The termina1 enzyme in the linoleoyl-CoA desaturase enzyme complex, delta-6-desaturase was implied in the control of cell proliferation in cancer cells. One of the aims of this study was to isolate the terminal enzyme. It was decided that in order to isolate this enzyme it was first necessary to isolate the entire complex and then to enzymatically solubilise the first two components of the complex i e cytochrome b5 reductase and cytochrome b5 from the complex resulting in a pure delta-6-desaturase . The first two components were isolated and purified using simplified and easily reproducible methodologies which could be utilised in the final purification of delta-6- desaturase. The entire enzyme complex, linoleoyl-CoA desaturase was also isolated in a pure form and this pure complex was used to attempt to isolate delta-6-desaturase. The terminal enzyme was isolated with some cytochrome b5 still bound to it. The methods used had proven to be successful and with some modifications should yield a pure enzyme. Zinc and GLA were known to play a role in the inhibition of cancer cell proliferation and zinc was hypothesised to inhibit cell growth by stimulating the activity of the linoleoyl-CoA desaturase enzyme complex which is involved in the regulation of cell proliferation. GLA is the product of the reaction that this enzyme complex catalyses and GLA has been shown to inhibit cancer ce ll growth. The effect of GLA on cell growth and linoleoyl-CoA desaturase activity was thus investigated. Results showed that both zinc and GLA inhibited cell growth and that the combined addition of zinc and GLA generally resulted in the inhibition of cell growth and the activation of linoleoyl-CoA desaturase activity in the BL-6 cells while having a less pronounced effect on the LLCMK cells. The results of this study support the hypothesis that zinc may be a cofactor of linoleoyl-CoA desaturase.

The transcription factor Six1 of the sine oculis homeobox family has been tied to skeletal muscle formation. Work completed thus far has allowed our research team to identify the precise mechanism by which Six1 regulates the expression of MyoD, a key myogenic gene, in muscle stem cells. Furthermore, loss-of-function of this protein, mediated by RNA interference, has implicated Six1 as essential towards normal myogenic differentiation. However, beyond Six1 and its involvement towards myogenesis, our data also suggests the transcription factor as a potential regulator of the cell cycle. Data from our lab shows that loss of Six1 expression significantly impairs primary myoblast proliferation and appears to impair satellite cell activation in response to muscle injury in vivo. Furthermore, loss of Six1 decreases the expression of key cell cycle genes. Combining functional genomics approaches such as ChIP-Seq and Gene Expression Profiling together with Gene Ontology Term Enrichment shows a significant representation for biological processes regarding the cell cycle and its regulation; these biological clusters contain a large subset of genes that are bound and modulated by Six1. In particular, Ccnd1 was found to display a similar expression pattern as Six1 in growing myoblasts and its expression was found to be directly controlled by Six1. Furthermore, Ccnd1 over-expression was sufficient to rescue the Six1-knockdown associated cell cycle phenotype. Together, these data suggest that in response to injury Six1 enhances the expression of the cell cycle gene Ccnd1 thus modulating myoblast proliferation for muscle regeneration.

Many animals, including zebrafish (Danio rerio), form social hierarchies as a result of competition for limited resources. Socially subordinate fish experience chronic activation of the hypothalamic-pituitary-interrenal (HPI) axis, leading to prolonged elevation of plasma cortisol, the glucocorticoid end-product of HPI axis activation. Elevated cortisol levels can reduce cellular proliferation and neurogenesis in the brain. Thus, the present study tested the hypothesis that social stress suppresses cellular proliferation in the brain of subordinate zebrafish via a cortisol-mediated mechanism. Cellular proliferation was assessed using the incorporation of 5-bromo-2'-deoxyuridine (BrdU), a thymidine analogue, as a marker. After 48 and 96 h of social interaction, significantly lower numbers of BrdU-positive cells were present in the forebrain of subordinate male zebrafish compared to dominant or control fish, suggesting a suppression of cellular proliferation in fish experiencing chronic social stress. Treatment of interacting male zebrafish with metyrapone, a cortisol synthesis inhibitor, attenuated the suppression of cellular proliferation in subordinate fish. Subordinate female zebrafish did not experience elevation of plasma cortisol or suppression of cellular proliferation in the forebrain. Collectively, these data provide evidence that cortisol plays a role in regulating cellular proliferation in the forebrain of male zebrafish during social interactions.

Sox9 est un facteur de transcription exprimé au cours du développement de l'intestin et son expression est maintenue à l'âge adulte dans trois populations cellulaires : les cellules souches, les cellules de Paneth, et les cellules tuft. L'inactivation de Sox9 dans l'épithélium intestinal embryonnaire entraîne, chez l'adulte, une hyperplasie des cryptes ainsi que l'absence de cellules de Paneth. Ce projet de thèse vise à déterminer le rôle de Sox9 dans les cellules de Paneth (dont la fonction est altérée chez les patients atteints de la maladie de Crohn), à identifier les mécanismes par lesquels Sox9 régule la prolifération et à proposer des cibles de Sox9 dans les cellules tuft. À l'aide de modèles murins d'inactivation de Sox9 au niveau de l'épithélium intestinal adulte, nous avons montré que la perte de ce facteur conduit à une augmentation de la prolifération dans les cryptes, confirmant ainsi que Sox9 régule négativement ce processus. Nos résultats indiquent que Sox9 est essentiel au maintien de l'identité des cellules de Paneth et nous proposons qu'il assure cette fonction en réprimant des gènes requis pour la différenciation des cellules de Goblet : Muc2 et Klf4. La perte de Sox9 dans les cellules de Paneth s'accompagne d'une réduction importante des molécules antimicrobiennes, ce qui entraîne une dysbiose intestinale. Dans un environnement spécifique (en présence du « mouse norovirus »), les souris déficientes en Sox9 présentent une perméabilité intestinale augmentée et une susceptibilité à l'inflammation accrue. Les dysfonctionnements des réponses antimicrobiennes et immunitaires dans notre modèle sont comparables à ceux observés chez les patients atteints de la maladie de Crohn, suggérant une implication potentielle de Sox9 dans cette pathologie. De plus, ces altérations pourraient expliquer l'augmentation de l'apparition des tumeurs observée chez les souris dont l'épithélium intestinal est déficient en Sox9, dans le contexte d'une mutation du gène suppresseur de tumeur Apc. Enfin, nous avons identifié des gènes potentiellement régulés par Sox9 qui pourraient expliquer son rôle dans le contrôle de la prolifération. Ces découvertes seront importantes pour mieux comprendre le processus du renouvellement de l'épithélium intestinal et identifier précisément le rôle de Sox9 dans le maintien de l'homéostasie et au cours du processus de la tumorigenèse intestinale. / Sox9 is a transcription factor expressed during the intestinal development and its expression is maintained throughout adult age in at least three populations of cells: stem cells, Paneth cells and tuft cells. Sox9 inactivation in the embryonic intestinal epithelium leads to crypts hyperplasia and to the loss of the Paneth cell lineage. The aim of this project is to determine Sox9 function in the adult intestinal epithelium, especially its role in Paneth cells (which function is altered in patients affected by inflammatory diseases such as Crohn disease), to identify how Sox9 controls proliferation and to propose molecular targets of Sox9 in tuft cells. Using mice models to inactivate Sox9 in adult intestinal epithelium, we could show that Sox9 is required to limit proliferation in the crypts, thus validating the hypothesis that Sox9 regulates negatively proliferation. Our results indicate that Sox9 is essential to maintain Paneth cells identity and we proposed that it ensures this function by repressing genes specific for Goblet cells differentiation: Muc2 and Klf4. Loss of Sox9 in Paneth cells is associated with a reduction of antimicrobial molecules which causes intestinal dysbiosis. In a specific environment (in presence of the « mouse norovirus »), Sox9-deficient mice have a defective intestinal permeability and are more susceptible to inflammation. The dysfunctions of the mucosal defences and of immunity responses in our model resemble those observed in Crohn patients, thus suggesting a potential implication of Sox9 in this pathology. In addition, these alterations could be responsible for the increased susceptibility of our deficient model to develop tumors in the context of a mutation of the tumor suppressor gene Apc. We started to unravel potential molecular targets of Sox9 that are involved in the control of proliferation, that will be important to better understand Sox9 function in the intestinal epithelium self-renewal and to identify precisely Sox9 function to maintain homeostasis and during intestinal tumorigenesis.

TRIM7 is a member of the TRIM (tripartite motif-containing) protein superfamily. This family has been implicated in many disorders such as genetic diseases, neurological diseases, and cancers. Little is known about the function of TRIM7 except that it interacts with glycogenin and may regulate glycogen biosynthesis. Recently, a yeast two-hybrid protein-protein interaction screen revealed the binding of TRIM7 to Sin1, a protein found in a complex with the mammalian target of rapamycin (mTOR) protein kinase. mTOR can form two complexes, mTORC1 and mTORC2, which are important for cell growth, differentiation, and survival. Sin1 is a core component of mTORC2 and is critical for mTORC2 stability and activity. It was confirmed by co-immunoprecipitation that TRIM7 associates with Sin1 and mTOR in cultured mammalian cells. Furthermore, it was demonstrated that TRIM7 is a phosphoprotein, although it was not directly targeted by mTOR in vitro. Similar to some other TRIM family proteins, it was demonstrated that TRIM7 has a ubiquitin E3 ligase function allowing it to autoubiquitinate both in vitro and in cells. The autoubiquitination of TRIM7 was dependent on its RING domain. Further characterization of TRIM7 indicated that it can both homo-oligomerise as well as hetero-oligomerise with other members of its sub-class of TRIM proteins and that it co-localises with them into discrete cytoplasmic loci. To determine the cellular function of TRIM7, a stable cell line expressing an shRNA directed against TRIM7 was generated. Successful knock down of TRIM7 was achieved and this led to an increase in the protein levels of components of the mTORC2 complex, including Sin1. This coincided with an increase in cell proliferation. In conclusion, this research identifies a novel role for TRIM7 as a ubiquitin ligase involved in regulating cell proliferation and provides a potential link between TRIM7 and the mTOR pathway, a major transducer of proliferative and cell survival signals.

Mastocitoma é uma das principais neoplasias cutâneas em cães, caracterizada por uma multiplicação anormal de mastócitos, com comportamento biológico muito variável. Os principais fatores prognósticos incluem grau histopatológico, marcadores de proliferação, como índice mitótico, Ki67 e AgNOR, além de estadiamento clínico. Diversos estudos concentram-se na avaliação da relação da apoptose com a oncogênese e seu papel no prognóstico. Em condições fisiológicas, a apoptose ocorre na maturação e senescência celular, mantendo a homeostasia dos diferentes tecidos, removendo do organismo células que tenham sofrido alguma mutação. A genética da apoptose pode ser interrompida frente à ocorrência de mutações, levando à perda do controle na proliferação celular, o que resulta no desenvolvimento de uma neoplasia. O presente estudo avaliou a ocorrência de apoptose por meio de ensaio TUNEL em mastocitomas cutâneos caninos, com o objetivo de testar sua relação com as graduações histopatológicas e o valor prognóstico quanto à sobrevida pós-cirúrgica, assim como compará-lo à expressão imuno-histoquímica de caspase 3 e Ki67. Quarenta e quatro mastocitomas cutâneos caninos, provenientes de 36 cães, foram submetidos à avaliação histopatológica para graduação quanto à diferenciação tumoral, à análise imuno-histoquímica para avaliação das expressões de Ki67 e caspase 3. A marcação positiva para TUNEL não mostrou relação com grau histopatológico, nem foi um bom indicador para sobrevida ou mortalidade em função da doença. Apesar disso, houve correlação positiva entre os índices apoptóticos. / Mast cell tumor is a very common neoplasm in dogs and characterized by an abnormal proliferation of mast cells, with variable biological behavior. The main prognostic factors include histological grade, proliferation markers, such as mitotic index, Ki67 and AgNOR, and clinical staging. Several studies focus in the relation of apoptosis and oncogenesis and its role in prognostication. In physiological conditions, apoptosis occurs due to aging and cell senescence, maintaining the homeostasis of different tissues by removing mutated cells. The genetics of apoptosis can be interrupted by mutations, leading to loss of control in cellular proliferation, and resulting in cancer development. This study evaluated the occurrence of apoptosis by TUNEL assay in canine cutaneous mast cell tumors, compared it with histopathological grading and the immunohistochemical expressions of caspase-3 and Ki67, as well as tested its prognostic value for post-surgical survival. Forty-four canine cutaneous mast cell tumors, from 36 dogs were submitted to histopathologic and immunohistochemical analyses. Positive staining for TUNEL showed no relation with histological grade, and was not considered a good indicator for survival or mortality. Nevertheless, a positive correlation between the apoptotic indexes was found.

Orientador: Hélio Amante Miot / Coorientador: Mariângela Esther Alencar Marques / Resumo: O carcinoma basocelular (CBC) é a neoplasia maligna mais comum entre os homens e sua incidência está aumentando em nosso meio. Pode se manifestar como lesão nodular, superficial, esclerodermiforme, micronodular e fibroepitelioma de Pinkus. O CBC é um carcinoma de baixa mortalidade, porém com elevada morbidade devido ao potencial destrutivo local e às elevadas taxas de reincidência. As diferenças evolutivas dos diversos tipos de carcinomas basocelulares não são bem definidas. A análise de fatores de proliferação e apoptose pode subsidiar o conhecimento sobre a fisiopatologia e as diferenças evolutivas dessas lesões. A literatura revela a escassez de investigações sobre a expressão diferencial de fatores de proliferação (Ki-67) e relacionados à apoptose (p53, survivina e NF-kB-p105) dos diferentes subtipos de carcinomas basocelulares e ainda tumores recidivados. O objetivo desse estudo é avaliar a expressão de marcadores de proliferação celular e apoptose em carcinomas basocelulares dos tipos nodular, superficial, esclerodermiforme e tumores recidivados. Foram selecionados subtipos histológicos únicos, como forma de tentar compreender o comportamento dos CBCs individualmente. As lâminas desses espécimes foram submetidas ao protocolo de marcação imuno-histoquímica. Foram estudadas 100 amostras, sendo 50 unidades de epiderme normal e as demais distribuídas entre cada subtipo tumoral. O padrão de marcação de cada marcador sobre os diferentes tipos de tecidos foi avaliado a partir ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Basal cell carcinoma (BCC) is the most common malignant neoplasm among men and its incidence is increasing in our country. It can manifest as nodular, superficial, sclerodermiform, micronodular and Pinkus fibroepithelioma lesions. BCC is a lowmortality carcinoma, but with high morbidity due to local destructive potential and high rates of recurrence. The evolutionary differences of the various types of basal cell carcinomas are not well defined. The analysis of proliferation and apoptosis factors may support knowledge about the pathophysiology and evolutionary differences of these lesions. The literature reveals the scarcity of investigations on the differential expression of proliferation factors (Ki-67) and related apoptosis (p53, survivin and NF-kB-p105) of the different subtypes of basal cell carcinomas and recurrent tumors. The aim of this study is to evaluate the expression of markers of cell proliferation and apoptosis in basal cell carcinomas of the nodular, superficial, sclerodermiform and recurrent tumors. Unique histological subtypes were selected as a way to attempt to understand the behavior of individual BCCs. The slides of these specimens were submitted to the immunohistochemical labeling protocol. A total of 100 samples were studied, being 50 normal epidermal units and the others distributed between each tumor subtype. The marking pattern of each marker on the different tissue types was evaluated from generalized linear models (GLMs) followed by Sidak post-hoc test, when necessary. Statiscally significant differences (p<0,01) in the immunostaining of different tissue types were found for the Ki-67, p53 and survivin markers, but not for the p105 marker (p=0,21). The Ki-67 marker was more expressed in sclerodermiform than in epidermal and nodular cells. Immunoblotting of p53 was less expressed in the epidermis than in superficial subtypes and relapses, and also less expressed in... (Complete abstract click electronic access below) / Doutor

Philosophiae Doctor - PhD / The blood-brain barrier (BBB) is a critical interface between the blood circulation and brain tissue which performs critical selection of circulating molecules that gain access to the brain tissue. Its unique ability to adjust to changes in the constituents of the blood circulation confer in the BBB a dynamic nature enabling changes in its properties to suit the homeostatic needs of the brain. Dysfunction of the BBB has been established to be pivotal to the initiation and/or maintenance of an array of neurological disorders, most of which involve the production of excess reactive oxygen species (ROS) and oxidative stress in their pathophysiology. Thus, clinical trials of exogenous antioxidant agents have been proposed and initiated, with most results being inconclusive. Extensive studies of the impact, capacity and plasticity of endogenous antioxidants in the cells that constitute the blood-brain barrier, especially the brain endothelial cells, therefore, became necessary for the rational choice, timing, and the mode of application of antioxidants in the management of oxidative stress-mediated neurological diseases.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13330号 / 論医博第2198号 / 新制||医||1044(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 長船 健二, 教授 羽賀 博典, 教授 山下 潤 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Salicylic acid and its concern have long been used for the management of pain, fever, and inflammation. Of this group, acetyl-salicylic acid, or aspirin (ASA) has proved to be the most potent in each case. Although hydrolysis, the first step in biotransformation of aspirin takes place very rapidly, many workers feel that the greater effectiveness is due to a unique action of the intact molecule; however, others disagree. In this study, the effects of aspirin and in vitro were compared in order to obtain a greater amount of information relating to the problem.