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The unfolded protein response is one mechanism utilized by endoplasmic reticulum (ER) to maintain the homeostasis between ER protein folding machinery and ER proteins. UPR is induced by three ER transmembrane sensors: IRE1, ATF6 and PERK. PKR-like ER kinase (PERK) can sense the ER stress signal through its luminal domain to activate its cytoplasmic kinase domain catalytic activity. PERK kinase domain belongs to the eIF2α family. Members in this family are activated after being autophosphorylated at their activation loops and then specifically phosphorylate eIF2α at its Ser51 position. Phosphorylation of eIF2α can shutdown the cytosol protein translation from the initiation stage. Studies on PERK luminal domain and kinase domain indicated that PERK may form un-clustered homo-dimer in its inactive form. The crystal structure of PERK kinase domain determined in this dissertation revealed an intermediate conformation between inactive conformation and active conformation. In this conformation, the activation loop in PERK kinase domain C-lobe has been phosphorylated at a conserved Thr residue, T980, and stabilized by this phosphorylation. In addition, helix 8 in PERK KD C-lobe is fixed through the interactions with stabilized activation loop and ready for substrate bindings. A "line-Up" model for PERK KD autophosphorylation was proposed in this dissertation base on the dimer orientation in this structure. This can be also used to explain mechanism of PERK pathway activation. The translocation of mitochondria matrix proteins is conducted by an IMM translocase TIM23. Tim44 is an essential, peripheral membrane protein in TIM23 translocation pathway, which associates with IMM through its C-terminal domain and recruits the import motor to the TIM23 channel. It can be localized either on the membrane or in the matrix. X-ray Crystallographic and biochemical studies on Tim44 CTD in this dissertation revealed that the N-terminal helices A1 and A2 in Tim44 CTD are amphipathic and critical for Tim44 membrane binding. Hydrophobic residues in A1 and A2 contribute to the hydrophobic interactions between Tim44 and IMM. Base on these data, a membrane-induced conformational change model has been proposed to explain the mechanism for Tim 44 membrane binding.

1 online resource (xi, 93 p.) : ill., digital, PDF file

Cell Biology;

Joint Health Sciences;

UPR PERK kinase domain Endoplasmic Reticulum eIF2α kinase protein translation TIM23 Tim44 mitochondria translocation peripheral membrane protein

UNRESTRICTED

Advisors/Committee Members: Sha, Bingdong, Chang, Chenbei<br>, Collawn, James<br>, Narayana, Sthanam V. L.<br>, Prevelige, Peter Edward.

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Metazoan cells are characterized with elaborate network of intracellular membranous compartments. These membranes allow the cell to spatially separate antagonistic processes and environments, and maintain sequential order of reactions necessary for maturation and secretion of biosynthetic cargo. The core transport machinery consists of coat proteins, tethering factors, SNAREs and small Ras-like GTPases. We have explored the function of the tethering factor p115 in organellogenesis and in secretory traffic. The p115-depleted system was utilized to explore structurefunction relationships within p115. Here, we analyzed the architecture of the Golgi after RNAi induced depletion of p115. We show that in p115 depleted cells Golgi disrupts in to dynamic perinuclear structures that retain the cis-trans polarity of the normal Golgi complex, and can reassemble after treatment with Brefeldin A and subsequent washout. We also characterized the impact of p115 depletion on the traffic and secretion of biosynthetic cargo. Our studies revealed that p115 depletion has a selective effect on cargo traffic - we observed ER retention of transmembrane VSV-G protein with no effect on trafficking of soluble proteins. The p115 tethering factor does not appear to have an enzymatic activity and acts through the interaction with its binding partners. The C-terminal coiled-coil region of p115 is critical for binding to Rab1 GTPase and SNAREs, and has been shown to be essential for p115 function in Golgi biogenesis. We used p115 deletion mutant to analyze the importance of CC3 and CC4 for p115 function. Expression of the p115/1-766 mutant, lacking CC3-4 region, in p115 depleted cells or in control cells disrupted Golgi in to scattered punctate fragments, which suggest a dominant-negative nature of the mutant. Interestingly, the p115/1-766 mutant supported ER exit of transmembrane VSV-G but arrested it in dispersed punctuate structures, without any detectable effect on transport of soluble cargo. Our studies provide the first evidence that the CC3-4 region is necessary for the function of p115. Combining our findings with the current knowledge on p115 functional domains, we propose a model where p115 not only increases the fidelity of SNARE-pin formation, but also functions in proteins sorting during ER exit of cargo proteins.

xi, 86 p. : ill., digital, PDF file

Cell Biology

Joint Health Sciences

p115 Golgi vesicular traffic

UNRESTRICTED

Advisors/Committee Members: Sztul, Elizabeth, Theibert, Anne Burton <br>, Collawn, Jim <br>, Kirk, Kevin L. <br>, Rayner, Julian C..

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The extraordinary capacity of Mycobacterium tuberculosis (Mtb) to adapt to environmental changes during infection contributes to its success as a pathogen. While the unique outer membrane (OM) of mycobacteria functions as a permeability barrier for toxic molecules, uptake of nutrients is required to sustain viability of Mtb. Whereas hydrophobic molecules can diffuse through membranes, uptake of small hydrophilic compounds is mediated by water-filled channels, porins. For example, Msp-like porins of M. smegmatis (Ms) were shown to be required for uptake of hydrophilic β-lactam antibiotics, which are also known to diffuse through porins in gram-negative bacteria. Because the structure of the OM of Ms and Mtb is similar, we hypothesized that disruption of an unknown porin in Mtb would increase resistance to β-lactams. A transposon library of M. bovis BCG (BCG) was screened for mutants with increased resistance to ampicillin. Mutations were found in genes required for the biosynthesis of the OM as well as two previously uncharacterized genes, rv0194 and rv3903c. Overexpression of rv0194 in BCG and Ms showed that Rv0194 functions as an ATP-binding cassette efflux pump that confers resistance to β-lactams, suggesting that Mtb has tripartite drug efflux pumps that span the entire cell envelope similar to that of E. coli. A mutation in rv3903c largely increased resistance to hydrophilic drugs and conferred resistance to nitric oxide both in vitro and in vivo. Uptake of glycerol was minimal in the rv3903c mutant resulting in decreased growth rate, which can be restored when grown on medium with the hydrophobic nutrient, oleic acid, as the sole carbon source. The recombinant N-terminal domain of Rv3903c purified from Ms and E. coli had a channel activity with a primary single conductance of 4.0±0.2 nS. Furthermore, it was able to complement the growth defect of the rv3903c mutant and an msp porin mutant of Ms. We suggest that Rv3903c is the first identified porin of Mtb in a new class of proteins with an unusual signal sequence and unique domain organization. Taken together, this work significantly advances our understanding of the transport mechanisms for nutrients and toxic compounds in mycobacteria.

1 online resource (ix, 145 p.) : ill., digital, PDF file.

Microbiology

Joint Health Sciences

UNRESTRICTED

Advisors/Committee Members: Niederweis, Michael, Bedwell, David M.<br>, Benjamin, William H.<br>, Dybvig, Kevin F.<br>, Stejn, Andries J.<br>, Turnbough, Charles L..

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Mycobacterium tuberculosis (Mtb) infects one third of the global population and causes approximately 2,000,000 Tuberculosis-related deaths annually. Mycobacteria are Gram positive organisms but contain a unique outer membrane (OM) which is functionally similar but structurally different from those of Gram negative bacteria. The mycobacterial OM presents an efficient permeability barrier towards hydrophilic solutes. Slow permeation kinetics of hydrophilic molecules through membranes and several discoveries of cell wall channel-forming proteins indicate that mycobacterial OMs are functionalized by proteins. MspA is the primary porin of M. smegmatis and mediates diffusion of small, hydrophilic nutrients and antibiotics across the OM. Unlike porins of Gram negative bacteria, MspA forms a large, octameric β-barrel and represents the founding member of a new class of OM proteins. Mtb does not encode Msp homologs and functions of Mtb OM proteins are unknown. Therefore, MspA serves to model transport of hydrophilic compounds across mycobacterial OMs. Here, the periplasmic L6 loop and the constriction zone were identified as determinants of transport through the MspA channel. The L6 loop affected transport of ions, monosaccharides, voltage-gating, expression, and channel stability, but was dispensable for porin function. Conversely, the constriction zone mutant MspA D90L was highly impaired for channel activity. As the role of the hydrophilic pathway across the OM during Mtb infection was unknown, we expressed MspA wt and D90L in Mtb during mouse infections to determine whether increased permeability would be beneficial due to increased nutrient uptake or detrimental due to toxic solute influx. Surface exposure of functional pores drastically reduced virulence, suggesting that toxic solute influx outweighs the benefits of increased nutrient uptake during infection. Because genes that reduce fitness are selected against in vivo, stable gene integration is required. Therefore, we constructed a new series of phage-based plasmids capable of multiple, stable integrations in mycobacterial chromosomes. When used in tandem, these vectors provide increased global expression of terminator-protected gene cassettes. Thus, this work describes how porins functionalize the hydrophilic pathway across mycobacterial OMs and defines low OM permeability as required for Mtb virulence. Additionally, new genetic tools were generated to enhance in vivo examination of genes in mycobacteria.

1 online resource (x, 185 p. : ill., digital, PDF file)

Microbiology;

Joint Health Sciences;

outer membrane mycobacterium porin nutrient uptake tuberculosis

UNRESTRICTED

Advisors/Committee Members: Niederweis, Michael, Dybvig, Kevin<br>, Marques, Guillermo<br>, Steyn, Andries<br>, Turnbough, Charles.

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Malignant gliomas are the most common and deadly form of primary brain cancer afflicting adults. Current treatment regimens, including surgical debulking, radiotherapy, and chemotherapy, have limited efficacy, and median patient survival remains only 14 months. Therefore, novel therapies must target different aspects of glioma biology. Two of the most striking features of this cancer are the unusual ability of glioma cells to robustly proliferate and migrate in the brain, and recent evidence suggests that ClC-3, a voltage-gated Cl- channel/transporter is implicated in both of these processes. We hypothesize that ClC-3 may facilitate proliferation and migration by promoting hydrodynamic shape and volume changes; as Cl- efflux occurs, water osmotically leaves the cytoplasm. These shape and volume changes are critical as, for example, a glioma cell divides into 2 daughter cells, or migrates through narrow extracellular spaces in the brain. In this dissertation, we assess upstream signaling to determine how ClC-3 is activated in the context of proliferation and migration. Using a combination of biophysical, biochemical, genetic, and imaging techniques, we identify several mechanisms suggesting that Ca2+/calmodulin-dependent protein kinase (CaMKII) regulates ClC-3 activity. We demonstrate that channels or ligands that increase [Ca2+]i also activate CaMKII, leading to downstream ClC-3 activation and promoting proliferation and migration. CaMKII regulation of ClC-3 is required for a critical cytoplasmic condensation checkpoint at the metaphase-anaphase transition, and inhibition of either protein leads to disrupted volume regulation and proliferation. Additionally, we found that bradykinin, a chemotactic peptide, increases glioma cell migration by activating CaMKII-dependent ClC-3 channels. Inhibition of ClC-3 or CaMKII completely blocked bradykinin-induced migration. We propose that CaMKII activation of ClC-3 is a critical mediator of cellular proliferation and migration and should be integrated into preexisting models. We speculate that [Ca2+]i may be a ""master regulator"" of both proliferation and migration by simultaneously controlling cytoskeletal proteins, kinases, and Ca2+-sensitive ion channels. Finally, our data suggest that targeting Cl- channels or bradykinin receptors on human glioma cells may be a novel therapeutic strategy for the management of malignant gliomas.

PhD

1 online resource (xi, 213 p.) :ill., digital, PDF file.

Neurobiology

Joint Health Sciences

bradykinin cell migration chloride channel ClC-3 Cl- channel

UNRESTRICTED

Advisors/Committee Members: Harald Sontheimer, Bedwell,David Kirk,Kevin Nabors,Burt Wadiche,Jacques.

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Sil1 is an Endoplasmic Reticulum (ER) localized protein. SIL1 was initially identified as a UPR-regulated gene. Later studies show Sil1 functions as the nucleotide exchange factor of ER lumenal Hsp70—Bip by directly interacting with the ATPase domain of Bip. Currently, the molecular mechanism how Sil1 catalyzes nucleotide exchange of Bip is still elusive. In this study we determine the complex structure of yeast S.cerevisia Sil1 and Bip (also called Kar2) ATPase domain at 2.3Å resolution by Single-anomalous dispersion (SAD) methods. The Sil1-Bip complex structure reveals that one sil1 molecule interacts with one Bip ATPase domain molecule to form the complex. Sil1 forms an elongated shape which is entirely comprised of α-helices including four Armadillo-like repeats. In the complex structure, Sil1 wraps around the subdomain IIb of the Bip ATPase domain and acts in a “clamp” model. The binding of Sil1 forces a ~13.5° outward rotation of subdomain IIb away from the nucleotide-binding cleft. The complex formation also induces a ~3.7° rotation of subdomain Ib to the opposite direction. These conformational changes open up the nucleotide-binding cleft of Bip ATPase domain, disrupt the hydrogen bonds between Bip ATPase domain and bound ADP, and eventually promote the ADP release and the subsequent ATP binding. A group of interface residues from Sil1 are mutated. Two single mutants H163A and E390A result in complete loss of Sil1-Bip interaction. All the other mutants compromise binding between two proteins to various extents. The ATPase activity assay results show all the Sil1 mutants lose the ability to stimulate the ATPase activity of Bip. The results are consistent with that of the reduced binding affinities between Sil1 mutants and Bip ATPase domain. Utilizing the structural and functional data, a reasonable mechanism for Sil1 functioning as the nucleotide exchange factor of Bip has been proposed.

1 online resource (xi, 78 p.) : ill., digital, PDF file.

Cell Biology;

Joint Health Sciences;

Sil1 Bip ATPase activity

UNRESTRICTED

Advisors/Committee Members: Sha, Bingdong, Chang, Chenbei<br>, Deivanayagam, Champion<br>, Lin, Fang-Tsyr<br>, Sthanam, Narayana.

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Cystic fibrosis (CF) is a lethal genetic disorder leading to pulmonary decline and premature death. The gene responsible for CF, the cystic fibrosis transmembrane conductance regulator (CFTR), serves as a chloride and bicarbonate channel situated at the apical cell surface of epithelia. The discovery of small molecules that augment channel gating of mutant CFTR (so-called ‘potentiators’) represents a major theme of CF research, and offers hope for new therapeutic interventions. High-throughput screening (HTS) provides a means to test millions of agents in an unbiased manner for CFTR potentiating properties. New agents identified by HTS have undergone extensive preclinical and clinical testing in CF individuals, and shown substantial progress toward clinical advancement. However, novel strategies are required to better understand CFTR potentiators and their applicability in the clinic. In this thesis, we pursued a detailed understanding of emerging CFTR potentiators with high relevance to therapeutic intervention. We describe a set of mechanistic experiments intended to functionally categorize CFTR potentiators based on their biochemical effects on the CFTR regulatory domain (RD), and their activity profiles in cell systems representative of the in vivo environment. Our findings indicate that CFTR potentiators acting independently of RD phosphorylation may be best tailored to rescue cAMP regulation of CFTR carrying the common ΔF508 mutation. In another series of experiments, we show that quercetin, a novel flavonoid, potentiates CFTR mediated anion transport in respiratory epithelia in vitro and in vivo, and may be useful to optimally detect rescue of ΔF508-CFTR. The present project therefore addresses two questions fundamental to both CFTR biology and CF therapeutic intervention: 1. How can knowledge of CFTR modulator mechanism aid in optimization of emerging CFTR potentiators and advance basic understanding of CFTR biology? 2. How can knowledge of a robust class of CFTR activators, the flavonoids, be used to identify compounds best able to activate and detect surface localized and functional CFTR in human subjects with cystic fibrosis? These findings address the increasingly recognized gap between identification of new therapeutic compounds for CF, and a better understanding of agents most suitable for basic and translational cystic fibrosis research.

1 online resource (xv, 115 p.) : ill., digital, PDF file.

Genetics

Joint Health Sciences;

CFTR Cystic Fibrosis potentiators quercetin regulatory-domain cAMP

UNRESTRICTED

Advisors/Committee Members: Sorscher, Eric J., Bedwell, David M.<br>, Dobrunz, Lynn E.<br>, Rowe, Steven M.<br>, Yoder, Bradley K..

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Alcoholic liver disease is a serious public health concern. In particular, the mitochondrion is a specific target of ethanol toxicity and much of the damage can be related to unregulated Ca2+ homeostasis and oxidative stress which are key players in the induction of the mitochondrial permeability transition pore (MPTP) within the organelle. The mechanism behind the induction of the MPTP remains elusive. Therefore, this body of work will provide insight on what effects chronic alcohol consumption has on mitochondrial dysfunction with an emphasis on the MPTP. Chapter 2: Assessment of mitochondrial dysfunction arising from treatment with hepatotoxicants provides a description of several biochemical assays used to assess mitochondrial function when exposed to toxicants such as ethanol. Chapter 3: Chronic ethanol consumption enhances sensitivity to Ca2+-mediated opening of the mitochondrial permeability transition pore and increases cyclophilin D in liver presents data which test the hypothesis that chronic alcohol consumption causes mitochondrial dysfunction leading to increased sensitivity to the induction of the MPTP and impairment of Ca2+ retention capacity. The most significant findings were isolated liver mitochondria from ethanol-fed rats had decreased Ca2+ retention capacity, increased sensitivity to Ca2+-mediated induction of the MPTP, and increased levels of Cyclophilin D. Chapter 4: Cyclophilin D gene ablation is not protective against mitochondrial dysfunction in alcoholic liver disease extends and expands on the results presented in Chapter 3. This chapter further investigated the role of Cyp D in alcohol-induced mitochondrial injury using a Cyp D null mouse model. In this study we observed steatosis in livers of both wild-type and Cyp D-/- mice fed the ethanol-containing diet. State 4 respiration (ADP-independent) was increased in liver mitochondria isolated from both ethanol-fed wild-type and Cyp D-/- mice. Lastly, liver mitochondria from ethanol-fed Cyp D-/- were more sensitive than control-fed Cyp D-/- to Ca2+-mediated MPTP induction. These findings suggest that Cyp D gene ablation is not protective against alcohol induced mitochondrial dysfunction. Together this information provides a more comprehensive understanding of the molecular events that contribute to chronic ethanol-induced mitochondrial dysfunction and damage. An understanding of which we propose will better guide future therapeutic approaches for alcoholic liver disease.

1 online resource (xi, 200 p. : ill., digital, PDF file).

Environmental Health Sciences;

Public Health;

alcohol liver mitochondria calcium MPTP

UNRESTRICTED

Advisors/Committee Members: Bailey, Shannon M., Ballinger, Scott W.<br>, Darley-Usmar, Victor M.<br>, Dickinson, Dale A.<br>, Landar, Aimee L.<br>, Lesort, Mathieu J..

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The pathophysiology of schizophrenia is complex and diverse, with many classes of receptors, neurotransmitters, and brain regions implicated in this illness. The many hypotheses proposed have yet to fully explain the heterogeneity of the genetic, postmor-tem, and clinical evidence. It is the goal of this dissertation to integrate the current hy-potheses of schizophrenia into a unified hypothesis of abnormal intracellular signaling and signal integration. Inconsistencies in genetic and postmortem findings suggest that the development of schizophrenia is multifaceted, and the heterogeneity of symptoms supports the hypothesis of any number of intracellular signaling abnormalities as the pre-cipitating factor in the development of this devastating illness. In this dissertation I ex-amined conserved intracellular signaling pathways for activation (via phosphorylation of kinases) and functional outcome (via phosphorylation of proteins with known functional consequences). Abnormal protein expression in the MAPK pathway includes decreased SynGAP, Rap2, JNK1/2, and PSD-95 in the anterior cingulate cortex (ACC). Abnormal activation, determined by phosphorylation, of the MAPK pathway includes decreased pThr183/Tyr185 JNK1/2 and pSer295 PSD-95 in the ACC in schizophrenia. Abnormal protein expression in the cAMP pathway includes increased RACK1, Fyn, and Cdk5 in the dorsolateral prefrontal cortex (DLPFC) in schizophrenia. Abnormal function, deter-mined by phosphorylation, of the cAMP pathway includes increased pTyr1336 NR2B and pSer295 PSD-95 in the DLPFC in schizophrenia. Signaling through G protein coupled receptors (GPCRs) is regulated by G protein-coupled receptor kinases (GRKs). I measured the protein and mRNA expression of GRKs 2-6 in the anterior cingulate cortex. I found increased transcript expression for GRK2 and GRK5, and increased protein ex-pression for GRK5. The abnormalities in GPCR regulation, coupled with alterations of signaling cascades downstream of these proteins suggest that dysfunction of intracellular signaling and signal integration contribute to the pathophysiology of schizophrenia.

1 online resource (xii, 158 p.) : ill., digital, PDF file.

Neurobiology

Joint Health Sciences

Schizophrenia signaling MAPK cAMP GRK phosphorylation

UNRESTRICTED

Advisors/Committee Members: McCullumsmith, Robert E., Sweatt, J. David<br>, Thiebert, Anne<br>, Cowell, Rita<br>, Meador-Woodruff, James H..

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Primary cilia are antenna-like organelles that extend from the surface of almost all mammalian cell types. They regulate many signaling pathways and sense physical and chemical changes in the extracellular environment. Defects in primary cilia cause several human disorders of different severity collectively called ciliopathies, including Nephronophthisis (NPHP), Joubert syndrome (JBTS), and Meckel-Gruber syndrome (MKS). Numerous MKS, JBTS and NPHP genes have been identified but in most cases of these ciliopathies the genetic defect is unknown. Despite the fact that NPHP, JBTS and MKS patients present with distinct clinical features, they have mutations in identical genes. This can be explained by different nature of mutations in these genes, genetic interactions with mutations in other cilia-associated genes and individual�s genetic background influence. This is supported by data in the nematode C. elegans showing that simultaneous mutations in nphp and mks genes cause more severe cilia defects than mutations in only a nphp or mks gene. Here, the C. elegans model system was utilized to study nphp-4 genetic interactions. First, we analyzed the pathogenicity of eight human NPHP4 missense mutations in the worm homolog. We show that the majority of these mutations produce cilia defective phenotypes. Interestingly, despite the recessive inheritance of NPHP, some of the nphp-4 mutations where able to enhance mks phenotypes being while in heterozygous state. Second, we performed a chemical mutagenesis screen to find mutations in novel genes that interactwith the nphp-4(tm925) null mutation to produce synthetic cilia defects and isolated nine independent loci. We identified mutations in the mks-5 and osm-3 genes that produced a nphp-4 dependent cilia phenotype. Since the other seven loci from the screen do not map to locations of known MKS/NPHP genes, their subsequent identification will likely provide novel candidates for human cilia related disorders. Overall these data expand our knowledge of genetic interactions in ciliopathies and further establishes C. elegans as a model to study these diseases.

1 online resource (ix, 143 p.) :ill., digital, PDF file.

Genetics;

Joint Health Sciences

Cilia, ciliopathies, C. elegans, genetic interactions, genetic screen

UNRESTRICTED

Advisors/Committee Members: Michael A. Miller, Additional advisors: Michael A. Miller, John L. Hartman, Kai Jiao, Scott Wilson..

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Growth factor signals often regulate similar cellular processes both during embryogenesis and in adult homeostasis. Stringent control of these signals ensures proper embryonic development and correct cell physiology in mature individuals. Recently, a family of two members of Tomoregulin (or transmembrane protein with epidermal growth factor-like and two follistatin domains [TMEFF]) was found to interact with transforming growth factor-[beta] (TGF-[beta]) and ErbB signaling pathways, both of which are implicated in development and in cancer biology in the adult. The function as well as the mechanisms of TMEFFs in modulating these two signals has not been elucidated in detail. In this dissertation, I investigate the activities of TMEFFs in modulating signaling by the of TGF-[beta] and ErbB pathways in a vertebrate model and in mammalian cell culture, respectively. In the first part, I showed that TMEFF1 selectively inhibits nodal but not activin signals in early Xenopus embryos through direct interaction with the nodal coreceptor Cripto. Accordingly, Cripto rescues the inhibition of the nodal signaling by TMEFF1 in Xenopus ectodermal explants. Furthermore, I showed that the Cripto-FRL1- Cryptic (CFC) domain in Cripto, which is essential for its binding to the type I nodal receptor ALK4, is also important for its interaction with TMEFF1. This study demonstrates for the first time that nodal signaling can be regulated by a novel mechanism of blocking the Cripto coreceptor. In the second part, I uncovered a novel association between both TMEFFs and all four ErbB family members in coimmunoprecipitation assays. The epidermal growth factor-like domain of TMEFF1 was dispensable for its association with ErbB4, suggesting that TMEFFs bind ErbB receptors in a manner unlike orthodox ErbB ligands. Functional analyses of the consequence of TMEFF/ErbB interaction revealed that TMEFFs do not affect ErbB signaling in tumor cell lines despite the direct physical association. TMEFFs may thus have subtle effects on ErbB-regulated processes in a cell type- and/or environmentdependent manner. In summary, my work identifies direct associations of TMEFFs with Cripto and ErbB receptor tyrosine kinases and suggests novel roles for TMEFFs in modulating TGF-[beta] and ErbB signaling during embryonic development and tumorigenesis.

x, 96 p. : ill., digital, PDF file

Cell Biology

Joint Health Sciences

UNRESTRICTED

Advisors/Committee Members: Chang, Chenbei, Frank, Stuart J. <br>, Justement, Louis B. <br>, Lin, Fang-Tsyr <br>, Serra, Rosa A..

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Cilia are evolutionarily conserved, membrane-bound, microtubule-based organelles found on a diverse array of cell types in eukaryotic organisms. Inherited diseases of cilia protein dysfunction include Nephronophthisis (NPHP), Joubert Syndrome (JBTS), Meckel-Gruber Syndrome (MKS), and Bardet-Biedl Syndrome (BBS). Important insight in the basic cell biological functions of BBS and NPHP proteins has been gained from analysis in the nematode Caenorhabditis elegans. My goal in this dissertation was to model MKS protein function in cilia biology utilizing the powerful genetic malleability of C. elegans. mks-1 and mks-3, the C. elegans homologs of two MKS-associated proteins in humans, were selected for this analysis. MKS-3 is a transmembrane protein of unknown function, and MKS-1 is a protein comprised of an uncharacterized motif called the B9 domain. Because the B9 domain is found in only two other proteins in C. elegans (and most other eukaryotes), the B9 proteins TZA-1 and TZA-2 were also included in this analysis. Lastly, the C. elegans homologs of NPHP proteins NPHP-1 and NPHP-4 were also analyzed in conjunction with the MKS/TZA proteins. Utilizing mutations in each of the genes encoding these proteins, we were able to develop a model in which the MKS/B9 proteins and the NPHP proteins form independent but functionally related complexes at the base of C. elegans cilia. Disruption of either complex alone by genetic mutation did not hinder cilia formation. However, simultaneous disruption of both complexes resulted in severe ciliogenesis defects. This functional requirement of either the MKS/B9 or the NPHP complex for cilia formation indicates that the two protein complexes serve similar yet distinct roles in maintaining cilia homeostasis. In the absence of a functional MKS/B9 complex, transmembrane proteins that normally localized only to the base of cilia (or just outside of the ciliary base) freely accessed the entire ciliary membrane. This data supports a model in which the MKS/B9 complex regulates ciliary membrane composition by selectively holding some transmembrane proteins at the base of cilia while blocking other transmembrane proteins from accessing the cilium. Overall, this dissertation provides insight into the related but distinct functions of MKS/B9 and NPHP proteins in cilia biology.

xv, 161 p. : ill., digital, PDF file

Cell Biology

Joint Health Sciences

Basal Body Cilia MKS NPHP C. elegans Transition Zone

UNRESTRICTED

Advisors/Committee Members: Miller, Michael A., Yoder, Bradley K.<br>Bell, P. Darwin<br>Marques, Guillero<br>Schwiebert, Erik M.<br>Sztul, Elizabeth S..

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The secretory pathway within a cell consists of series of membrane compartments connected by shuttling secretory vesicles containing cargo that travel from endoplasmic reticulum (ER) though ER-to-Golgi intermediate compartment (ERGIC), the Golgi apparatus, and endosomal compartment to the plasma membrane (PM). The Golgi specific brefeldin A (BFA)-resistant factor 1 GBF1 has been identified as an important factor involved in traffic between ER and Golgi and within the Golgi apparatus. GBF1 belongs to a family of guanine nucleotide exchange factors (GEFs) that stimulate the exchange of GDP for GTP on small GTPases, ADP-ribosylation factors (ARFs). The exchange of the nucleotide activates ARFs and activated ARFs interact with variety of effectors including coat proteins and lipid modifying enzymes. In order to initiate the coat protein complex I (COPI) recruitment cascade, GBF1 must associate with membranes, but the membrane dynamic of GBF1 and its role in Golgi biogenesis were unknown. A central focus of this study was to explore membrane association of GBF1, the molecular events facilitated by GBF1, and to determine how interaction with lipid flippases integrates GBF1 activity with other traffic events. We found that GBF1 cycles rapidly between the cytosol and membranes and is stabilized on membranes when in a complex with ARF-GDP. Using siRNA mediated depletion we showed that GBF1 is necessary for membrane association of COPI. Although a secretory pathway capable of trafficking of soluble proteins in GBF1- depleted cells is maintained, GBF1-mediated ARF activation and COPI recruitment is required for trafficking of transmembrane proteins. The GBF1 catalytic domain (Sec7) is sufficient to catalyze nucleotide exchange by itself; therefore it was highly probable that the remaining part of GBF1 plays a regulatory role. We generated GBF1 mutants impaired in binding to putative lipid flippase and explored its cellular behavior and influence on Golgi morphology. Our results suggest that these interactions may be important for the modulation of GBF1 activity. The findings presented here further describe GBF1as a key molecule required for Golgi biogenesis and cargo trafficking, and will serve as a paradigm for other GEFs.

1 online resource (xvi, 157 p. : ill., digital, PDF file)

Cell Biology;

Joint Health Sciences;

GBF1 GEF coat ARF COPI

UNRESTRICTED

Advisors/Committee Members: Sztul, Elizabeth, Collawn, Jim<br>, Marques, Guillermo<br>, Theibert, Anne<br>, Woods, Anne.

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Tim44 is a peripheral membrane protein and a component of the TIM23 translocon on the matrix side. It is well established that Tim44 tethers the presequence associated motor (PAM) complex to the Inner Mitochondrial Membrane (IMM), through its C-Terminal Domain (CTD). This study focuses on understanding the high resolution structure of Tim44 CTD and the molecular basis for its membrane anchoring mechanism. The crystal structure of Tim44 CTD revealed that it exists as a single domain. The N-terminal amphipathic helices A1 and A2 protrude away from the main body of Tim44 CTD. These two flexible helices have been tested for their putative role in membrane anchoring by functional analyses. Biochemical studies were conducted using mutant forms of Tim44 CTD. Deletion and site directed mutants of Tim44 in the A1 and A2 helical region were used to study if the membrane binding ability is hindered. The deletion mutants included two constructs in which either a part of the A1-A2 helical region or the entire A1-A2 helical region was removed. The results showed that the A1 and A2 helices are required for Tim44 CTD to bind membranes. The point mutants included three constructs in which three conserved residues within A1 and A2 helices were mutated. Hydrophobic point mutations compromised the membrane binding ability. These investigations along with other structural data suggested that the A1-A2 helical region undergoes a conformational change along a hinge during membrane binding. This hinge is present in the loop that follows the A2 helix and is composed of two conserved glycine residues. This conformational change exposes the hydrophobic residues of the A1 and A2 helices to the IMM. In a soluble form these helices conceals the hydrophobic residues towards the protein core but during membrane binding, these residues seem to get exposed and interact with the membrane. We propose a mechanism by which the A1 and A2 helices turn along the glycine hinge either to conceal or to expose their hydrophobic side-chains based on the nature of the surrounding environment. The studies enumerated in this dissertation reveal the structure of Tim44 CTD at an atomic resolution. The membrane anchoring region of Tim44 CTD has been revealed using functional studies. Utilizing the structural and the functional data, a plausible mechanism for Tim44 CTD membrane binding has been proposed and tested.

xii, 92 p. : ill., digital, PDF file

Cell Biology

Joint Health Sciences

Tim44 Mitochondria Translocon Peripheral Membrane Protiein Crystal Structure

UNRESTRICTED

Advisors/Committee Members: Sha, Bingdong, Chang, Chenbei<br>Collawn, James<br>Deivanayagam, Champion<br>Sthanam, Narayana.

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Insulin resistance and hyperglycemia are common findings in patients following acute illnesses or injuries, such as surgical trauma, thermal injury, infection, hemorrhage, and sepsis. Insulin has three main target tissues: liver, skeletal muscle, and adipose tissue. Insulin resistance in the liver results in an increase in gluconeogenesis. In skeletal muscle, insulin resistance causes a decrease in insulin-stimulated glucose uptake. However, little is known about the contribution that adipose tissue makes to the insulin resistant state. The goals of the research presented in this dissertation were to: 1) determine whether insulin resistance developed in adipose tissue following acute trauma and hemorrhage; 2) characterize the expression of adipose-derived cytokines and adipokines following trauma and hemorrhage; and 3) determine whether there were long-term effects on insulin signaling and the expression of adipose-derived cytokines and adipokines after resuscitation and recovery. A rat model of surgical trauma and hemorrhage was used to achieve these goals. Insulin signaling via the insulin receptor/insulin receptor substrate-1/Akt pathway was examined. There was an acute onset of insulin signaling defects in adipose tissue as early as 15 min following hemorrhage which persisted for at least 90 min. During the hemorrhage period, there were no consistent and persistent alterations in the adipose-derived cytokines and adipokines that were examined. At the end of the 24 h recovery period, adiponectin mRNA was notably decreased and IL-1β mRNA was increased. The studies in this dissertation provide evidence for the acute onset of adipose tissue insulin resistance following trauma and hemorrhage. Additional evidence is provided that adipose tissue-derived cytokines and adipokines do not play key roles in the development of insulin resistance, but may in fact play important roles in maintaining insulin resistant states. Hyperglycemia in critically ill patients is associated with a poor outcome. Controlling hyperglycemia has been demonstrated to reduce morbidity and mortality. Thus, understanding the role of adipose tissue in insulin resistance may assist in the development of therapeutic interventions to manage insulin resistance following acute injury.

1 online resource (xv, 120 p. : ill., digital, PDF file)

Biochemistry and Molecular Genetics;

Joint Health Sciences;

insulin resistance adipose tissue trauma and hemorrhage glucose transport adipokines

UNRESTRICTED

Advisors/Committee Members: Messina, Joseph L., Engler, Jeffrey A.<br>, Hardy, Robert W.<br>, Ryan, Thomas M.<br>, Wood, Philip A..

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The molecular chaperones have special functions in mitochondrial protein translocation. For some mitochondrial preproteins, the protein targeting from the cytosolic ribosome to the surface of the mitochondrion is carried out through chaperones/Tom70p pathway. This network is connected by the proteins including heat shock protein Hsp40-Hsp70 and mitochondrial receptor Tom70p. However, a deep understanding of the interactions of these cytosolic proteins is greatly hampered by the lack of high-resolution structures of the proteins involved. Structural studies of the proteins Hsp40-Hsp70 and Tom70p have been undertaken using single-crystal X-ray diffraction method. First, the crystal structure of the Ydj1 Cterminal fragment, which comprises the Ydj1 domain III and C-terminal dimerization motif, indicates that the dimerization motif of type I Hsp40 Ydj1 differs significantly from that of yeast type II Hsp40. In the structure, the domain IIs of Ydj1 monomers that contain the zinc-finger motifs point directly against each other. The modeled full-length Ydj1 dimer structure reveals that a large cleft is formed between the two monomers, which is presumably the docking site for Hsp70. Second, the crystal structure of yeast Hsp40 Sis1 peptide-binding fragment complexed with the Hsp70 Ssa1 C-terminus provides a structural basis for the mechanism by which the conserved C-terminal EEVD motif in Hsp70 regulates Hsp40–Hsp70 interaction. The Ssa1 extreme C-terminal eight residues, G634PTVEEVD641, form a [Beta]-strand with the domain I of Sis1 peptide-binding fragment. The Ssa1 C-terminus binds Sis1 at the site where Sis1 interacts with the nonnative polypeptides. Third, the crystal structure of yeast Tom70p, which is a major surface receptor in the mitochondrial translocase of the outer membrane (TOM) complex, provides insights into the mechanisms by which Tom70p receives the mitochondrial protein precursors. In the structure, Tom70p forms a homo-dimer. Each subunit consists primarily of tetratricopeptide repeat (TPR) motifs, which are organized into a righthanded superhelix. The TPR motifs in the N-terminal domain of Tom70p form a peptidebinding groove for the C-terminal EEVD motif of Hsp70, whereas the C-terminal domain of Tom70p contains a large pocket that may be the binding site for mitochondrial precursors. Collectively, these structural studies presented in this dissertation help elucidate the mechanisms underlying how Hsp40 facilitates the preprotein delivery to Hsp70 and how Tom70p receives the mitochondrial preproteins from Hsp70. Taken together, the studies will contribute to our understanding of the mechanisms of mitochondrial preprotein targeting, the initial step of mitochondrial protein translocation.

x, 99 p. : ill., digital, PDF file

Cell Biology

Joint Health Sciences;

Crystal Structure Molecular Chaperone Hsp40 Hsp70 Translocon Tom70

UNRESTRICTED

Advisors/Committee Members: Sha, Bingdong, Chang, Chenbei<br>, Collawn, James F.<br>, Narayana, Sthanam V. L.<br>, Smith, Craig D..

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Growth hormone (GH) is a powerful promoter of postnatal longitudinal bone growth in mammals. GH signaling is mediated exclusively by the GH receptor (GHR), and is dependent upon activation of the tyrosine kinase JAK2 (Janus kinase 2). Cellular sensitivity to GH, then, is determined by the surface abundance of full-length GHR pro-tein. Proteolysis of the GHR ectodomain is known to occur. This process liberates the GHR extracellular domain (ECD), leaving a GHR “remnant” that remains bound to the plasma membrane. GHR proteolysis is induced by a variety of stimuli, including plate-let-derived growth factor (PDGF), high concentrations (20%) of serum and PMA (a phorbol ester). Inducible proteolysis of the GHR may result in acute desensitization of cells to GH by limiting the availability of cell-surface receptors. In this dissertation, we focused our studies on the fate of the remnant protein generated by inducible proteolysis. We found that the remnant protein is subjected to additional proteolysis within the TMD by [Gamma]-secretase, resulting in a soluble GHR fragment (the GHR stub). The GHR stub is highly labile and degraded by the proteasome. GH treatment, known to prevent inducible proteolysis of the GHR, did not promote stub formation. N-terminal sequenc-ing of purified stub verified that γ-secretase cleavage occurs within the GHR TMD. Us-ing confocal microscopy, we show that GHR stub can be localized to the nucleus. Pre-liminary data from confocal microscopy suggests that nuclear accumulation of the stub may be enhanced by deleting the distal 85 amino acids of GHR. Finally, attempts to pre-vent [Gamma]-secretase-mediated processing of the GHR remnant were not successful, perhaps due to promiscuous nature of this protease.

viii, 104 p. : ill., digital, PDF file)

Cell Biology

Joint Health Sciences

Growth Hormone Receptor Gamma-secretase Proteolysis

UNRESTRICTED

Advisors/Committee Members: Frank, Stuart J., Collawn, Jim<br>, Fuller, Gerald M.<br>, Lin, Fang-Tsyr<br>, Schweibert, Erik.

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Glioblastoma Multifrome is the most common and aggressive of the primary brain tumors. These tumors express multiple members of the Epithelial Sodium Channel (ENaC)/Degenerin (Deg) family, associated with a basally active amiloride sensitive cation current. We hypothesize that this glioma current is mediated by a hybrid channel composed of a mixture of Epithelial Sodium Channel (ENaC) and Acid Sensing Ion Channel (ASIC) subunits. To test this hypothesis we made dominant negative cDNAs for ASIC1, αENaC, and γENaC. D54-MG glioma cells transfected with the dominant negative constructs for ASIC1, αENaC, or γENaC showed reduced protein expression for each of the specific subunits and a significant reduction in the amiloride-sensitive whole cell current as compared with untransfected D54-MG cells. We also show an interaction between ASIC1, αENaC, and γENaC on the plasma membrane of D54-MG glioma cells using co-immunoprecipitation and in transfected rat primary astrocytes using Total Internal Reflection Fluorescence (TIRF) microscopy. Knocking down ASIC1, αENaC, or γENaC also significantly inhibited glioma cell migration compared to cells without subunit knockdown consistent with the hypothesis that these subunits play an important role in glioma biology. Using immunohistochemisty we show a higher expression level of ASIC1, αENaC, and γENaC in GBM biopsy tissue compared with non-neoplastic brain tissue. To understand the reason for this increase in expression of ASIC1 and ENaC subunits in GBM cells and tissue compared with primary astrocytes and non-neoplastic brain tissue, we used TIRF microscopy to image live glioma cells under acidic conditions (pH 6.0), similar to the acidic environment found in the core of a glioblastoma lesion.We found an increase in the plasma membrane localization of ASIC1 in D54-MG glioma cells when cells were grown at pH 6.0 compared to pH 7.4. Using High Resolution Clear Native electrophoresis we also found that ASIC1 forms a complex with ENaC subunits in D54-MG glioma cells that migrate at ≈ 480 kDa. These data suggest that different ENaC/Deg subunits can combine to form a hybrid channel that likely underlies the amiloride-sensitive current seen in human glioma cells.

1 online resource (x, 138 p. : ill., digital, PDF file)

Neurobiology;

Joint Health Sciences;

GBM ASIC ENaC TIRFM

UNRESTRICTED

Advisors/Committee Members: Benos, Dale J., Hablitz, John J.<br>, Lester, Robin A.J.<br>, McMahon, Lori L.<br>, Theibert, Anne B..

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Unrestricted cell proliferation and suppression of cell death are two essential events for tumor development. My dissertation research involves two proteins, 14-3-3 &tau and EDD which are involved in diverse pathways related to these two fields in recent studies. Previous study demonstrates that 14-3-3ô regulates p21 degradation. Up-regulation of 14-3-3ô is seen in breast cancer and is correlated with the down-regulation of p21 in breast cancer. Amplification or overexpression of EDD was observed in breast cancer and ovarian cancers. Illustrating the new roles of these two proteins in proliferation and cell death will advance our knowledge in tumorigenesis and help us develop right strategies for cancer treatment. Our present study uncovered a new role of EDD in cell cycle progression. Silencing of EDD induces phosphorylation of p53 at Ser15 and the transcription of p53 target genes without activation of DNA damage response. Brdu incorporation assay reveals that depletion of EDD is able to induce the G1/S arrest via p53. On the other hand, overexpression of EDD prevents p53 phosphorylation at Ser15 and the induction of p53 target genes during DNA damage and this effect does not require its E3 ligase activity. We also demonstrated that EDD can directly interact with p53 and the inhibition of p53 phosphorylation by ATM relies on this interaction. Thus, through the association with p53, EDD actively inhibits p53 phosphorylation by ATM, and plays a role in ensuring smooth G1/S progression.As to my second project on 14-3-3ô, we demonstrated that 14-3-3ô regulates the autophagy through Beclin 1, an essential protein in autophagy. We also confirmed that E2Fs regulate Beclin 1 and E2F1 mediated the modulation of Beclin 1 by 14-3-3ô. Futhermore, we revealed that tenascin-C, an anti-adhesive extracellular matrix protein for detachment (a process which causes autophagy), induces Beclin 1 through 14-3-3ô, thus placing the tenascin-C/14-3-3ô/E2F1/Beclin 1 regulation in a physiological context.

PhD

1 online resource (xiii, 88 p.) :ill., digital, PDF file.

Cell Biology

Joint Health Sciences

14-3-3 autophagy Cell Cycle EDD p53

UNRESTRICTED

Advisors/Committee Members: Lin Weei-Chin, Stuart,Frank Bingdong,Sha Ruppert,Susan Lin,Fang-Tysr.

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O-linked beta-N-acetylglucosamine (O-GlcNAc) is a dynamic and ubiquitous posttranslational modification of serine and threonine residues on nuclear and cytoplasmic proteins. O-GlcNAc has emerged as an important regulator of cellular processes such as cell signaling, transcription, translation, apoptosis, and cell cycle regulation, among others. O-GlcNAc is thought to be a contributor to pathologies such as hyperglycemia and insulin resistance. O-GlcNAc has been viewed as an indicator of cellular energy levels and is associated with diabetic complications under nutrient excess. Other studies have shown that a variety of stress stimuli increase the levels of protein O-GlcNAc in mammalian cells, and this increase is associated with cytoprotection. Inhibiting O-GlcNAcylation decreased cell survival in response to stress, while increased O-GlcNAcylation augmented cell survival. Many of these studies demonstrated that this cytoprotection was associated with mitochondrial proteins.Compared to the nucleo-cytoplasm, much less work has been done in elucidating the potential role of O-GlcNAc in the mitochondria. In cardiac myocytes chronically exposed to high levels of glucose, complexes of the mitochondrial respiratory chain were shown to be O-GlcNAc modified. However these modifications were associated with impaired mitochondrial respiratory function. Hyperglycemia also induces mitochondrial superoxide production, which was shown to increase hexosamine biosynthesis and O- GlcNAcylation. Reactive oxygen species production can also lead to mitochondrial respiratory dysfunction and loss of mitochondrial membrane potential. It is possible that O-GlcNAcylation of respiratory chain complexes is involved in cardioprotection. While these results may seem contradictory, O-GlcNAcylation may simply be a constitutive element in non-pathological cellular functions, including oxidative phosphorylation, and perturbations in homeostatic O-GlcNAc signaling may lead to cell and mitochondrial dysfunction.Despite the current progress, the effect of O-GlcNAc on the many functions that mitochondria perform is still relatively unknown. It is increasingly apparent that mitochondrial proteins are O-GlcNAc modified and that this modification appears to modulate the function of these proteins.

MS

1 online resource (xv, 60 pages) :illustrations

M.S.University of Alabama at Birmingham2013.

Joint Health Sciences

Glycosilation, Hexosamine Biosynthesis, Mitochondria, O-GlcNAc, Reactive Oxygen Species

UNRESTRICTED

Advisors/Committee Members: John C. Chatham, Ballinger, Scott W. Darley-Usmar, Victor M. McMahon, Lori L. Young, Martin E..

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Numerous disorders are characterized by the presence of cystic lesions within the kidney. The proteins associated with these disorders often localize to cilia, and improper formation or signaling from the cilum has been established as a causative factor leading to cyst formation. In this dissertation my goal was to determine whether the invertebrate nematode Caenorhabditis elegans could be utilized as a malleable system to identify pathways involved in human cystic disorders. In particular I wanted to assess the func-tion of two proteins mutated in the human cystic kidney disorder Nephronophthisis, Nephrocystin-1 and Nephrocystin-4. The corresponding genes nphp1 and nphp4 were initially selected for study because they both have clear homologs in the nematode, nph-1 and nph-4, respectively. Additionally, both nph genes exhibited the presence of X-box sequences in their promoters, suggesting regulation by the X-box binding protein DAF-19, a transcription factor which regulates many cilia specific genes in C. elegans. My analysis determined that both nph-1 and nph-4 were expressed in ciliated sensory neurons and were indeed regulated by DAF-19. Subsequently, both NPH proteins were shown to co-localize specifically to the transition zone at the base of cilia, a region analogous to the mammalian basal body. Mutants of nph-1 and nph-4 were characterized to determine the function they may be playing at this cellular region. Although the cilia in these mutants were formed normally, defects in the cilia-mediated signaling processes of chemotaxis behavior, lifespan regulation, and locomotion in the presence of food were observed. Additionally, the NPH-4 protein was found to be required for NPH-1 protein localization. ii Interestingly, a specific Nephrocystin-4 patient mutation was identified that could rescue this localization of NPH-1, but could not rescue the decreased locomotory behavior seen in nph-4 mutant worms. Locomotory behavior is thought to result from a signal received by the cilia that transmits downstream to EGL-4, the C. elegans homolog of cGMP-dependent Protein Kinase G. Analysis of nph mutants along with egl-4 mutants indicated that the EGL-4 protein likely also functions downstream of the NPH proteins. Overall, this dissertation successfully established C. elegans as a fruitful model to study Nephronophthisis protein function.

1 online resource (xiii, 123 p. : ill., digital, PDF file)

Cell Biology;

Joint Health Sciences;

cilia cystic kidney disease nephronophthisis transition zone

UNRESTRICTED

Advisors/Committee Members: Yoder, Bradley K., Caldwell, Kim A.<br>, Miller, Michael A.<br>, Schwiebert, Erik M.<br>, Sztul, Elizabeth S..

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Porphyromonas gingivalis is a main causative agent for adult chronic periodontitis and immunization with its virulence factor Hemagglutinin B (HagB) provides protection against infection. Toll-like receptors (TLRs) recognize various microbial products and are crucial in eliciting and regulating the innate and adaptive immune responses to infections. The objective of this dissertation was to investigate the cellular mechanisms that influence the innate and adaptive immune response to HagB and P. gingivalis, focusing on the role of TLR signaling in the response. We started with investigating the ability of HagB to activate dendritic cells (DC), the most efficient antigen-presenting cell in priming naive CD4 T cells, and the role TLR signaling played in this response. Our results showed that HagB induced DC maturation in a TLR4 dependent manner and required adaptor molecules MyD88 and TRIF for an optimal response. Second, we investigated the requirement of TLR4 signaling in shaping the CD4 T cell response to HagB. Our results showed that HagB immunization primed a CD4 T cell response that responded to P. gingivalis stimulation by IFN-? production. TLR4 signaling shaped the type of CD4 T cell and antibody response induced and regulated the expression of transcription factors, T-bet, GATA3, and Foxp3, and the IL-2/STAT5 signaling pathway. MyD88 and TRIF played differential regulatory roles downstream of TLR4 in shaping the CD4 T cell response. Third, we investigated the role of Interleukin 10 (IL-10) in inhibiting the CD4 T cell response to P. gingivalis. Our results showed that CD4+CD25- T cells produced IL- 10 and upregulated Foxp3 in response to P. gingivalis in a TLR2 dependent manner. Upon neutralization of IL-10, CD4+CD25- T cells produced substantial amounts of IFN-? and upregulated T-bet. The results from these studies suggest that the immune response to HagB and P. gingivalis is tightly regulated by inflammatory signals mediated by TLR4 and IFN-?, and inhibitory signals mediated by TLR2 and IL-10. The proper understanding of the mechanisms governing the balance between these inflammatory and inhibitory pathways can provide us with necessary information for effective design of strategies to fight periodontal diseases.

1 online resource (xi, 194 p.) :ill., digital, PDF file.

Microbiology

Joint Health Sciences

Toll-like receptors, P. gingivalis, Hemagglutinin, CD4 T cells

UNRESTRICTED

Advisors/Committee Members: Suzanne M. Michalek, Additional advisors: Peter D. Burrows, Charles O. Elson, Jenny Katz, Robinna G. Lorenz, Moon Nahm..

▼ The proton dependent oligopeptide transporters (POT family) are members of the Major Facilitator Superfamily of secondary active transporter proteins. They use the transmembrane proton gradient to drive the uptake of di- and tripeptides into the cytoplasm. Members of the family are highly conserved in pro- and eukaryotic genomes, and in humans they are responsible for the oral absorption of many drug families, including -lactam antibiotics. Recently, the crystal structures of PepTSo and PepTSt, two prokaryotic homologues of the human proteins PepT1 and PepT2, captured the proteins in two distinct conformations, providing insight into the structural aspects of the transport mechanism. A protocol was designed for functional liposome reconstitution of POT proteins, and transport assays were conducted to characterise their substrate specificity, pH dependence and kinetic properties. Using site-directed mutagenesis, we identified binding site residues involved in peptide recognition and proton translocation, and distinguished between the two roles by comparing protein activity in proton- and peptide-driven conditions. We also investigated the roles of key residues in the conformational transitions that accompany the transport cycle, using data from biochemical assays, molecular dynamics simulations and modeling, as well as electron paramagnetic resonance measurements. In addition, several bacterial POT members were screened for crystallisation, in order to assess their stability and crystal diffraction quality in different detergents. Further work was performed with bacterial POT homologues YdgR and GkPOT, including binding studies using NMR spectroscopy and assaying drug transport in vivo and in vitro. Together, the data establish bacterial POTs as model systems for studying the mammalian oligopeptide transporters, and a mechanistic model for peptide transport is proposed.

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Low density Lipoprotein receptor Related 6 (LRP6) functions as an essential coreceptor for Wnt/β-catenin signaling as pathway activation, reflected by cytosolic β- catenin stabilization and TCF/LEF-1 transactivation, requires Glycogen Synthase Kinase 3β (GSK3β)-mediated phosphorylation of multiple PPP(S/T)P motifs within the membrane anchored LRP6 intracellular domain. Additionally, LRP6 undergoes a proteolytic cleavage event resulting in the formation of a soluble LRP6 intracellular domain (LRP6-ICD). LRP6-ICD can function within the Wnt/β-catenin pathway by interacting with GSK3β and attenuating the phosphorylation of GSK3β substrates such as cytosolic β-catenin as well as stimulate TCF/LEF-1 activity. However, LRP6-ICD’s role(s) within the Wnt/β-catenin pathway and the mechanism(s) that govern LRP6-ICD activity are not clear. The cytosolic function of LRP6-ICD has been examined, but its possible function in the nucleus remains unexplored. A better understanding of LRP6- ICD biology will improve our understanding of the regulation of the Wnt/β-catenin pathway. The objective for the first part of the project was to determine if LRP6-ICD requires GSK3β-mediated phosphorylation of its PPP(S/T)P motifs to function within the Wnt/β-catenin pathway, similar to LRP6. Results show LRP6-ICD is functionally distinct from LRP6 as LRP6-ICD functions within the pathway in the absence of PPP(S/T)P phosphorylation and functions as a pathway modulatory protein not as a primary activating component. We also showed LRP6-ICD modulates the pathway by functioning as a true GSK3β effector protein capable of directly attenuating GSK3β kinase activity. In the second part, we analyzed a potential role for LRP6-ICD in the nucleus. Our findings showed LRP6-ICD is a nucleocytoplasmic protein that differentially regulates Wnt/β-catenin pathway activity depending upon its localization and proteinprotein interaction. We validated the hypothesis that cytosolic LRP6-ICD positively modulates Wnt/β-catenin activity through GSK3β and enhanced cytosolic β-catenin stabilization. Surprisingly, nuclear LRP6-ICD negatively modulates the pathway by interacting with and attenuating Amino-terminal Enhancer of Split (AES), a positive Wnt/β-catenin modulatory protein. To summarize, our studies reveal that LRP6-ICD differentially modulates the Wnt/β-catenin pathway in a context and localization dependent manner. Our studies also provide further insight concerning the growing role modulatory proteins play in the Wnt/β-catenin pathway and mechanistic diversity of the pathway.

1 online resource (xi, 139 p. : ill., digital, PDF file)

Cell Biology;

Joint Health Sciences;

LRP6 LRP6-ICD AES Beta-catenin GSK3

UNRESTRICTED

Advisors/Committee Members: Johnson, Gail V.W., Benveniste, Etty<br>, Frank, Stuart J.<br>, Lesort, Mathieu<br>, Murphy-Ullrich, Joanne E..

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Posttranslational modifications of histones regulate important chromatin and cellular functions. Among them, ubiquitination of histone H2A is correlated to transcriptional repression, such as HOX gene silencing and X chromosome inactiviation. Little was known about the removal of ubiquitin from histones, the enzyme(s) involved and its function in chromatin dynamics. We have identified the protein Ubp-M (USP16) to be the H2A- and nucleosome-specific deubiquitinase. We also demonstrated that Ubp- M-mediated H2A deubiquitination is involved in cell cycle progression to M-phase, HOX gene expression, and posterior development in Xenopus laevis. Furthermore, we have also purified USP12 and USP46 which contain an Ubp-M independent deubiquitinase activity for both uH2A and uH2B. USP12 and USP46 each form a complex with the WD40 repeat-containing protein WDR48, which is required for the deubiquitinase activity. USP12 and USP46 regulate HOX gene expression and gastrulation during Xenopus laevis development. These studies will contribute to the understanding of the regulatory mechanisms of H2A and H2B ubiquitination and deubiquitination, and their biological functions.

1 online resource (x, 90 p.) : ill., digital, PDF file.

Biochemistry and Molecular Genetics;

Joint Health Sciences;

histone H2A ubiquitination deubiquitination Ubp-M

UNRESTRICTED

Advisors/Committee Members: Wang, Hengbin, Chang, Chenbei<br>, Chow, Louise<br>, Ruppert, J. Michael<br>, Townes, Tim M..

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In the nervous system, PI 3-kinase has been implicated in neuronal differentiation. My studies have focused on a candidate neuronal PI 3-kinase target centaurin alpha-1, which binds PtdIns(3,4,5)P3, and is an Arf6 GAP. Centaurin alpha-1 is localized in dendrites, dendritic spines and synapses, and is required for neuronal differentiation and spine morphogenesis. In dissociated neuronal cultures, expression of centaurin alpha-1 enhances dendritic branching, and increases dendritic filopodia, lamellipodia and spine-like protrusions. Expression of centaurin alpha-1 GAP inactive mutant or knocking down centaurin alpha-1 levels using siRNA leads to inhibition of dendritic outgrowth and branching. Manipulations of centaurin alpha-1 also disrupt spine morphogenesis in organotypic brain slice cultures. The effects of centaurin alpha-1 on dendritic development are dependent on it functioning through regulation of Arf6. The constitutively GTPbound mutant Arf6, which reduces dendritic branching on its own, is able to reverse the effects of centaurin alpha-1 overexpression. Conversely, expression of the GDP-bound mutant, Arf6, which enhances branching and outgrowth on its own, can prevent the loss of dendrites induced by centaurin alpha-1 GAP inactive mutant expression or siRNA knock down. Arf6 has been shown to regulate Rac1, and both Arf6 and centaurin alpha- 1 have been proposed to regulate ERK; both Rac1 and ERK have been implicated in neuronal differentiation. Thus, I examined whether centaurin alpha-1 modulates neuronal Rac1 and ERK, to identify candidate downstream effectors of centaurin alpha-1 and Arf6 in neuronal differentiation. ERK and Rac1 activation are enhanced in centaurin alpha-1 over-expressing neurons and this activation is dependent on centaurin alpha-1 GAP activity. As a regulator of Arf6, centaurin alpha-1 has emerged as a candidate to participate in PI 3-kinase regulated Arf6 pathways that control dendritic differentiation and spine morphogenesis.

xiv, 160 p. : ill., digital, PDF file

Neurobiology

Joint Health Sciences

Centaurin alpha-1 P13 kinase Arf6 Dendrites Rac 1 ERK1/2

UNRESTRICTED

Advisors/Committee Members: Theibert, Anne, Floyd, Candice <br>, Pozzo-Miller, Lucas <br>, Roth, Kevin <br>, Wilson, Scott.

▼ Nicotinic acetylcholine receptors are pentameric ligand-gated ion channels. These receptors are present in the central and peripheral nervous systems where they mediate fast synaptic transmission by allowing the flux of cations through the plasma membrane. The heteropentameric α4β2 subtype is the most abundant nicotinic receptor in the brain and it's the focus of my dissertation project. This receptor is involved in learning, memory formation, mood, attention and reward. Its dysfunction has been linked to neurodegenerative diseases and mental illnesses including schizophrenia, Alzheimer's disease, epilepsy, Parkinson's disease and nicotine addiction. Because of its key role in the brain and connection to diseases, I sought to understand the basic principles underlying gating, subunit assembly, ligand recognition and ion selectivity of the human α4β2 nicotinic acetylcholine receptor. I developed multiple biochemical and biophysical methods that enabled the crystallization of the α4β2 receptor. The expression system and assay for stoichiometry I developed are applicable to a broad range of soluble and membrane proteins. I leveraged these methods to obtain crystals of the receptor that, after extensive optimization, diffracted X-rays to beyond 4 Å resolution. This result provided the first high-resolution structure of a nicotinic acetylcholine receptor. Co-crystallization with the agonist nicotine revealed principles of ligand selectivity among the different classes of subunit interfaces; specifically, I was able to explain high-affinity nicotine binding to the α-β subunit interfaces and its exclusion at β-β and β-α interfaces. Nicotine stabilized the receptor in a non-conducting, desensitized conformation. I showed that the constriction point in the permeation pathway was formed at the selectivity filter located at the cytosolic end of the pore. In addition, I used the high-resolution structure as a template to perform site-directed mutagenesis to examine the mechanisms of ligand recognition and channel gating. I elucidated the ligand exclusion mechanism at the β-β and β-α interfaces and proposed a potential role for these interfaces in the allosteric gating mechanism. In summary, these structural and functional studies have provided information on the basic principles of the high-affinity nicotine interactions, the architecture of allosteric sites and the permeation pathway, principles of subunit assembly, and increase our understanding of the mechanism of channel desensitization. Advisors/Committee Members: Jiang, Youxing, Hibbs, Ryan E., Kavalali, Ege T., Thomas, Philip J..

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Human cytomegalovirus, a ubiquitous human pathogen, establishes a persistent infection in the infected host. HCMV assembly takes place in the nucleus and cytoplasm of infected cells by a complex and incompletely defined process. The viral DNA is enclosed by the capsid, which is surrounded by a proteinaceous tegument, which is covered by a cell-derived envelope studded with viral glycoproteins. The assembly pathway and protein interactions required for formation of the tegument layer and the function of most of the proteins in the tegument remain poorly understood. In this study, we investigated the functions of an essential abundant tegument protein, pp150, in the assembly of the virus. This protein is thought to bind to capsids and play an important role in the cytoplasmic maturation of HCMV. However, a major issue that remains unexplained is the mechanism by which this protein is localized to the cytoplasmic assembly compartment (AC), the putative site of virus maturation. The cellular and viral proteins that interact with pp150, and thus contribute to its function are also unknown. We conducted a series of yeast two-hybrid screens to identify potential interactors of pp150. Among the many potential interactors we found, we concentrated on a cellular protein Bicaudal-D1 and a viral tegument protein ppUL25. The interaction between pp150 and BicD1 was confirmed by co-immunoprecipitation, co-localization and proven to be direct using Fluorescence Resonance Energy Transfer. BicD1 was found to be essential for specifically trafficking pp150 to the cytoplasmic viral assembly compartment (AC). We found that pp150 interacted with the C-terminal coiled-coil domain of BicD1, which also interacts with the GTPase Rab6. Next, we found that Rab6 also form part of the pp150-BicD1 complex and plays a role in trafficking of pp150, potentially by addressing the pp150-BicD1 complex to the Rab6-associated membranes. Also, the AC depends on the microtubule network and the dynein molecular motor for its formation/maintainence. Finally, we confirmed that the pp150 directly interacted with ppUL25. This interaction, combined with the BicD1-Rab6-Dynein-dependent trafficking of pp150 may explain how ppUL25 gets trafficked to the AC, and provides evidence that tegumentation of HCMV involves nucleation of tegument subunits that utilize host cell proteins to target to the AC.

1 online resource (xiv, 164 p.) :ill., digital, PDF file.

Microbiology

Joint Health Sciences

UNRESTRICTED

Advisors/Committee Members: William J. Britt, Additional advisors: Terje Dokland, Casey D. Morrow, Elizabeth Szutul, Sunnie Thompson..

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The ability to differentially manipulate available genetic information in order to generate diverse cellular identities represents an innovation of complex multicellular eukaryotic organisms. Cis-acting modules that regulate transcription play extremely important roles in ensuring lineage-specific expression of genes that define cellular identities. In our studies, using Interferon-gamma (IFN-gamma, a cytokine encoded by the gene Ifng), a cytokine encoded by T, NK and NKT cells as a model, we have identified and characterized the roles of key cis regulatory elements that facilitate T lineage specific induction as well as repression of Ifng transcription. In particular, our studies have highlighted the role of RelA, an NF-kappa B family member in driving acute Ifng transcription in response to reactivation signals. These studies have led to the recognition of multiple conserved non-coding sequences (CNS) in the Ifng locus including CNSs -34, -22, +46 and +54 that positively modulate Ifng transcription by co-recruiting STAT4 and RelA. To gain further insight into the actions of these complex cis-regulatory modules in induction of Ifng transcription in vivo, we have generated mice that carry a targeted deletion of a candidate enhancer CNS-22 that has been demonstrated to recruit multiple key trans factors including T-bet, STAT4, Runx3 and RelA to transactivate Ifng transcription. Th1, Tc1 and NK cells derived from CNS-22-/- mice are severely compromised in their ability to secrete IFN-gamma in response to IL-12 and IL-18, but also show less prominent defects in IFN-gamma induction in response to TCR reactivation signals as well. Using CNS-22 as a candidate enhancer, we have also explored some of the fundamental mechanisms that facilitate enhancer actions of CNS-22. Specifically, we demonstrate that activation induced local hyperacetylation of regions that flank CNS-22 plays an essential role in driving inducible transcription of Ifng. Lastly, we extend our findings to other distal enhancers and demonstrate stimulus-responsive activation of Ifng expression is associated with inducible hyperacetylation of local micro-domains that flank multiple enhancers that activate Ifng transcription.

PhD

1 online resource (x, 114 p.) :ill., digital, PDF file.

Microbiology

Joint Health Sciences

cytokine enhancers epigenetics gene regulation helper T cell Th1 cell

UNRESTRICTED

Advisors/Committee Members: Casey T. Weaver, Hatton, Robin D. Klug, Christopher A. Benveniste, Etty N. Ryan, Thomas M. Justement, Louis B..