L'altération de glycosylation est l'une des caractéristiques du cancer du sein. Ainsi le défaut de glycosylation affecte différentes protéines glycosylées responsables des différents processus cancéreux. Les canaux SOC (Store operated channels) constituent la voie majeure de l'entrée du calcium dans les cellules et sont impliqués dans la prolifération, la migration et la survie des cellules cancéreuses du sein. CO-OCS est un nouvel inhibiteur de la glycosylation avec plus de sélectivité vis-à-vis des α-glucosidases, et montre des activités anticancéreuses des cellules cancéreuses du sein, sans affecter les cellules mammaires normales. L'objectif de ma thèse est d'étudier les mécanismes moléculaires par lesquels CO-OCS induit ses effets anti-tumoraux. CO-OCS inhibe la migration des cellules cancéreuses à fort potentiel métastatique. Cet effet anti-migratoire est dû à une réduction de l'expression de la β1-intégrine, de Stim1, et de l'activation des voies de signalisation FAK et ERK1/2 par CO-OCS. Dans les cellules cancéreuses peu invasives, CO-OCS diminue la prolifération et augmente la mortalité de ces cellules en affectant l'expression de 3 protéines : Stim1 et Orai1 : protéines N-glycosylées au niveau du réticulum endoplasmique (RE), et GRP78, protéine de stress du RE. Ainsi en supprimant complétement l'expression de Stim1, CO-OCS réduit la prolifération en accumulant les cellules dans les phases G1 et G2/M du cycle cellulaire. Alors que la réduction de l'expression de GRP78 et d'Orai1 par le CO-OCS augmente respectivement l'apoptose et la nécrose. Par ailleurs, l'invalidation de Stim1 atténue l'effet apoptotique induit par CO-OCS. CO-OCS réduit aussi le contenu calcique du RE. Cette réduction du calcium réticulaire est due à une fuite de calcium par le Translocon. En effet, l'Anisomycine, inhibiteur du Translocon, restore de contenu calcique réticulaire et antagonise l'apoptose induite par le CO-OCS. En conclusion, CO-OCS induit une accumulation de protéines mal-repliées dans le RE induisant ainsi un stress réticulaire. Trois cibles du CO-OCS ont été identifiées : l'expression de Stim1 favorise la prolifération tandis que celle d'Orai1 et de GRP78 protègent respectivement les cellules de l'apoptose et de la nécrose induites par CO-OCS. De plus, en diminuant l'expression de GRP78, CO-OCS induit une fuite du calcium du RE par le Translocon / Alteration in glycosylation pattern is one of the hallmarks of breast cancer. The levels and the abnormal expressions of glycan were found in breast cancer patients. Glycosylation defect can affect different glycosylated proteins which are implicated in cancerogenesis. Changes in intracellular Ca2+ levels can regulate different cellular processes. SOC channels are implicated in breast cancer proliferation, migration and survival. CO-OCS is a new glycosylation inhibitor with more selectivity toward theα- glucosidases exhibited anti-cancer activities in breast cancer cells without affecting the normal mammary cells. The objective of my thesis is investigating the related molecular mechanisms by which CO-OCS induced its anti-tumour effects.CO-OCS impaired breast cancer migration through decrease β1-integrin expression and the activation of FAK and ERK1/2 signalling pathways. CO-OCS also induced anti-migratory effect via Stim1 protein expression down-regulation leading to inhibition of SOCE. Additionally, CO-OCS affected the expression of both Orai1 and Stim1 proteins leading to anti-proliferative effects and cell cycle arrest in G1 and G2/M phase respectively. Moreover, CO-OCS affected the expression of Stim1 at the protein level without affecting its transcript level. GRP78 implicated in CO-OCS apoptotic death. The expression of Stim1 regulated the apoptosis induced by CO-OCS via modulating GRP78 expression. Orai1 down-regulation promoted CO-OCS necrotic effect. CO-OCS induced ER- calcium depletion due to increase in ER calcium leak via the Translocon; Anisomycin (Translocon inhibitor) decreased the apoptosis induced by CO-OCS. In conclusion, these results show that in breast cancer, by targeting Stim1, Orai1 and GRP78, CO-OCS reduced cell proliferation and induced apoptosis and necrosis cell death. Stim1 favours CO-OCS apoptotic effect while Orai1 protected from necrosis induced by CO-OCS. The inhibition of Translocon decreased CO-OCS apoptotic cell death via restoring the ER calcium homeostasis

Dans les cardiomyocytes, la concentration intracellulaire de Ca2+ doit être finement régulée pour maintenir l’homéostasie calcique. La protéine Stromal interaction molecule 1 (STIM1), qui joue un rôle important dans le maintien des niveaux intracellulaires de Ca2+ des cellules non excitables en opérant l’entrée capacitative de Ca2+ (SOCE), est aussi présente dans les cardiomyocytes. Plusieurs études démontrent que STIM1 et le SOCE jouent un rôle important dans le développement de l’hypertrophie des cardiomyocytes. De plus, récemment, un nouveau rôle de STIM1 a commencé à émerger. STIM1 pourrait moduler l’expression de certains canaux calciques à la membrane plasmique en régulant leur trafic intracellulaire. Le but de l’étude était d’identifier des partenaires d’interaction de STIM1 dans l’optique de révéler le mécanisme par lequel STIM1 induit l’internalisation d’un canal calcique voltage-dépendant. Les protéines recueillies par le pull-down à partir des lysats de coeurs de rats avec une colonne d’affinité composée du domaine ERM de STIM1, ont été analysées en spectrométrie de masse. La protéine Muscle related coiled-coil (MURC), une protéine de la famille des Cavin, a été retenue comme partenaire d’interaction potentiel de STIM1. Comme elle est exprimée dans les cardiomyocytes et dans les cellules musculaires squelettiques, qui sont des cellules où la régulation de la signalisation calcique est primordiale pour le bon fonctionnement des tissus et qu’elle semble interagir avec STIM1, qui est un acteur important de la signalisation calcique, notre objectif s’est élargi et nous avons investigué sur l’implication de MURC dans la signalisation calcique en général. Nous avons donc confirmé par co-immunoprécipitation que le domaine ERM de STIM1 interagissait avec MURC. Puis, par des essais d’imagerie calcique, nous avons démontré que la surexpression de MURC pouvait provoquer différentes réponses dans différents types cellulaires en fonction de l’activation de la mobilisation calcique. En effet, nous avons observé une augmentation du SOCE qui est indépendante de la voie RhoA/ROCK dans les cellules HEK293T, une diminution de l’entrée de calcium médiée par un récepteur (ROCE) qui est pourrait être dépendante de la voie RhoA/ROCK dans les cellules T6.11 et une diminution de l’activation de RhoA de façon dépendante de l’activation du SOCE dans les cardiomyocytes HL-1. Nous avons aussi montré que MURC pouvait interagir à la membrane plasmique avec les protéines Orai1, qui sont les protéines formant les canaux CRAC (Ca2+ release-activated Ca2+) du SOCE et ce de façon dépendante de l’activation de STIM1. Enfin, les résultats de cette étude suggèrent que MURC est un partenaire d’interaction de STIM1 impliqué dans la signalisation calcique. En effet, MURC peut moduler l’activation de RhoA, ce qui pourrait induire l’internalisation de canaux calciques. De plus, son interaction avec STIM1 et Orai1 pourrait notamment faire un pont facilitant l’interaction entre STIM1 et Orai1 ce qui aurait pour effet d’augmenter le SOCE et possiblement contribuer à augmenter l’hypertrophie des cardiomyocytes.

Indiana University-Purdue University Indianapolis (IUPUI) / Store-operated Ca2+ entry (SOCE) is involved in the maintenance of endoplasmic reticulum (ER) Ca2+ levels. The SOCE involves Stromal Interaction Molecule 1 (STIM1), distributed throughout the ER, and Orai1 channels, dispersed on the plasma membrane. SOCE is activated by the depletion of ER Ca2+ causing STIM1 to induce ER expansion and recruits Orai1 channels thus replenishing ER Ca2+. We reported downregulation of STIM1 in human islets from donors with type 2 diabetes (T2D) and in INS-1 β-cells treated with cytokines, and loss of STIM1 expression impairs β-cell SOCE, ER stress, and reduced insulin secretion. However, the regulatory mechanisms of STIM1 downregulation are unknown. To test this, actinomycin D and cycloheximide chase assay was performed to define whether IL-1β treatment impacted STIM1 mRNA or protein half-life. IL-1β had no impact on mRNA or protein decay. MicroRNAs (miRNAs), a class of small non-coding RNAs can regulate gene expression post-transcriptionally by binding to complementary regions in the 3’ untranslated region (UTR) of target mRNAs, affecting mRNA stability and translatability. The objective of this study was to establish miRNA regulation of STIM1 expression and altered SOCE. To identify potential miRNA candidates, RNA sequencing was done in human islets, treated with IL-1β and IFN-γ for 24 hrs. A total of 20 miRNAs were differentially expressed using a FC value of ≥ 1.5 and a p value of < 0.05. Of these, two miRNAs (miR-146a-5p and miR-4640-5p) were predicted by TargetScan to bind the 3’UTR of STIM1.To validate these findings, INS-1 β-cells, and human islets were treated with or without IL-1β. Only miR-146a-5p was upregulated in both systems. Consistent with inverse correlation, INS-1 β-cells transfected with miR-146a-5p mimic showed reduced STIM1 expression. To test whether miR-146a-5p inhibition preserves STIM1 expression, INS1 cells were treated with miR-146a-5p inhibitor along with IL-1β and inhibition of miR-146a-5p led to partial preservation of STIM1 expression. Future studies will test the effect of miR-146a-5p mimics and inhibitors on SOCE. The results indicate that the stress induced by IL-1β leads to induction of miR-146a-5p, which may then target STIM1 mRNA. Such studies could enable broader implementation of miRNA in βcell dysfunction.

Calcium ions have important roles in cellular processes including intracellular signaling, protein folding, enzyme activation and initiation of programmed cell death. Cells maintain low levels of calcium in their cytosol in order to regulate these processes. When activation of calcium-dependent processes is needed, cells can release calcium stored in the endoplasmic reticulum (ER) into the cytosol to initiate the processes. This can also initiate formation of plasma membrane channels that allow entry of additional calcium from the extracellular milieu. The change in calcium levels is referred to as calcium flux. A key protein involved in initiation of calcium flux is Stromal Interaction Molecule 1 (STIM1), which has recently been identified as a sensor of ER calcium levels. STIM1 is an ER transmembrane protein that is activated by a drop in ER calcium levels. Upon activation, STIM1 oligomerizes with a plasma membrane protein, ORA1, to form calcium-selective plasma membrane channels. Dysregulation of calcium flux has been reported in cancers, autoimmune diseases and other diseases. STIM1 is a promising target in drug discovery due to its key role early in calcium flux. Here we review the involvement and importance of STIM1 in diseases and we discuss STIM1 as a viable target for drug discovery using computational chemistry methods to rapidly identify new molecules to target STIM1. Herein, computational techniques were used to understand the mechanistic role of STIM1 and virtual screening is in process to discover potential inhibitors of STIM1 activity. Also mutational analysis on STIM1 was performed computationally to see the effect it had on the protein computationally. It has been found that tumor cells and tissues, compared to normal cells, have higher levels of copper and possibly other metal ions. This presents a potential vulnerability of tumor cells that can serve as a physiological difference between cancer cells and normal cells and allows design of compounds that selectively target tumor cells while sparing normal cells. Recently we have identified compounds that have potential to inhibit the proteasome in tumor cells and induce cell death by mobilizing endogenous tumor copper resulting in in cellulo activation of the compound. These compounds hence act as pro-drugs, becoming active drugs in tumor cells with high copper content but remaining essentially inactive in normal cells, thereby greatly reducing adverse effects in patients. Such use would be of significant benefit in early detection and treatment of cancers, in particular, aggressive cancers such as pancreatic cancer which is usually not detected until it has reached an advanced stage. Six compounds were identified following virtual screening of the NCI Diversity Set with our proteasome computer model followed by confirmation with a biochemical assay that showed significant inhibition of the proteasome by the compounds in the presence of copper ions. In a dose response assay, NSC 37408 (6, 7-dihydroxy-1-benzofuran-3-one), our best compound, exhibited an IC50 of 3µM in the presence of 100 nM copper. Chagas’ Disease, a parasitic disease caused by the parasite Trypanosma Cruzi, is endemic to Latin America. The disease manifests itself in a short acute phase and a long chronic phase. Current treatments are effective only in the acute phase and are not used in the chronic phase due to toxicity of the drugs. Hence a new drug discovery approach was chosen for this disease. Cruzain is the major etiologic enzyme involved in the disease and is only present in the parasite. It is also an enzyme expressed by the parasite in both phases. Herein, a novel peptoid library containing hydromethylketones was constructed and screened against a virtual structure of cruzain. The peptoids thus found through this drug discovery effort can be used as potential drug candidates against cruzain. Computational techniques will help achieve a high degree of specificity and aid in proposing assays for determining compounds with high activity Alzheimer disease is the most common form of dementia. Its pathogenesis incorporates many potential targets for treatment. Among the targets identified, Apolipoprotein E4 (apoE4) is especially interesting due to its catalytic role in the degradation and clearance of amyloid beta (Aβ), a risk factor for Alzheimer disease. ApoE exists in 3 isoforms which directly impact its functionality in the body. There are characteristic structural differences between them. In ApoE4 ionic interactions exist between Arg-61 and Glu-255 residues, unlike the other isoforms. Hence interruption of this interaction by inhibitors may change the structure of apoE4 to a more linear structure as observed in the other isoforms. Virtual screening of the NCI diversity set on an energy minimized protein virtual structure was performed to identify potential small molecule inhibitors and to gain further understanding of interactions that can be targeted to inhibit this protein. From the top ligands in the NCI diversity set, a peptide library was designed to target the protein. Previous research has indicated that liquid assisted grinding (LAG) is efficient and reliable for cocrystal formation when compared to solvent crystallization and dimethyl formamide is the best solvent for grinding. Herein, we report the comparison of four screening processes: Slurry, solvent crystallization, LAG and dry grinding. Thirty-eight crystal forms containing the Narom··· COOH, Narom···OH supramolecular heterosynthons were screened in the process, and it was observed that slurry methodology is as efficient and reliable in forming cocrystals as solution crystallization. Twenty-four new crystal forms were also isolated herein. LAG was found to be more efficient as compared to dry grinding and was successful in the formation of twenty-five crystal forms of the thirty-eight screened. Dimethyl formamide still remains the best solvent for LAG. All our slurry experiments were performed in water and it was found that water can be used reliably for this method for compounds within a wide range of solubility, thereby increasing the versatility and usability of this method for future screening procedures.

Increases in cardiac afterload caused by disease conditions results in remodeling of heart structure by hypertrophy and alterations in the molecular regulation of contractile performance. These adaptations can be regulated by various Ca2+-dependent signaling processes. STIM1 is an important regulator of Ca2+ signaling in different cell types by sensing endoplasmic reticular Ca2+ levels and coupling to plasma membrane Orai channels. The role of STIM1 in heart is not well understood, given the robust Ca2+ regulatory machinery present within cardiac myocytes. Previous reports indicate that STIM1 may play a role in regulation of cardiac hypertrophy. The goal of this work is to understand how STIM1 can affect contractile Ca2+ regulation in normal and diseased myocytes. We induced cardiac hypertrophy by slow progressive pressure overload in adult cats. Isolated adult feline ventricular myocytes (AFMs) exhibited increased STIM1 expression and activity, which correlated with altered Ca2+ handling. Use of BTP2 to block Orai channels resulted in a reduction of action potential (AP) duration and diastolic spark rate of hypertrophied myocytes, without affecting myocytes from sham-operated animals. Overexpressed STIM1 in cultured AFMs caused persistent Ca2+ influx that resulted in increased diastolic spark rates and prolonged APs, similar to myocytes from banded animals. STIM1 mediated Ca2+ influx could load the sarcoplasmic reticulum and activated CaMKII, which increased spark rates and lead to spontaneous APs. Importantly, STIM1 operated by associating with Orai channels because these effects could be blocked with either BTP2 or with a dominant negative Orai construct. Prolonged Ca2+ entry through this pathway eventually causes cell death. In conclusion, the work presented in this thesis establishes a role for STIM1-Orai in contractile Ca2+ regulation. / Biomedical Sciences

The extensive relationship between modulation of intracellular Ca2+ content and the control of cell proliferation (Boynton et al. 1974; Whitfield et al. 1979; Berridge and Irvine 1984), differentiation (Bridges et al. 1981; Holliday et al. 1991) and death (Orrenius et al. 2003) has led to much examination into the relationship between Ca2+ signaling pathways and the onset of various pathological conditions, including cancer, cardiac hypertrophy, immunodeficiency, neurodegeneration. Control of Ca2+ signals is achieved via an extensive combination of pumps, channels and exchangers which regulate the concentration of Ca2+ within not only the cytosol but also all intracellular compartments. Accordingly, a great deal of research has focused on the mechanisms which regulate these channels and pumps, and recently the primary mechanism for Ca2+ influx in non-excitable cells has been identified. This process, termed Store-operated calcium entry (SOCe), is a key evolutionarily conserved mechanism whereby decreases in endoplasmic reticulum Ca2+ content (sensed by the ER Ca2+ sensor, STIM1) leads to the influx of Ca2+ across the plasma membrane through the Orai family of Ca2+ channels. However, many questions remain about how this Ca2+ signaling pathway is regulated. In this thesis, I provide evidence regarding the transcriptional and molecular mechanisms regulating SOCe. Initial studies in my thesis work aimed to identify some of the key events leading to dysregulation of Ca2+ homeostasis in the kidney specific pediatric malignancy, Wilms' Tumor. I found that STIM1 expression levels and SOCe signals are significantly reduced in Primary Wilms' Tumor samples. Subsequent analysis of these phenomena led me to the finding that STIM1 expression is under the control of the transcription factors Wilms' Tumor Suppressor 1 (WT1) and Early Growth Response 1 (EGR1). Subsequent investigations were carried out with the purpose to assess how activation of the EGR1 transcription factor alters long term Ca2+ signals. Indeed, I found that receptor-mediated activation of EGR1 leads to induction of STIM1 expression and increases in SOCe. However, unexpectedly through these analyses, I propose a novel role for STIM1 that STIM1 interacts with the Plasma Membrane Ca2+ ATPase (PMCA) through its C-terminal proline-rich domain and reduces PMCA-mediated Ca2+ clearance, effectively creating local, augmented Ca2+ gradients. This coordinated control of Ca2+ entry and exit from the cell has wide-ranging implications for Ca2+ signaling in multiple cell types. / Biochemistry

La myopathie à agrégats tubulaire (MTA) est une maladie rare caractérisée par la présence, dans la biopsie musculaire, d’« agrégats tubulaires ». Nous avons retrouvé 15 cas de MTA parmi 13987 biopsies musculaires réalisées dans les 35 dernières années dans notre service. Parmi ces cas, se trouvait une famille de trois patients qui ne pouvaient être inclus dans aucun groupe clinique connu de myopathie à agrégats tubulaires, car ils étaient asymptomatiques avec une hyperCKémie isolée et un test de contracture positif.Nos travaux ont mis en évidence des mutations hétérozygotes du gène STIM1. Ces mutations localisées dans la partie du gène qui code le domaine intraluminal EF-hand, entrainent une activation constitutive de STIM1 avec exacerbation du mécanisme SOCE et en conséquence l’augmentation de l’influx de Ca2+, démontré dans des myoblastes transfectés. Nous avons également mis en évidence la mutation p.Arg304Trp du gène STIM1, comme cause du syndrome de Stormorken. Cela augmente le spectre phénotypique des mutations de ce gène.Nos résultats de l’analyse protéomique montrent que la protéostase dans la MTA est dérégulée, car le profil du protéome est différent chez les patients et dans les agrégats tubulaires microdisséqués par laser, en comparaison a des contrôles. Les protéines enrichies s’avèrent appartenir à des voies biologiques impliquées dans l’homéostase ionique, les systèmes de membranes de la triade et de l’exosome et dans le métabolisme mitochondrial.Nos travaux ouvrent des perspectives pour mieux comprendre la physiopathologie de la myopathie à agrégats tubulaires et ainsi pouvoir proposer des solutions thérapeutiques efficaces. / Myopathy with tubular aggregates (MTA) is a rare disease characterized by the presence of tubular aggregates in muscle biopsy. We found 15 cases of MAT in our registry including 13987 muscle biopsies performed over 35 years. Among them, there was a family of three patients that did not fit any of the previously described clinical groups of MTA, since they were asymptomatic with isolated hyperCKemia and positive contracture test. Our works revealed heterozygous mutations in the gene STIM1. These mutations localized in the gene portion that codes for the intraluminal EF-hand domain, leads to a constitutive activation of STIM1 with SOCE mecanism enhancement and consequent increase of the Ca2+ entry which was demonstrated using transfected myoblasts. We also revealed the mutation p.Arg304Trp in the coding sequencing of the CC1 domain of STIM1, as the cause of Stormorken syndrome. This fact increases the phenotypical spectra of the mutations for this gene.The results of our proteomic analysis show that the proteostasis in MTA is disturbed, because the proteome profile is different in total muscle of the patients and in their tubular aggregates when compared to controls. The enriched proteins belong to the biological pathways linked to ionic homeostasis, membrane systems of the triads and the exosome, and to the mitochondrial metabolism.Our works bring perspectives for the continuation of our studies, in order to better understand the physiopathology of the myopathy with tubular aggregates and propose efficacious therapeutic solutions.

Melanoma is the deadliest form of skin cancer. Prognosis for early stage melanoma patients is excellent, and surgery is often curative for these patients. However, once patients have presented with invasive disease, the average 5-year survival rate drops significantly from over 90% to between 10 and 15%. Several therapies have been developed to target a commonly mutated oncogene BRAF, or its downstream effectors. Unfortunately, while these treatments show robust initial response, most patients relapse within a year. Moreover, therapy-resistant tumors are often more invasive and metastatic. Therefore, it is important to investigate the molecular mechanisms underlying melanoma invasion and metastasis, and to prevent melanoma cell dissemination and metastatic progression. Invadopodia are proteolytic membrane protrusions used by metastatic cancer cells to degrade the extracellular matrix and to facilitate cancer cell invasion and metastasis. In my thesis research I have focused on protein fucosylation and store-operated calcium entry, two separate mechanisms involved in invadopodial regulation. Post translational modifications of proteins are essential for their structure and function. Many cell surface proteins require modifications such as glycosylation for protein-protein interactions, cell adhesion, and signal transduction. Fucosylation is a form of glycosylation that adds L-fucose on glycan structures of proteins. There is evidence indicating that fucosylation plays an important but cancer-type and branching dependent role in cancer progression. Emerging evidence indicates that the fucose salvage pathway and protein fucosylation are altered during melanoma progression and metastasis. Here, we report that the fucose salvage pathway inhibits invadopodia formation and extracellular matrix degradation by promoting α(1,2) fucosylation of cell surface proteins. The activation of the fucose salvage pathway decreases invadopodia numbers and inhibits the proteolytic activity of invadopodia in WM793 melanoma cells. Inhibiting fucokinase, one of the critical enzymes in the fucose salvage pathway, in melanoma cells abrogates L-fucose-mediated inhibition of invadopodia, suggesting dependence on the fucose salvage pathway. The inhibition of invadopodia formation by L-Fucose treatment or fucokinase overexpression could be rescued by treatment with α(1,2), but not α(1,3/4) fucosidase, implicating an α(1,2) fucose linkage-dependent inhibitory effect. The ectopic expression of FUT1, an α(1,2) fucosyltransferase, is sufficient to inhibit invadopodia formation and ECM degradation. Our findings indicate that the fucose salvage pathway can inhibit invadopodia formation, and consequently, invasiveness in melanoma via α(1,2) fucosylation. Re-activation of this pathway in melanoma could be useful for preventing melanoma invasion and metastasis. Calcium is a critical second messenger involved in a multitude of biological processes from cell proliferation to muscle contraction. In melanoma, previous studies have found that activation of the store operated calcium entry (SOCE) channel promotes tumor invasion and metastasis, in vitro and in xenograft models. The expression levels of STIM1, an essential component of the store operated calcium channels, has been found to increase with later stages of melanoma. In melanoma cell lines, the over expression of STIM1 enhances invadopodia number whereas STIM1 knockdown inhibits invadopodia formation. Similarly, gelatin degradation activity is enhanced with STIM1 overexpression and abrogated with STIM1 knockdown, implicating STIM1 as an important factor in the regulation of invadopodia formation and melanoma invasion. Though the studies published have shown a significant role of STIM1 in tumor progression, a robust transgenic animal model has not yet been established. Here, we developed a novel transgenic mouse model which, upon 4-hydroxytamoxifen (4OHT) treatment, induces the BRAFV600E mutation and PTEN, STIM1, and STIM2 deletions in melanocytes via an inducible Cre-lox system. Our investigation found that the loss of STIM1 exacerbates tumor growth and results in tumor formation significantly more quickly than STIM1 wild type mice. Whereas PCR analysis of 4OHT-treated skin showed deletion of STIM1 and PTEN, immunohistochemical staining of these genes in tumors did not convincingly demonstrate complete deletion. Therefore, it remains to be determined whether the effects we observed are due to STIM1 and STIM2 loss. These findings need to be corroborated in the future. Our studies focus on two important mechanisms required for melanoma progression and metastasis. We found that α(1,2) fucosylation is able to inhibit invadopodia formation, and melanoma cell invasion. The reestablishment of α(1,2) fucosylation in melanoma could potentially be exploited to inhibit melanoma metastasis. Additionally, early evidence points to STIM1 having a tumor suppressive role in melanoma oncogenesis and tumor growth based on the transgenic mouse model. Although the phenotype is unexpected, further investigation of this model will likely provide important insight for the complicate roles of SOCE in melanoma initiation and progression.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第15408号 / 工博第3287号 / 新制||工||1495(附属図書館) / 27886 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 森 泰生, 教授 濵地 格, 教授 跡見 晴幸 / 学位規則第4条第1項該当

Antigen presentation to T cells results in their activation through T Cell Receptor (TCR) stimulation, resulting in sustained elevation of cytosolic Ca2+ concentration critical for T cell activation. Sustained Ca2+ signals are important for the activation of Nuclear Factor of Activated T cells (NFAT), which is a key regulator of T cell activation through its transcriptional control of genes in multiple process including cytokine production, proliferation and differentiation(Rao, Luo, & Hogan, 1997). Recently it was shown that Stromal Interaction Molecule 1 (STIM1) inhibits plasma membrane Ca2+/ATPase 4 (PMCA4) function during T cell activation resulting in sustained elevation of Ca2+ signals(Ritchie, Samakai, & Soboloff, 2012). This interaction requires upregulation of both STIM1 and PMCA4. In this thesis, I hypothesize that changes in Ca2+ signals arising from transcriptional changes of STIM1 and PMCA are important for the efficient activation of T cells. In the first part of this thesis, I assess the transcriptional regulation of STIM1 and PMCA4. My in vitro studies show that expression of both proteins is regulated by the EGR family members, EGR1 and EGR4. Additionally, transcriptional regulation of PMCA inhibition by EGR1 and EGR4 is required for efficient activation of T cells. Interestingly, whereas significant roles for EGR1, EGR2 and EGR3 in T cell development and function have been established, a role for EGR4 has not, hitherto been elucidated. In the second half of this thesis, using qPCR, I reveal that EGR4 expression is stimulated by TCR engagement in primary double positive, CD4 and CD8 positive murine T cells. Further, EGR4-null mice exhibit shifts in early thymic development, although this does not affect the relative number of double or single positive T cells in the thymus. Interestingly, EGR4-null primary T cells exhibit normal Ca2+ entry, but fail to exhibit activation-induced inhibition of Ca2+ clearance. Although not all subsets of EGR1 and EGR4 null primary T cells exhibited decreased STIM1 expression, significant defects in proliferation, migration and/or cytokine production were observed upon stimulation in all populations, albeit to different extents. These findings reveal a two-faceted role in which EGRs regulate T cell development and function through both Ca2+-dependent and independent methods. I believe that these findings have important implications towards the general understanding of transcriptional control of Ca2+ signaling, as well as having a possible impact in the quest to advance therapies targeting immunological disorders. / Biochemistry