Thesis (Ph. D.)--University of Chicago, Pritzker School of Medicine, Department of Molecular Genetics and Cell Biology, June 2002. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Chicago, Committee on Neurobiology, December 2002. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Chicago, Dept. of Molecular Genetics and Cell Biology, June 2003. / Includes bibliographical references. Also available on the Internet.

L’ion calcium est un second messager qui intervient dans de nombreux processuscellulaires dont la prolifération, la différenciation et l’apoptose. Ainsi, l’homéostasiecalcique constitue un point central de régulation de la signalisation cellulaire. En effet, laconcentration calcique cytosolique de calcium subit des oscillations, qui suivant leuramplitude ou leur fréquence, vont être capables d’activer spécifiquement certains facteursde transcription. La régulation de ces oscillations implique entre autres les ATPases de typeSERCA (Sarco/Endoplasmic Reticulum Calcium ATPase) qui accumulent le calcium dansle réticulum endoplasmique. L’objectif de ce travail de thèse a été l’étude des SERCAs aucours de la différenciation lymphocytaire B et dans l’épithélium du plexus choroïde ; ceci,afin de mieux comprendre le profil d’expression de ces pompes et les mécanismes derégulation impliqués.Au cours de la différenciation de lignées de leucémie aiguë lymphoblastique (LAL) nousavons observé que l’expression de l’isoforme SERCA2 restait stable ou augmentaitlégèrement alors que celle de l’isoforme SERCA3 était toujours fortement induite, pouvantatteindre des niveaux observés dans les cellules lymphoïdes matures. Nous avons égalementobservé que l’inhibition de l’activité des SERCAs altère la différenciation cellulaire qui estdépendante de la voie des PKC. Ces données indiquent que SERCA3 pourrait être utiliséecomme marqueur de la différenciation lymphocytaire B. Une régulation de l’expression desSERCAs a également été mise en évidence au cours de la différenciation de l’épithélium duplexus choroïde normal ou tumoral. SERCA3 est fortement exprimée dans l’épithéliumnormal, mais on retrouve une baisse ou une perte de son expression dans l’épithéliumtumoral, cette baisse est corrélée à la perte de la différenciation selon le grade des tumeurs.L’étude de l’expression des SERCAs dans les cellules primaires du plexus choroïde traitépar des agents cyto-différenciateurs (acides gras à chaîne courte), montre que ladifférenciation est associée à une surexpression de SERCA3. SERCA3 peut donc égalementêtre un marqueur de la différenciation de l’épithélium du plexus choroïde.L’ensemble de ce travail a montré que la différenciation cellulaire est associée à la régulationde protéines impliquées dans la régulation de l’homéostasie calcique : les SERCAs. On peutainsi proposer SERCA3 comme un nouveau marqueur phénotypique utile pour l’analyse dela différenciation du plexus choroïde normale et néoplasique, ainsi que pour celle de ladifférenciation lymphoïde pré-B leucémique. / Cellular calcium is involved in a multitude of biological processes including thecontrol of cell proliferation, differentiation and programmed cell death, and constitutestherefore a keconcentration calcique cytosolique de calcium subit des oscillations, qui suivant leuramplitude ou leur fréquence, vont être capables d’activer spécifiquement certains facteursde transcription. La régulation de ces oscillations implique entre autres les ATPases de typeSERCA (Sarco/Endoplasmic Reticulum Calcium ATPase) qui accumulent le calcium dansle réticulum endoplasmique. L’objectif de ce travail de thèse a été l’étude des SERCAs aucours de la différenciation lymphocytaire B et dans l’épithélium du plexus choroïde ; ceci,afin de mieux comprendre le profil d’expression de ces pompes et les mécanismes derégulation impliqués.Au cours de la différenciation de lignées de leucémie aiguë lymphoblastique (LAL) nousavons observé que l’expression de l’isoforme SERCA2 restait stable ou augmentaitlégèrement alors que celle de l’isoforme SERCA3 était toujours fortement induite, pouvantatteindre des niveaux observés dans les cellules lymphoïdes matures. Nous avons égalementobservé que l’inhibition de l’activité des SERCAs altère la différenciation cellulaire qui estdépendante de la voie des PKC. Ces données indiquent que SERCA3 pourrait être utiliséey element in cell signaling. Calcium levels vary in a dynamic mannerdepending on the state of activation of the cell, and can display oscillations the amplitudeand frequency of which can convey specific signals to various transcription factors.Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA enzymes) accumulate calciumfrom the cytosol into the endoplasmic reticulum (ER). By modulating the spatiotemporalcharacteristics of calcium signals and oscillations, SERCA pumps constitute an importantand unique point of control of calcium-dependent cell activation. In this work weinvestigated SERCA expression during early B lymphoid differentiation and in normal,tumoral and hyperplastic choroid plexus epithelial cells.We have shown that SERCA3 expression is markedly increased during thepharmacologically induced differentiation of immature B acute lymphoblastic leukemiacells, whereas the expression of the simultaneously expressed SERCA2 isoform is notmodified significantly. SERCA3 expression during this differentiation process can reachlevels observed in mature B lymphoid cells, and is dependent on the activation of proteinkinase C. Moreover, the direct pharmacological inhibition of SERCA-dependent calciumtransport interferes with the differentiation process.Our investigations on the choroid plexus show, that whereas SERCA3 is highly expressedin normal choroid plexus epithelium, expression is strongly decreased in benign choroidplexus tumors and is lost in carcinoma, whereas expression is retained in hyperplasia. Inaddition, treatment of primary normal choroid plexus epithelial cells by short chain fattyacid-type cell differentiation-inducing agents in vitro leads to the induction of SERCA3expression.Our observations when taken together indicate that ER calcium homeostasis is remodeledduring the differentiation of immature B lymphoid cells and in the choroid plexus due to theinduction of SERCA3 expression. We show that a cross-talk exists between SERCA functionand the control of differentiation in B cells, that SERCA3 constitutes a new phenotypicmarker for the study of early B cell differentiation, and that the lack of SERCA3 expressionmay be useful for the identification of choroid plexus tumors.

>Magister Scientiae - MSc / Cancer is defined as the abnormal growth of genetically mutated or perturbant cells. Nicotine is a known cancer promoter and an apoptotic suppressor. This alkaloid acts on the nicotinic acetylcholine receptors which affects the ubiquitin-proteasome protein degradation pathway and ultimately hinders apoptosis. The endoplasmic reticulum (ER) is an interconnecting organelle which synthesises proteins and its quality control processes ensures the proper protein folding, post-translational modifications and conformation of secretory and trans-membrane proteins. Studies demonstrated that the antibiotic, Tunicamycin (Tm) and the sesquiterpene lactone, Thapsigargin (Tg) causes ER stress and consequently cellular arrest. Tm interferes with N-glycosylation of newly synthesised proteins triggering the unfolded protein response, while Tg inhibits intracellular Ca2+ ATPases resulting in increased cytosolic Ca2+. Studies showed that these compounds have potential pro-apoptotic effects. The combinatorial effects of nicotine, Tm and Tg may produce antagonistic or synergistic effects and provide a therapeutic tool against breast cancer. The aim of the study was to determine the apoptotic effects of nicotine, Tm, and Tg on human breast carcinoma (MCF-7) at various time intervals and further to elucidate whether selected ratios of their combinations resulted in synergistic or antagonistic effects.

No description available.

Endoplasmic reticulum (ER) stress plays an important role in the pathogenesis of obesity-related inflammation and insulin resistance in adipose tissue. However, the mechanisms responsible for induction of ER stress are presently unclear. Proper ER redox state is crucial for oxidative protein folding and secretion and impaired protein folding in ER leads to induction of unfolded protein response and ER stress. However, while ER redox state is more oxidizing compared to the rest of the cell, its regulation is poorly understood. In order to determine the effects of ER redox state on development of ER stress and insulin resistance, several fluorescence-based sensors have been developed. However, these sensors have yielded results that are inconsistent with each other and with earlier non-fluorescence-based studies. In this study we attempted to develop and characterize a sensitive tool to study the ER redox state in adipocytes in real-time by targeting a new generation of redox-sensitive green fluorescent protein (roGFP) to ER. The roGFP1-iL sensor targeted to the ER is termed ‘eroGFP1-iL’ by convention. The ER-targeting eroGFP1-iL construct contains the signal peptide from adiponectin and the ER retention motif KDEL and has a midpoint reduction potential of -229 mV in vitro in oxidized and reduced lipoic acid. Despite having a midpoint reduction potential that is 50 mV higher than the previously determined midpoint reduction potential of the ER, eroGFP1-iL was found capable of detecting both oxidizing and reducing changes in the ER. In an attempt to determine the mechanisms by which roGFP1-iL detects oxidizing changes, we found that, first, glutathione mediated the formation of disulfide-bonded roGFP1-iL dimers with an intermediate excitation fluorescence spectrum resembling a mixture of oxidized and reduced monomers. Second, glutathione facilitated dimerization of roGFP1-iL, which in effect shifted the equilibrium from oxidized monomers to dimers, thereby increasing the molecule’s reduction potential compared with a dithiol redox buffer like lipoic acid. From this study, we concluded that the glutathione redox couple in ER significantly raised the reduction potential of roGFP1-iL in vivo by facilitating its dimerization while preserving its ratiometric nature, which makes it suitable for monitoring oxidizing and reducing changes in ER with high reliability in real-time. The ability of roGFP1-iL to detect both oxidizing and reducing changes in ER and its dynamic response in glutathione redox buffer between approximately -190 and -130 mV in vitro suggest a range of ER redox potential consistent with those determined by earlier approaches that did not involve fluorescent sensors. Our primary aim in developing eroGFP1-iL as a redox-sensing tool was to be able to assess whether redox changes represent an early initiator of ER stress in obesity-induced reduction in high molecular weight (HMW) adiponectin in circulation. Hypoxia is a known mediator of redox changes. We found that oligomerization of HMW adiponectin was impaired in the hypoxic conditions observed in differentiated fat cells. The redox-active antioxidant ascorbate was found capable of reversing hypoxia-induced ER stress. Lastly, we demonstrated that changes in ER redox condition is associated with ER stress response and is implicated in the mechanism of action of the insulin-sensitizing agent troglitazone and desensitizing agent palmitate. Using the redox sensing property of eroGFP1-iL, palmitate was found to be an effective modulator of redox changes in the ER and troglitazone was found to cause oxidizing changes in the ER. The action of palmitate in causing aberrant ER redox conditions was associated with aberrant HMW adiponectin multimerization. Palmitate-induced ER stress was ameliorated by troglitazone. Taken together, the data suggest a potential role of ER redox changes in ER stress and impaired protein folding in adipocytes.

Homeostatic synaptic plasticity (HSP) regulates synaptic strength in response to changing neuronal firing patterns. This form of plasticity is defined by neurons’ ability to sense and over time integrate their level of firing activity, and to actively maintain it within a defined range. For instance, a compensatory increase in synaptic strength occurs when neuronal activity is chronically attenuated. However, the underpinning cellular mechanisms of this fundamental neural process remain poorly understood. We previously found that during activity deprivation, HSP leads to an increase in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptor function as well as a shift in subunit composition from Ca2+-impermeable GluA2-containing AMPA receptors to Ca2+-permeable GluA2-lacking AMPA receptors not only at synapses, but also at extrasynaptic sites. Neurons therefore appear to be actively enhancing Ca2+ entry, possibly as a compensatory mechanism in response to a prolonged Ca2+ deficit. To test whether the homeostatic response may, at least in part, be mediated by internal Ca2+ stores, we depleted endoplasmic reticulum (ER) Ca2+ stores by using the Sarco/endoplasmic reticulum Ca2+ ATPases (SERCA) pump blocker cyclopiazonic acid (CPA) for a prolonged period. Interestingly, we have found that prolonged Ca2+-store depletion leads not only to an increase in synaptic strength per se, but also a cell-wide increase in synaptic Ca2+-permeable GluA2-lacking AMPARs. This increase in Ca2+ influx following periods of inactivity is conceptually highly reminiscent of a store-operated response, whereby cells re-establish their calcium levels following Ca2+ store depletion using cell surface Ca2+ channels. Our results suggest that neurons use synaptic receptors as means to regulate store Ca2+ levels, thus significantly expanding our understanding of the repertoire used by neurons to modulate cellular excitability.

published_or_final_version / abstract / Biochemistry / Master / Master of Philosophy

Abstract ORIENTIA TSUTSUGAMUSHI ANKYRIN REPEAT-PROTEIN FAMILY TARGETING OF THE HOST ENDOPLASMIC RETICULUM By Lauren VieBrock, B.S. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University Virginia Commonwealth University, 2015 Director: Jason A. Carlyon, Ph.D. Professor Microbiology and Immunology Scrub typhus is an understudied, potentially fatal febrile illness, which poses threat to one billion people annually in the Asia-Pacific region. The host-pathogen interactions that facilitate the intracellular survival of the etiologic agent, Orientia tsutsugamushi, are not well understood. The Orientia tsutsugamushi genome encodes a large number of ankyrin repeat-containing proteins (Anks), key virulence factors for other intracellular pathogens, as well as components for Type I (T1SS) and Type 4 secretion systems (T4SS), commonly used to deliver them. We sought to characterize the roles of the Anks in O. tsutsugamushi infection. In this study, we demonstrated that O. tsutsugamushi expressed all 20 anks and the genes for the T1SS, for which they are substrates. Many ectopically expressed Anks displayed a tropism for the host endoplasmic reticulum (ER). These results suggest the importance of the Anks and the ER to Orientia tsutsugamushi pathobiology. We demonstrated that O. tsutsugamushi tightly associated with the ER and induced ER stress and defects in protein secretion of its host cells. Therefore, we hypothesized that the ER-tropic anks expressed during the initial hours of infection are critical for establishing infection and do so by interacting with specific host cell targets to modulate host cell function to benefit intracellular survival. ER-tropic Ank4 was detected as expressed early in infection and was further characterized for its contribution to the alterations of the ER during infection. Bat3 was identified as a target of Ank4, and Ank4 expression correlated with a decrease in Bat3 protein levels, induction of ER stress, and defects in protein secretion. These effects were Ank4 F-box dependent, implicating polyubiquitination and proteosomal degradation of Bat3. As Ank4 colocalized with Bat3, a chaperone component of ER-associated degradation (ERAD) of misfolded proteins, ERAD function was measured in cells expressing Ank4. In an F-box dependent manner, Ank4 expression resulted in decreased degradation of a model substrate and indicated inhibition of the ERAD pathway. Similarly, we demonstrated that in O. tsutsugamushi infection, Bat3 levels were significantly reduced early in infection and ERAD degradation was inhibited. After several days of infection however, Bat3 levels and ERAD degradation had both recovered, suggesting temporal modulation of ERAD in infection. Taken together, these data suggest that O. tsutsugamushi has a large capacity to disrupt the host ER, exemplified by Ank4 mediated ERAD dysfunction by depletion of host Bat3.