminal of Pma1 may be involved in theinteraction with Rim8. In yet another experiment, we mutatedRim8 so as to find which areas of the adaptor werevital for Pma1 internalization.Our results showed that other players tested in the Rim pathway(vma2∆rim20∆and vma2∆vps23∆)werenot required for Pma1 internalization. In addition we observed that mutations in Rim8 that compromise its function in the Rim pathway still allow Pma1 internalization, even though they show synthetic growth phenotypes with vma2∆ mutations. Two-hybrid assay could not detect thesites of interaction between Rim8 and Pma1 and newstrategies will be employed to determine these sites. Changes in electrophoretic mobility of Rim8 suggested that Rim8 undergoes posttranslational modifications, and showed differences in vma2∆mutants and WT mutants.
    • STUDYING RETINA DEVELOPMENT USING SMALL MOLECULE BMP INHIBITORS AND MAKING STABLE MOUSE EMBRYONIC STEM CELL LINES

      Viczian, Andrea; Keshvani, Caezaan (2013)
      Age related macular degeneration results in loss ofconephotoreceptors. Studies have not been able to efficientlytransplant cone cells, possiblydue to their limited numbersand lack of information about their development in the mammalian retina.Our lab has discoveredthat BMP signal inhibition is important for eye developmentin the frogas well as generating retinal cone photoreceptorsin mouse embryonic stem cell cultures. The frog, Xenopus laevis,can grow eyeswithin a few days; it is also amenable to transplantation and genetic alteration. In this study, two small molecule BMP inhibitors, LDN193189 and Dorsomorphin, causedshortened tails(dorsalized) phenotypein Xenopusembryos as well asexpansionofthe neural plate in neural stage embryos. This suggests that these chemical inhibitors can be used in place of the standard BMP antagonist, Noggin, in cell culture experiments. Mouse embryonic stem cell cultures treated with Noggin have been used to generate retinal progenitors that generate cone photoreceptorsin our lab. In order to further study cones indetail,we needed to enrich the population of cone photoreceptors from retinal progenitors by marking these cells in-­‐vitro. We generated stable mouse embryonic stem cells expressing a promoter that drives expression in rod/cone progenitor cells upstream from the mCherry fluorescent protein. This construct also contained aubiquitous promoterdriving an antibiotic resistance genefor antibiotic selection. In future studies, these stable ES celllines could be used to differentiate into rod/cone progenitors selected specifically by FAC sorting. Small molecule BMP inhibitors could be used in place of Noggin and analyzed in the generation of conecells.
    • EVALUATION OF THE ROLE OF THE CENTRAL PAIR IN CHLAMYDOMONAS REINHARDTII FLAGELLAR MOTILITY

      Mitchell, David; Smith, Brandon (2013)
      Cilia and flagella are essential for the function of nearly all eukaryotes. This organelle is made up of nine outer doublet microtubules and two central singlet microtubules to form the canonical (9+2) ciliary structure. Cilia and flagella use this structure, as well as several protein complexes, such as the outer and inner dynein arms, the radial spokes, and the proteins that decorate the central pair to propagate the bending that produces motion. Flagellar motion is highly regulated, and each of these structures is necessary to regulate the dynein arms that generate the motile force. The central pair is one of the least understood of these structures. To date there are two major impediments hindering our understanding of the central pair: a lack of understanding as to how distinct central pair structures work in concert, and a general lack of available central pair mutant strains in the model organism Chlamydomonas reinhardtii. In order to further our understanding of how the central pair functions I have used multiple strategies. Firstly I have used previously characterized central pair mutants to study both structural interactions within the central pair and how the double mutant affects motility regulation. Secondly I provide evidence that a potential central pair mutant, H2, is indeed a central pair mutant and affects the C2b projection. Lastly I will attempt to characterize a new Chlamydomonas mutant, 10B5. Together these analyses will demonstrate that double mutants can have an additive effect on the structure of the central pair, and that double central pair mutants do not appear to suppress one another, but are at least ivepistatic to the most severe phenotype. I will also show evidence that 10B5 is not a central pair mutant, but with further study it may offer new insight into motilityregulation.
    • 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.
    • 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.
    • SELF-ASSOCIATION IS A COMMON PROPERTY IN BOVINE, MOUSE AND XENOPUSARRESTIN

      Calvert, Peter; Qiu, Shuang (2014)
      The light-driven translocation of arrestin from rod inner segment to outer segment was indicated to involve free diffusion with binding affinityto light-activated phosphorhodopsin, however, it is still debatable how arrestin is excluded from the dark-adapted outer segment. Previousstudies demonstrated that bovine visual arrestin had the property of self-association. The self-association propertyof both wild-type and mutated purified visual arrestin of several species (bovine, mouse and Xenopus laevis) was studiedby performing analytical ultracentrifugation experimentswhich providedthe oligomer formation information and association constants.Theself-association parameters of purified bovine and mousevisual arrestinwere investigated and compared with other studies. Results showed that arrestin of both species could self-associate, forming dimersin a concentration-dependent manner, but tetramerswere not detected at the highest concentrations examined.Xenopus arrestin was shown to self-associateas well, existing in a monomer-dimer equilibrium with the dimer dissociation constantKD,dim=80.8μM, which suggestedthat self-association was also a feature of Xenopus visual arrestin. Interestingly, homologous mutations (F78A/Y84A/F193A)of Xenopusarrestin, which were supposed to beconstitutive monomers, failed to completely disrupt the oligomerizationof Xenopusarrestinwith the dimer dissociation constantKD,dim=200.7μM , indicating that these regions were not conserved in amphibian visual arrestin, includingXenopuslaevis. The percentage of the dimer was higher than that of monomer at physiological concentrations in all species of arrestins tested.
    • Domain swapping, fragment complementation, and self-assembly in engineered chimeric proteins.

      Loh, Stewart; Craft, Matthew (2014)
      Domain swapping is a form of protein oligomerization in which two or more identical proteins reciprocally exchange parts of their tertiary structure. The structure of the domain swapped proteins is identical to the structure formed by the monomer except for the hinge region linking the two domains. Domain swapping provides the cell with a way to control complex assembly, alter enzyme kinetics and specificity. Domain swapping can also be used to reconstitute enzyme function or as a form of molecular recognition. Only a small number of proteins are known domain swap, and the forces behind domain swapping are not well understood. Much more need to be understood abouthow and why proteins domain swap, before it would be possible to reliably engineer proteins to do so. In an effort to understand the thermodynamic forces that drive domain swapping, the goal of this project was to induce domain swapping and investigate the effects different hinge regions have on an otherwise identical domain swapped structure. To accomplish this we inserted ubiquitin (Ub) into five surface loops of ribose binding protein (RBP), a protein that does not naturally domain swap. The presenceof ubiquitin puts conformational strain on RBP and vice versa, where the folding of one causes the other to unfold. The entropic penalty for having unfolded domains can be relieved by domain swapping, allowing all protein domains to be folded. Using gelfiltration and circular dichroism we determined that our RBP-Ub (RU) fusion proteins domain swap. This domain swapping is dependent upon the conformational strain caused by a folded Ub, and can be reversed by the addition of a flexible glycine linker. Using our RU system we provide the first evidence that proteins with non-identical hinge regions can domain swap to form stable, functional oligomers. Finally we present a physical model that explains the ability of different RU’s to domain swap, which provides at least some of the criteria required of domain swapping proteins.
    • Preclinical Development of Anti-Cancer Drugs from Natural Products.

      Huang, Ying; Sun, Qing (2014)
      Cancer has been and will continue to be the common concern in the United States and worldwide. As a conventional treatment to fight cancer, new anti-cancer drugs with more efficiency and less toxicity are extremely required. In this study, we have identified two novel compounds with anti-cancer properties from two traditional Chinese medicinal plants. One is Lappaol F that was extracted from the seeds of the plant Actium Lapp L., which has been used in China for centuries as anti-viral and anti-bacterial medicine. Another is M-9 that was extracted from the stem of Marsdenia tenacissima,a plant that has been applied to treat inflammation and cancer in China. Our results showed that Lappaol F inhibited cancer cell growth by regulating a series of cell cycle related proteins and inducing cell cycle arrest at G1 and/or G2 phase. p21 played a critical role in Lappaol F-induced cyclin B1 and cyclin-dependent kinase 1 (CDK1) suppression as well as G2arrest. Lappaol F also induced cell death in a number of cancer cells through caspases activation. Lappaol F-mediated cell growth inhibition was p53-independent. Notably, results from animal studies showed that Lappaol F effectively inhibited tumor growth in vivo, while being well tolerated by the mice. Thus, Lappaol F has a strong potential to be developed as a novel anti-cancer chemotherapeutic. Our studies showed that M-9 successfully sensitized several tumor cells but not non-tumorigenic cells to paclitaxel (Taxol) treatment. Additionally, M-9 reversed chemotherapeutic resistance in a number of multidrug resistant cells. Further results suggested that M-9 functioned, at least to a certain extent, via inhibiting drug efflux by competitively binding to P-glycoprotein (P-gp), a protein that accounts for multidrug resistance. Importantly, results from the in vivostudies demonstrated that M-9 strongly enhanced Taxol-induced growth suppression against xenografts derived from HeLa cells. Moreover, mice tolerated the treatment of Taxol and M-9 well. Therefore, M-9 is a novel chemosensitizer candidate to overcome P-gp-mediated multidrug resistance. Taken together, our studies provide a solid basis for further development of these two compounds as anti-cancer remedies.
    • MITOCHONDRIAL ELECTRON TRANSPORT CHAIN ACTIVITY IN SYSTEMIC LUPUS ERYTHEMATOSUS

      Perl, Andras; Doherty, Edward (2014)
      Systemic lupus erythematosus (SLE) is an autoimmune disorder, characterized by T cell and B cell dysfunction. SLE mitochondria have been shown to be dysfunctional with increased mass, mitochondrial potential, decreased ATP, elevated reactive oxygen species (ROS) and reactive nitrogen species (RNS) concentrations, and altered Ca2+ stores. Drug treatments that target the mitochondria have shown efficacy in treating SLE. Here we have investigated electron transport chain (ETC) activity in SLE, to better understand the causes of mitochondrial dysfunction in SLE. We have found that mitochondrial complexes I and IV of the ETC have elevated respiration in SLE compared to healthy controls after both overnight resting and anti-CD3/CD28 stimulation. We have also shown that SLE complex I is resistant to NO inhibition of respiration. SLE peripheral blood lymphocytes (PBL) have increased S-nitrosylation (SNO) while immunoprecipitated complex I had decreased SNO of proteins compared to healthy controls. The drug Nacetylcysteine (NAC) was able to inhibit complex I activity in SLE, and was found to reduce the amount of complex I protein NDUFS3 after 15 minutes as measured by western blotting. These results have led us to the conclusion that SLE mitochondrial complex I is in an active form which is resistant to SNO and is driving the production of ROS and RNS that are associated with SLE. The drug NAC is able to inhibit complex I respiration which may have therapeutic efficacy by reducing the ROS and RNS stress in SLE.
    • Rational Design of Protein-Based Biosensors Using Engineered Binding-Induced Conformational Switches

      Loh, Stewart; ZHENG, HUIMEI (2014)
      Biosensor development continues to be driven by the growing need to accurately detect and monitor analytes with many biotechnology, clinical, agriculture, and military applications. With their well-established capacity for molecular recognition, proteins are the go-to choice of binding elements in many conventional sensor designs. Switchable proteins offer the potential of integrating analyte binding and signal transduction within a single molecule, thus reducing the need for complex and expensive detection equipment and opening the door to miniaturization and in vivo applications. The principal challenge is that the majority of natural binding proteins do not undergo a large-scale change in conformation upon target binding. This work describes two complementary protein design strategies for the rational conversion of ordinary binding proteins into ligand induced conformational switches for biosensing purposes. In the first approach, we applied the Alternate Frame Folding (AFF) mechanism to the human sulfiredoxin (hSrx) and the fibronectin (FN3) monobody scaffold towards the creation of an ATP biosensor and a customizable biosensor platform, respectively. In a second novel approach, the Protein Fragment Exchange (FREX) mechanism was demonstrated in a proof-of principle study that converts the FN3 scaffold into a biosensor, capable of genetic encoding and application in mammalian cells. While these designs were based on well established principles of protein folding and thermodynamics, the results obtained from these studies also offer important insights regarding protein sequence-structure-function relationships.
    • The role of Src homology 2 domain containing 5' Ionsitol phosphatase-1 in mesenchymal stem cells.

      Kerr, William; Iyer, Sonia (2014)
      Based on earlier findings, some of our initial questions were, as follows: Does SHIP1 have role in the bone marrow niche in maintaining HSC homeostasis and function? Is SHIP1 expressed in mesenchymal stem cells? Does SHIP1 play a functional role in MSC biology? Is SHIP1 deletion in myeloid lineage or mesenchymal lineage sufficient to cause osteoporosis? During the course my graduate thesis work, we have answered these questions and unraveled that SHIP1 is at the nexus of several molecular pathways controlling mesenchymal stem cell biology. In the first aim, we show that under conditions that drive osteolineage differentiation by MSC, SHIP1 limits MSC proliferation and facilitates osteoblast development by repressing the USP1/Id2 axis. The UPS1/Id2 axis was recently shown to promote ‘stemness’ in MSC. Our findings identify a novel SHIP1/USP1/Id2 circuit that controls MSC proliferation and lineage commitment and thus link inositol phospholipid signaling to control of MSC self-renewal and multi-lineage potential by USP1/Id2. Our findings are the first to show that SHIP1 can promote lineage commitment in a population of mesenchymal stem/progenitor cells rather than simply acting as an inhibitor of cell survival and function in differentiated cells. Our findings also provide cellular and molecular explanations for how SHIP1 influences MSC biology, osteolineage development, adipogenesis and osteoporosis. We also provide some very compelling data that represent the first demonstration of in vivo modulation of MSC development and function by small molecule targeting of a cell-signaling pathway that has important metabolic implications. Our analysis of OSXCreSHIPflox/flox mice indicated that as these mice age they lose both bone mass and body fat, we hypothesized then that treatment of aged adult mice with a selective small molecule inhibitor of SHIP1 might achieve the same outcome – reduction of bone mass and body fat. Thus, chapter 2 and 4 includes a series of studies that rather convincingly demonstrate pharmacologic inhibition of SHIP1 significantly reduces both body fat and bone mass in older mice. In addition, in chapter 4, we explore the therapeutic potential of SHIP1/2 inhibition in diabetes and obesity. As previous work by others has shown that SHIP2-/- mice are resistant to diet-induced obesity and diabetes. This led us to hypothesize that pan-SHIP1/2 inhibition (SHIPi), if tolerated, might reduce obesity and improve blood glucose control during aging and/or with consumption of a high fat diet. When paired with our genetic studies in OSXCreSHIP1flox/flox mice these pharmacological studies demonstrate unequivocally that SHIP proteins are molecular targets in both obesity and osteopetrotic diseases. Moreover, we demonstrate for the first time that targeting of cell signaling in the adult MSC compartment can achieve significant metabolic changes that could have translational application in both obesity and osteopetrotic diseases. At this juncture this work represents a potent blend of novel basic and applied findings that have profound implications for regulation of mesenchymal stem cell fate, bone biology as well as for treatment of obesity and bone diseases. In chapter 3 we demonstrate that intracellular signaling by SHIP1 in MSC is demonstrated to have a critical role in the control of HSC output during aging and this increases our understanding of how myeloid bias occurs in aging, and thus could have implications for the development of myeloproliferative disease in aging.
    • THE ROLE OFNORMAL AND ONCOGENICJANUS KINASE 2 IN HEMATOPOIETIC STEM CELLS

      Mohi, Golam; AKADA, HAJIME (2014)
      During my Ph.D. training, I first experimentally proved that the expression of oncogenic Jak2V617F wassufficient to induce MPNs and transformed only HSCs into CSCs for developing MPNs. Thus, it is criticalto understand the role of both normal and oncogenic Jak2 in HSCs to find the mechanism to cureJak2V617F-positive MPNs. Therefore, I have mainly studied two major questions in Jak2:1) The role of normal Jak2 in hematopoietic stem cells for adult hematopoiesis2) The role of oncogenic form of Jak2, Jak2V617F, in cancer stem cells for MPN developmentFirst question has not been addressed since 1998, because conventional Jak2 knock-out mice wereembryonic lethal. Thus, I hypothesized that Jak2 plays a pivotal role in adult hematopoietic stem cellmaintenance. I successfully prove that Jak2 is the one of key regulators of HSCs. Conditional Jak2 deletionin mice caused an irreversible HSCs impairment. My data strongly suggest that Jak2 plays a critical role inthe maintenance of quiescence, survival and self-renewal of adult HSCs.Second question has been studied after the discovery of a somatic point mutation, Jak2V617F, in a majorityof patients with MPNs in 2005. I hypothesized that this oncogenic mutation confers unique properties inCSCs maintenance for MPNs development. Surprisingly, I found that the site of leukemogenesis shiftedfrom BM to spleen, and spleen became the major source of CSCs for Jak2V617F-positive MPNs. The age-associated progressive expansion of CSCs was seen in spleen. Splenic-CSCs were capable to propagateMPN disease and possessed a greater proliferative advantage than BM-CSCs. The Jak2V617F-CSCsestablished a positive-feedback mechanism with CD169+ macrophage progenitors. Depletion of CD169+macrophage progenitors reduced the number of Jak2V617F-CSCs. Gene profiling revealed that splenic-CSCs have distinct gene expression compared to BM-CSCs. Together, I demonstrated that Jak2V617F-CSCs are maintained in spleen for long-term MPN progression.By utilizing gene analysis data from two projects, I discovered a set of unique genes/pathways regulated byonly Jak2V617F but not by wildtype Jak2. All together, my Ph.D. researches provided the potential genetarget a novel therapy for Jak2V617F-positive MPNs.
    • 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.
    • Functional Studies of Tumor Suppressor ECRG2 in the Regulation of Cancer Cell Death and Drug Resistance

      Huang, Ying; Lucchesi, Chris (2015)
      Esophageal Cancer Related Gene 2 (ECRG2) is a newer tumor suppressor whose mRNAhas previously been shown to be decreased in multiple human malignanceswhencompared to normal/adjacent tissues.Of importance, ECRG2 has previously been revealedto possess tumor suppressive attributes,including the ability to induce cell death in cancer cells. However, how ECRG2 is able to activate the apoptotic machinery has yet to be elucidated. In the present study,we highlight multiple angles that ECRG2 leverages in order to sensitize cancer cells to apoptosis. Moreover, we report for the first time,that ECRG2 protein expression in lung cancer patientsamples is lost/decreased in upwards of 90% ofthecancer tissues evaluatedcompared to normal tissue. Additionally, a single somatic point mutant found in patient tumor samples, V30E, was shown to lose tumor suppressive abilities and acquired resistance against multiple anticancer drugs. Our results demonstrate that ECRG2is upregulated in response to DNA damage, andis capable of inducing the activation of both caspase cascades (intrinsic and extrinsic) leadingto cancer cell death. We further illustratedthat ECRG2-mediated cell deathwasattributed to a reduction in the levels of apoptosis inhibitor, X chromosome-linked inhibitor of apoptosis protein (XIAP). ECRG2 was revealedto regulate XIAP protein levels via RNA-binding protein,human antigen R(HuR).We further highlight that ECRG2 causes increased HuR ubiquitination,subsequently leading to its degradation. Thus, we demonstrate that ECRG2 sensitizescancer cells to apoptosis through the downregulation of HuR, and consequent downregulation of XIAP. Next, we have identified ECRG2 as a potent positive regulator of death receptor 5 (DR5) gene expression.ECRG2-mediated upregulation of DR5 was shown to be facilitated through the upregulation of tumor suppressor p53 and transcription factors ATF3 and NFⱪB. Together, in a cooperative fashion, increased levelsof p53, ATF3 and NFⱪB stimulateDR5 gene expression. Contrastingly, silencing of ECRG2 not only decreased the levels of NFⱪB and DR5, but also resulted in decreased cancer cell sensitivity to genotoxic stressandTRAIL treatment. Collectively,our work establishes that ECRG2 is capable of inducing apoptosis in cancer cells by increasing the expression of pro-apoptotic proteins, while also negatively influencing anti-apoptotic proteins.Further, the loss of ECRG2’stumor suppressive abilities, as wehaveshownby the loss of ECRG2in lung patient tumor samples,and through the somatic point mutantV30E, illuminates possible mechanismsin which cancer cells can acquiremultiple drug resistance.
    • 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.