Abstract The mitochondrial oxidative phosphorylation system (OXPHOS) generates energy but also deleterious reactive oxygen species (ROS). Changes in the cytoskeleton, composed mainly of microfilaments, microtubules and intermediate filaments, have been observed in OXPHOS deficiency. The 3243A>G point mutation in mitochondrial DNA (mtDNA) leads to mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), which is the most common mitochondrial disease. Interestingly, mitochondrial aberrations have been demonstrated in patients with a mutation in NOTCH3, the genetic cause of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Randomization of vimentin intermediate filament direction and length together with slower population growth was observed in myoblasts with 3243A>G, with no difference in the amount of apoptotic cell death. Upon complex IV inhibition (with or without the microtubule-depolymerizing compound nocodazole) or a lack of mtDNA (ρ0) in osteosarcoma cells the vimentin network collapsed perinuclearly, forming thick bundles, whereas complex I inhibition led to thinner vimentin network bundles. Furthermore, the amount of vimentin was increased in ρ0 cells. Mitochondria accumulated around the nucleus upon complex IV inhibition and in ρ0 cells. Analysis of the total proteome revealed that specific OXPHOS deficiencies led to changes in the expression of cytoskeletal proteins and proteins involved in apoptosis, OXPHOS, glycolysis and oxidative stress response. Muscle histochemical and genetic analysis showed ragged red fibres and cytochrome c oxidase-negative fibres to be associated with 5650G>A in a patient with R133C in NOTCH3 and 5650G>A in MTTA. Immunolabelling of cells with R133C and 5650G>A revealed a sparse tubulin network with asters and less abundant mitochondria by comparison with control cell lines. Comparison of nucleotide diversity between CADASIL pedigrees and controls showed increased mtDNA sequence variation in the CADASIL patients. Also maternal relatives in two CADASIL pedigrees differed from each other in their mtDNA. These findings suggest that defects in OXPHOS lead to selective changes in the vimentin network, which may have a role in the pathophysiology of mitochondrial diseases. They also suggest a relationship between NOTCH3 and mtDNA, and establish the pathogenicity of 5650G>A. The overall results emphasize that a deficiency in the energy converting system together with oxidative stress can lead to cytoskeletal changes.

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京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22144号 / 医博第4535号 / 新制||医||1039(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 秀一, 教授 生田 宏一, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Melanoma remains an aggressive form of skin cancer with limited treatment options. Novel methods to treat primary tumors and prevent metastatic disease can lead to improved survival for those diagnosed with melanoma. Through this work, we have evaluated the antineoplastic effects of Rhodiola crenulata (R. crenulata) root extracts on B16-F10 melanoma both in vitro and in vivo. In this study, we observed that R. crenulata treatment resulted in an increased cell death as well as a reduced cell growth, proliferation and migration in vitro. Additionally, we observed that R. crenulata decreased the expression of integrin β1 and vimentin, and increased expression of E-cadherin upon in vitro treatment. Further, we observed in a topical R. crenulata based cream therapy, a more radial growth pattern of tumors as well as a reduced mitotic activity and increased tumor necrosis. Markedly, we observed that mice supplemented with R. crenulata orally in their drinking water also displayed reduced establishment of metastatic foci in a disseminated model of melanoma. Collectively, these findings reveal that R. crenulata exhibits striking anti-tumorigenic and anti-metastatic properties, and that this extract may increase survival and harbor potential novel adjuvant therapy for the treatment of melanoma.

Irrespective of plentiful efforts to enhance primary prevention and early detection, the number of melanoma cases in the United States has increased steadily over the past 30 years, thus greatly affecting public health and the economy. We have investigated the effects of five novel aPKC inhibitors; 2-acetyl-1,3-cyclopentanedione (ACPD), 3,4-Diaminonaphthalene-2,7-disulfonic acid (DNDA), [4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1T) along with its nucleoside analog 5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S) and 8-hydroxy-1,3,6-naphthalenetrisulfonic acid (ζ-Stat) on cell proliferation, apoptosis, migration and invasion of two malignant melanoma cell lines compared to normal melanocyte cell lines. Molecular docking data suggested that both ACPD and DNDA specifically bind to protein kinase C-zeta (PKC-ζ) and PKC-iota (PKC-ι) while both ICA-1 compounds specifically bind to PKC-ι, and ζ-Stat showed a high affinity towards PKC-ζ. Kinase activity assays were carried out to confirm these observations. Results suggest that PKC-ι is involved in melanoma malignancy than PKC-ζ. Both isoforms promote the activation of nuclear factor (NF)-κB and protein kinase B (AKT) thereby supporting survival and progression. In addition, we demonstrated that PKC-ι induced the metastasis of melanoma cells by activating Vimentin, and PKC-ι inhibition downregulated epithilial-mesencymal transition (EMT), while inducing apoptosis. Of note, PKC-ἱ specific inhibitors downregulated the expression of both PKC-ι and phosphorylated PKC-ι, suggesting that PKC-ι plays a role in regulating its own expression in melanoma. We also report the underlaying mechanisms of the transcriptional regulation of PKC-ι (PRKCI gene) expression in melanoma. c-Jun, interferon-stimulated gene factor 3 (ISGF3), paired box gene 3 (PAX3), early growth response protein 1 (EGR1) and forkhead box protein O1 (FOXO1), which bind on or near the promoter sequence of the PRKCI gene, were analyzed for their role in PKC-ι regulation in SK-MEL-2 and MeWo cell lines. We silenced selected transcription factors using siRNA, and the results revealed that the silencing of c-Jun and FOXO1 significantly altered the expression of PRKCI. The levels of both phosphorylated and total PKC-ι increased upon FOXO1 silencing and decreased upon c-Jun silencing, suggesting that c-Jun acts as an upregulator, while FOXO1 acts as a downregulator of PRKCI expression. We also used a multiplex ELISA to analyze multiple pathways other than NF-κB that were affected by treatment with PKC-ι inhibitor. The silencing of NF-κB p65 and PKC-ι by siRNA suggested that the regulation of PKC-ι expression was strongly associated with FOXO1. In addition, we observed a significant decrease in the mRNA levels of both interleukin (IL)-6 and IL-8, with a significant increase in the levels of IL-17E and intercellular adhesion molecule 1 (ICAM-1) upon the knockdown of expression of PKC-ι in both cell lines. This suggested that PKC-ι expression was affected by these cytokines in an autocrine manner. Overall, the findings of this study suggest that PKC-ι inhibition suppresses its own expression, diminishing oncogenic signaling, while upregulating anti-tumor signaling, thus rendering it an effective novel biomarker for use in the design of novel targeted therapeutics for melanoma.

<p>Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin protein. Recent research demonstrates that post-translational modifications of huntingtin could be an important determinant of mutant huntingtin’s toxicity in HD. In particular, phosphorylation at residues serine 13 and 16 within the first 17 amino acids of huntingtin (N17), have been shown to be critical modulators of mutant huntingtin’s toxicity and localization, and can be triggered by stress. This project aims to study how phosphorylation within N17 alters the interactome at this site and what physiological stress results in the nuclear localization of N17 and huntingtin. The initial search to identify potential interactors was conducted through an affinity chromatography assay using a wild type striatal cell line derived from knock in mouse model of HD. Fluorescent lifetime imaging microscopy (FLIM) to determine Fӧrester resonance energy transfer (FRET), co-immunoprecipitation and co-immunofluorescence assays were then used to validate real interactors of N17. Analysis from this project has validated two previously unidentified interactors of N17; SET, a small nucleo-oncoprotein, and vimentin, a type 3 intermediate filament. Both interactors have suggested two potentially novel roles for N17 within huntingtin, in cell cycle regulation and intermediate filament dynamics. Finally, smart screening techniques using stress-inducing compounds reveal that the sensitivity of N17 to stress and its subsequent nuclear localization can be attributed in part to activation of oxidative stress pathways. Data shown here can be expanded upon to elucidate how huntingtin function and response to cell stress are regulated and mediated via N17 and potentially how this mechanism is disrupted within HD.</p> / Master of Science (MSc)

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La protéine kinase dépendante de l’ADN (DNA-PK) est une sérine-thréonine kinase qui est un élément essentiel dans la voie de réparation de l’ADN endommagé par recombinaison non-homologue (non-homologous end-joining; NHEJ). DNA-PK est également impliquée dans de nombreux processus cellulaires autre que la réparation de l'ADN. Plusieurs travaux ont montré que les protéines impliquées dans la réparation des dommages de l'ADN tels que BRCA-1, MRN-11, PARP-1 et également de DNA-PK jouent un rôle important dans la métastase du cancer. Dans ce travail, nous nous sommes concentrées sur le rôle de DNA-PK dans les métastases du mélanome. Dans un premier temps, en utilisant les molécules Dbait 32Hc comme un moyen d'activer DNA-PK dans le noyau et le cytoplasme, nous avons identifié plusieurs nouvelles cibles cytoplasmiques de DNA-PK, dont la vimentine. Nous avons montré que DNA-PK phosphoryle la vimentine sur Ser459 et que cette forme phosphorylée est la plupart du temps située au niveau des protrusion cellulaires des cellules migratrices. Nous avons ensuite démontré que la vimentine-Ser459-P induite par le traitement de Dbait32Hc participe à l'inhibition de l'adhésion et la migration cellulaire. Ainsi, cette approche a conduit à l'identification de nouvelles cibles cytoplasmiques de DNA-PK et a révélé un lien entre la signalisation des dommages de l'ADN et le cytosquelette. Ensuite, nous avons montré que DNA-PK joue un rôle important dans la migration et invasion cellules en régulant la sécrétion des facteurs associés à la métastase. Nous avons montré que l'absence ou l’inhibition de DNA-PK conduit à une régulation négative des facteurs pro-métastatique sécrétés et à la régulation positive de facteurs anti-métastatiques sécrétés tels que les inhibiteurs des métalloprotéinases matricielles. Nous avons confirmé le rôle de DNA-PK in vivo dans l'implantation de la tumeur primaire et dans la formation des métastases. Ainsi, nos études ont évalué le rôle de DNA-PK sur le contrôle du microenvironnement de la tumeur par le contrôle de la sécrétion de facteurs importants pour la métastase. En résumé, nos résultats mettent en évidence l'importance de la DNA-PK comme cible de traitement anti-métastatique. / The DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase, which is a critical component of the DNA-damage repair pathways through non-homologous end-joining (NHEJ). Besides DNA repair, it is also involved in numerous cellular pathways. Emerging results show that proteins involved in DNA damage repair such as BRCA-1, MRN-11, PARP-1 and also DNA-PK could play a role in cancer metastasis. In the current study, we demonstrated the role of DNA-PK in melanoma metastasis. Firstly using Dbait 32Hc molecules as a tool for specifically activating DNA-PK in a nucleus and cytoplasm, we identified several new cytoplasmic targets of DNA-PK including vimentin. We established that DNA-PK phosphorylates vimentin on Ser459 and that this phosphorylation was mostly located at cell protrusions of melanoma migratory cells. Following this, we confirmed that vimentin-Ser459-P induced by Dbait 32Hc treatment participates to the inhibition of cell adhesion and migration. Thus, this approach led to the identification of downstream cytoplasmic targets of DNA-PK and revealed a connection between DNA damage signaling and the cytoskeleton. Secondly, we show that DNA-PK plays an important role in cell migration and melanoma cell invasion through the regulation of secretion of metastasis-associated factors. Absence or inhibition of DNA-PK leads to down-regulation of pro-metastatic secreted factors and up-regulation of anti-metastatic secreted factors such as inhibitors of matrix metalloproteinases. We confirmed in vivo, that DNA-PK is required for efficient primary tumor implantation and metastases formation. Thus, our studies demonstrate for the first time that DNA-PK acts on tumor microenvironment by controlling secretion of important factors for cell migration and invasion. In summary, our findings highlight the importance of DNA-PK as a target of anti-metastatic treatment.

Charles University, Faculty of Pharmacy in Hradec Králové, Department of Biological and Medical Sciences The University of Alcalá, Faculty of Medicine, Department of biomedicine and biotechnology Student: Zuzana Chvojanová Supervisor: PharmDr. Miroslav Kovařík, Ph.D. Consultant: María Isabel Arenas Jimenéz Title of the diploma thesis: Study of the variations in the expression of different adhesion and cytoskeletal proteins of podocytes (E-Cadherin, Podocin, Vimentin) due to Bisphenol A Bisphenol A (BPA) is one of the most widespread compounds in the world, producing over 6 billion metric tons per year. It is widely used as part of polycarbonate plastics and epoxy resins, from which reusable plastic bottles, food boxes and some medical equipment are made. It is also used to coat the inner layer of the cans. Previous studies have shown that BPA contributes to many chronic diseases in the human body, such as kidney disease - diabetic nephropathy. Podocytes - terminally differentiated cells of the Bowman's capsule in glomerulus - are an integral part of the filtration barrier, where they play an important role in preventing the plasmatic proteins from penetrating to the urine. Therefore, in this study, we looked at the effect of BPA on these cells and their particular proteins, using both in vivo and...