Thesis (Ph. D.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains viii, 173 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Iron is an essential trace element for almost all living organisms. Iron overload in cells and tissues, however, leads to their disruption. Most oftenly damaged are parenchymatic organs such as the liver, pancreas and heart. The aim of this thesis was to create cellular in vitro models for the investigation of effects of excess iron on hepatocytes and pancreatic beta cells and on these models to investigate cellular processes which lead to cellular damage during iron overload. We focused on examining the presence of oxidative and endoplasmic reticulum stress and the activation of apoptotic cell death. For our experiments, we used HEP-G2 cell line which represents human hepatocytes and NES2Y cell line which represents human pancreatic beta cells. To study the mechanisms of cellular damage during iron overload, we used two approaches by which we observed both acute and long-term effects of high levels of iron on damage of the tested cell lines. When studying the acute effect of excess iron on the cells, we applied high doses of iron (using 15 mM ferric citrate in medium) that led to the activation of cell death in hours. Long-term effects of iron overload were tested on cells regularly cultivated in the presence of 50 μM and 100 μM ferric citrate over a period of several months. Iron concentrations...

L'athérosclérose est une pathologie artérielle complexe à évolution lente définie par un remodelage de la paroi vasculaire, associée à des réactions inflammatoires, à des processus de prolifération, de mort cellulaire et à une accumulation de lipides oxydés tels que les oxystérols (dérivés oxydés du cholestérol) dont le rôle est fortement suspecté dans le développement de l'athérome.<br /><br />Le travail réalisé a été effectué sur des cellules monocytaires humaines U937 et THP-1, aortiques de rat A7R5 et carcinomateuses humaines MCF-7 (déficientes en caspase-3). Différents oxystérols, présents en quantité importante dans les lésions athéromateuses, ont été utilisés (7-cétocholestérol (7KC), 7Β-hydroxycholestérol, 25-hydroxycholestérol, cholestérol-5Α, 6Α-epoxide, cholestérol-5Β, 6Β-epoxide) ainsi que du cholestérol.<br /><br />La première partie du travail a montré que la mort cellulaire induite par le 7KC est un phénomène complexe présentant des caractéristiques apoptotiques accompagnées d'une synthèse de structures multilamellaires cytoplasmiques appelées corps myéliniques visualisés par microscopie électronique à transmission. La synthèse rapide de ces structures précède les effets cytotoxiques : chute du potentiel membranaire mitochondrial, augmentation de la perméabilité à l'iodure de propidium et modifications de la morphologie nucléaire (condensation, fragmentation, gonflement des noyaux). L'isolement de ces structures par ultracentrifugation après coloration à la monodansylcadavérine (fluorochrome lysosomotropique acide) ou au Nile Red (fluorescence jaune en présence de lipides neutres et rouge en présence de lipides polaires) a permis de montrer qu'elles sont riches en lipides polaires (sphingomyéline, phosphatidylcholine) et en cholestérol et qu'elles accumulent le 7KC. Au sein de ces corps myéliniques, une co-localisation du 7KC avec les lipides polaires a été démontrée par la technique de FRET réalisée par microscopie confocale mono- et bi-photonique. Les caractéristiques morphologiques et biochimiques des figures myéliniques ont permis d'établir que le 7-cétocholestérol est un puissant inducteur de phospholipidose.<br /><br />Compte tenu des modifications lipidiques spatiotemporelles, quantitatives et qualitatives, induites par le 7KC et révélé par le Nile Red, la seconde partie du travail a précisé les relations entre la mort cellulaire, la synthèse de corps myéliniques, l'accumulation de lipides polaires et l'activité caspase. Avec les différents oxystérols étudiés, les corps myéliniques ne sont observés qu'avec des composés cytotoxiques (7KC, 7Β-hydroxycholestérol, cholestérol-5Β, 6Β-epoxide) et leur synthèse est indépendante d'activité caspase. En revanche, l'accumulation de lipides polaires induite par le 7KC est inhibée en présence de z-VAD-fmk (inhibiteurs de caspases large spectre) et de z-VDVAD-fmk (inhibiteur de caspase-2). Certaines caspases et en particulier la caspase-2 pourraient contribuer à l'accumulation de lipides polaires.<br /><br />La troisième partie du travail a conduit à étudier les effets de la Vitamine-E (VitE) sur la mort cellulaire induite par le 7KC en raison de ses propriétés anti-apoptotiques et anti-oxydantes. La VitE protège de la mort cellulaire induite par le 7KC. Les effets protecteurs pourraient en partie être dus à la capacité de la VitE à maintenir fonctionnelle la voie PI3-K/c-Akt en s'opposant aux déphosphorylations de PDK-1 et de c-Akt, et en préservant l'activité PI3-K. Par ailleurs, la VitE s'oppose aux modifications lipidiques au niveau de la membrane cytoplasmique et à l'accumulation de lipides polaires. La VitE réduit aussi la dégradation de la pro-forme de la caspase-2L et augmente les taux d'ARNm correspondants.<br /><br />Ces travaux montrent des relations entre la mort cellulaire induite par des oxystérols et le métabolisme des lipides. Ils révèlent aussi que la VitE protège de la mort induite par le 7KC en agissant au niveau de la voie PI3-K/c-Akt. Par ailleurs, la VitE s'oppose aux modifications lipidiques membranaires et cytoplasmiques associées à la mort cellulaire induite par le 7KC.

Cancer is the second leading cause of death among men and women after heart disease. Though our knowledge associated with the complexities of the cancer network has significantly improved over the past several decades, we have only recently started to get a more complete molecular understanding of the disease. To better comprehend signaling pathways that prevent disease development, we focused our efforts on investigating endogenous tumor suppression networks in controlling effectors of cancer cell survival and proliferation. Survivin is one such effector molecule that controls both cell proliferation and survival. In order to identify how this protein is overexpressed in cancer cells as opposed to normal cells, we looked at signaling molecules that negatively regulate this inhibitor of apoptosis protein. PTEN and caspase 2 are two of the identified proteins that utilize their enzymatic activity to suppress tumor growth by inhibiting downstream cell survival effectors, namely survivin. PTEN uses its phosphatase activity to suppress the PI3K/AKT pathway and maintain cellular homeostasis. In the absence of AKT activity, FOXO transcription factors are able to target downstream gene expression and regulate cell proliferation and survival. Here we have identified survivin as a novel gene target of FOXO, which binds to a specific promoter region of survivin and suppresses its transcription. Alternatively, caspase 2 uses its catalytic activity to suppress survivin gene expression by targeting the NFκB pathway. Caspase 2 acts by cleaving a novel substrate known as RIP1 that prevents NFκB from entering the nucleus, thus inhibiting target gene transcription. Interestingly, survivin is known to be a direct gene target of NFκB that controls cancer cell survival. In our investigation, we found that survivin is downregulated upon caspase 2 activation via the NFκB pathway, resulting in decreased cell cycle kinetics, increased apoptotic threshold and suppressed tumor growth in mice. These studies conclude that survivin is a common effector molecule that is regulated by tumor suppressors to maintain cellular homeostasis. However, upon deactivation of the tumor suppressor pathway, survivin is deregulated and contributes significantly to disease progression. These observations may lead to potential therapeutic implications and novel targeting strategies that will help eradicate harmful cancer cells and spare surrounding healthy cells; often the most persistent problem of most conventional chemotherapy.

Centrosomes are microtubule-based organelles composed of two centrioles and peri-centriolar material, involved in the formation and organization of the mitotic spindle, serving as microtubule-organizing center and involved in ciliogenesis. Supernumerary centrosomes are detrimental for cell physiology and activate the PIDDosome, a multi-protein complex that serves as a platform for the activation of Caspase-2, composed of: PIDD1, RAIDD and Caspase-2 itself. Caspase-2’s preferred cleavage site based on peptide screening is VDVAD, however Caspase-2, when activated via the PIDDosome, cleaves its bona fide substrate MDM2 (negative p53 regulator) in the FDVPD sequence. Here, I present evidence for VDVADase activity in apoptotic cells lacking Caspase-2, which suggests that this cleavage site is not Caspase-2 specific when the Caspase-2 activation occurs via the PIDDosome. In order to investigate if the mode of activation of Caspase-2 determines its substrate specificities I performed a Caspase-2 rescue experiment and introduced several mutations affecting the Caspase-2 autoproteolytic-processing. Furthermore, I present evidence that exogenous Caspase-2 is able to form the PIDDosome and cleaves MDM2 but when key autoproteolytic sites are mutated no MDM2 cleavage is detectable. Supernumerary centrosomes also accumulate upon overexpression of PLK4 (a kinase regulator of the centriole duplication). Immunofluorescence images of cells overexpressing PLK4 were taken following the centrioles quantification over time. Consequently, a large amount of image data was accumulated, which necessitated the development of a semi-automated pipeline for centrioles counting. This pipeline was generated using the image processing and analysis tool ImageJ and the deep learning segmentation tool MitoS together with the pretrained MitoSegNet model, which was finetuned to count centrioles stained against different centrosomal epitopes, namely Centrin 1, γ-Tubulin and ANKRD26. This semi-automated method of centrioles quantification is easy to use, reproducible and faster than manual quantification. Using this pipeline to quantify centrioles in p53, SCLT1 or ANKRD26 lacking cells we demonstrate accumulation of supernumerary centrosomes in these cells similar to parental cells. / I centrosomi sono organelli cellulari a base di microtubuli, composti da due centrioli e dal materiale pericentriolare che li circonda. I centrosomi sono coinvolti nell'organizzazione dei microtubuli, nella formazione del fuso mitotico e nella ciliogenesi. I centrosomi soprannumerari sono dannosi per la fisiologia cellulare e attivano il PIDDosoma, un complesso multiproteico, composto da PIDD1, RAIDD e Caspasi-2, che funge da piattaforma per l'attivazione della caspasi stessa. Il sito preferenziale di proteolisi di Caspasi-2 è stato individuato tramite screening peptidico nella sequenza VDVAD. Nonostante ciò, quando attivata tramite il PIDDosoma, Caspasi-2 scinde il suo substrato di elezione MDM2 (regolatore negativo di p53) a livello della sequenza FDVPD. In questa tesi presento evidenze di attività VDVAD-asica in cellule apoptotiche prive di Caspasi-2, suggerendo che questo sito di taglio non sia specifico di Caspasi-2 quando la sua attivazione avviene tramite il PIDDosoma. Al fine di indagare se la modalità di attivazione della proteasi determina le sue specificità di substrato, ho eseguito esperimenti di complementazione di Caspasi-2 facendo uso di diversi mutanti che influenzano il suo processamento autoproteolitico. Inoltre, presento prove che Caspasi-2 esogena è in grado di assemblare il PIDDosoma e proteolizzare MDM2 ma quando i suoi siti chiave di autoproteolisi sono mutati non è rilevabile il taglio di MDM2. I centrosomi soprannumerari si accumulano anche in caso di sovraespressione di PLK4 (chinasi regolatrice della duplicazione dei centrioli). Immagini di immunofluorescenza di cellule che sovraesprimono PLK4 sono state acquisite seguendo la cinetica di accumulo dei centrioli nel tempo. Di conseguenza, l’ingente mole di dati generati ha reso necessario lo sviluppo di una procedura semiautomatica per la conta dei centrioli. Questa pipeline è stata generata utilizzando il programma di elaborazione e analisi di immagini ImageJ e il programma di segmentazione basato su deep learning MitoS, insieme al modello MitoSegNet, che è stato affinato per la conta dei centrioli evidenziati tramite immunofluorescenza diretta contro diversi epitopi centrosomiali, ossia: Centrin 1, γ-Tubulina e ANKRD26. Questo metodo semiautomatico di quantificazione dei centrioli è facile da usare, riproducibile e più veloce della quantificazione manuale. Utilizzando questa procedura per quantificare i centrioli nelle cellule prive di p53, SCLT1 o ANKRD26, dimostriamo che l'accumulo di centrosomi soprannumerari in queste cellule è simile a quello riscontrato nelle cellule parentali.