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Prostanoids, Diabetes and the Brain: unveiling a pathophysiological role for 15-deoxy-delta-12, 14-prostaglandin J2 in diabetes-related encephalopathy and cerebrovascular injurySeshadri, Swathi January 2008 (has links)
Amongst a host of diabetes-related changes in the central nervous system (CNS) the cerebral microvesssels remain a susceptible target for detrimental effects of the disease associated with altered barrier transport and function of the cerebral microvasculature. While oxidative stress and the activation of COX enzymes emerge as two separate pathogenic mechanisms in a variety of CNS-related diseases, a common link between these presumably distinct processes is the oxidation of arachidonic acid and subsequent formation of bioactive prostaglandins. With basal levels of the parent compound, Prostaglandin D2, exceeding those of other prostaglandins in the brain, the liberation of 15-deoxy-?12, 14-Prostaglandin J2 (15d-PGJ2) may in turn be warranted. Irrespective of whether cerebrovascular dysfunction is a cause or consequence of diabetes-related encephalopathies, the current understanding of the molecular mechanisms leading to this detrimental process is limited. The present thesis attempts to characterize the role of one such arachidonic acid metabolite, 15d-PGJ2, as a potential mediator of neurovascular degeneration. The findings in effect unveil a pathophysiological role for 15d-PGJ2 in a deleterious state of untreated diabetes; corroborated by the Streptozotocin-induced mouse model of diabetes. With a near 8-fold increase in 15d-PGJ2 levels and concomitant reductions of cortical vessel density within the diabetic brain, pharmacological inhibition with the selenocompound SeCl4 appeared to partially rescue cortical capillary density subsequent to reductions in 15d-PGJ2. Furthermore, intracerebroventricular (ICV) administration of pathophysiological doses of 15d-PGJ¬2 in vivo and treatment of brain explants and aortic rings ex vivo / Les plus fréquentes complications reliées au Diabète, au niveau du système nerveux central, sont associées aux microvaisseaux cérébraux; cible fragile et compromise. Les effets délétères du diabète causent un endommagement à la fonction de transport de la barrière hémato-encéphalique et des cellules endothéliales. L'augmentation du stress oxydatif et l'activation des cyclooxygenases jouent un rôle primordial dans la génèse de ces complications et entraînent la formation subséquente des prostaglandines. Les mécanismes moléculaires associés à la dysfonction de l'endothélium vasculaire cérébral demeurent indéterminés. Le contenu de cette thèse tentera de caractériser le rôle de la prostaglandine 15-deoxy-?12,14- J2 (15d-PGJ2), une potentielle médiatrice de la dégénérescence neuromicrovasculaire. Nos données révèlent un rôle pathologique de la 15d-PGJ2 dans un modèle animal de diabète induit à la streptozotocine. Les niveaux de 15d-PGJ2 augmentent de 8 fois chez les souris diabétiques par rapport aux souris contrôles et semblent induire une dégénérescence microvasculaire cérébrale. L'inhibition de la formation de 15d-PGJ2 dans ce modèle par SeCl4 prévient partiellement l'induction de ce phénomène. Par ailleurs, l'injection cérébroventriculaire de 15d-PGJ2 in vivo, le traitement d'explants cérébraux et d'anneaux aortiques ex vivo confirment les propriétés anti-angiogéniques. Finalement, nous démontrons que 15d-PGJ2 induit une surproduction d'espéces oxygénées activées suivie d'une mort cellulaire apoptotique indépendante des récepteurs DP1/DP2 et PPAR?. À travers cette thèse, nous avons réussi à dévoiler de nouveaux mécanismes induisant les lésions microvasc
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Human leukotriene C4 synthase : a unique homodimeric and phisphoregulated glutathione S-transferaseAli, Ambereen January 1994 (has links)
Human Leukotriene C$ sb4$ synthase is a membrane-bound glutathione S-transferase that catalyzes the first committed step leading to the biosynthesis of the pro-inflammatory mediators, the cysteinyl leukotrienes (LTC$ sb4,$ LTD$ sb4$ and LTE$ sb4$). Using chromatographic separation and photoaffinity labelling techniques, LTC$ sb4$ synthase was found to be a unique enzyme distinct from all other known glutathione S-transferases. An 18 kDa membrane polypeptide was specifically labelled in the microsomal membranes of myelocytic cell lines containing LTC$ sb4$ synthase activity (U937 and THP-1), with a photoaffinity derivative of LTC$ sb4$ (azido (I$ sp{125}$) -LTC$ sb4$) and was identified as a candidate for being LTC$ sb4$ synthase or a subunit thereof. LTC$ sb4$ synthase was subsequently purified to homogeneity from THP-1 cells and the purified preparation contained only one polypeptide which had a molecular mass of 18 kDa. By gel filtration chromatography, the native molecular mass of LTC$ sb4$ synthase was determined to be approximately $39 pm3$ kDa. It was therefore concluded that LTC$ sb4$ synthase is enzymatically active as a homodimer. The sequence of the N-terminal 35 amino acids of purified human LTC$ sb4$ synthase was determined and was found to be unique, composed primarily of hydrophobic amino acids and containing a protein kinase C (PKC) consensus sequence. Further analysis of the potential phosphoregulation of LTC$ sb4$ synthase in neutrophilic and eosinophilic HL-60 cells demonstrated that cysteinyl leukotriene biosynthesis, but not non-cysteinyl leukotriene biosynthesis, was specifically attenuated by phorbol ester-mediated activation of PKC. In eosinophilic HL-60 and THP-1 cells this decrease in cysteinyl leukotriene production was demonstrated to be due to non-competitive inhibition of LTC$ sb4$ synthase activity. Concomitant with the PKC-mediated decrease in cysteinyl leukotriene biosynthesis, an increase in prostanoid biosynthesis occurred. Based
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Traditional Chinese herbal anthelmintics : their history and laboratory evaluationChan, Lena January 1980 (has links)
No description available.
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MBD2 transforms normal cells into highly invasive cancer cells by causing DNA demethylation and the activation of pro-cancerous genesAndrews, Stephen January 2010 (has links)
The epigenome, comprised of the chromatin and the DNA methylome, sets up and maintains gene expression patterns. Disruption of the epigenome and its components is a hallmark of cancer, particularly global DNA hypomethylation. The existence of a bona fide DNA demethylase has long been disputed. This thesis focuses on studying methylated DNA-binding protein-2's (MBD2) role in DNA demethylation and cancer. / MBD2 was concurrently classified as a DNA demethylase and a transcriptional repressor. The effects of MBD2 overexpression and depletion in normal and cancerous cells on the methylome strongly supports MBD2's role in participating in the DNA demethylation reaction, as do demethylase assays with purified MBD2 and with nuclear extracts prepared from cells that are either over-producing or under-producing MBD2. / Global DNA hypomethylation is a hallmark of cancer. We therefore tested whether increased MBD2 expression could transform a normal cell into a cancerous cell. Over-production of MBD2 in normal cells, including primary human fibroblasts, results in highly invasive and migratory cancer cells with hypomethylated genomes. MBD2 depletion in several cancer cell lines blocks cellular transformation and results in silencing and methylation of MBD2 target genes. / MBD2's ability to transform cells led us to study whether MBD2 mediates transformation by well-characterized oncogenes such as the proto-oncogene RAS. MBD2 depletion from cells transformed by RAS resulted in an increase in global DNA methylation and a reduction of transformation. / Microarray gene expression analysis revealed novel targets of MBD2 and genes that had not yet been classified as oncogenic, such as the dual function cytokine Il-33. We show here that Il-33 signaling is capable of turning a normal cell to become invasive and that Il-33 can act as a chemo-attractant for normal cells to migrate. Furthermore, abrogation of the cytokine effect of Il-33 in cancer cell by using a recombinant decoy receptor as a pharmacological inhibitor results in an abrogation of the cell's transformed phenotype. / Taken together, the data presented in this thesis show that MBD2 is involved in the DNA demethylation reaction and that MBD2, through DNA demethylation and activation of pro-metastatic genes, can transform normal cells into highly metastatic cancer cells. This thesis supports investigating pharmacological inhibitors of DNA demethylation as anti-metastatic drugs. / L'épigénome, composé de la chromatine et ADN methylome, met en place et maintient les profils d'expression génique. Des perturbations de l'épigénome et de ses composantes est une caractéristique de cancer, particulièrement hypométhylation de l'ADN. L'existence d'un ADN demethylase a longtemps été contestée. Cette thèse vise à étudier le rôle de MBD2 dans la déméthylation de l'ADN et le cancer. / MBD2 a été classé séparément mais en même temps comme étant un demethylase ADN et un répresseur transcriptionnel. Les effets de la surexpression d'MBD2 et l'appauvrissement dans les cellules normales et cancéreuses sur le methylome appuie fermement le rôle MBD2 en tant que participation à l'activité de déméthylation de l'ADN comme le font des tests de demethylase sur MBD2 purifiée et sur des extraits nucléaires préparés à partir de cellules qui sont soit en situation de surproduction ou de sous-production MBD2. / L'hypométhylation global de l'ADN est une des caractéristiques du cancer. Nous avons donc testé si une élévation d'MBD2 pourrait transformer une cellule normale en cellule cancéreuse. La surproduction de MBD2 dans les cellules normales, y compris les fibroblastes humains primaires, donne comme résultats des cellules cancéreuses très envahissantes et migratrices dont les génomes sont hypo-méthylées. L'appauvrissement d'MBD2 dans plusieurs lignées cellulaires cancéreuses bloque la transformation cellulaire et à comme effet atténuer et de la méthylation de gènes cibles d'MBD2. / La capacité remarquable MBD2 à transformer des cellules nous a amené à étudier si MBD2 médiatise la transformation de certains oncogènes bien caractérisés tels que le proto-oncogène ras. Appauvrissement d'MBD2 dans des cellules transformées par RAS a entraîné une augmentation globale de la méthylation de l'ADN et une réduction globale de la transformation. / L'analyse Microarray de l'expression génique ont révélé de nouvelles cibles de MBD2 et des gènes qui n'avaient pas encore été classée comme oncogène, tels que la double fonction de cytokines IL-33. Nous montrons ici que la signalisation l'IL-33 est capable de transformer une cellule normale en cellule envahissante et que l'IL-33 peut agir comme un chimio-attractant en permettant à des cellules normales de migrer. En outre, le phénotype transformé induit par l'IL-33 peut être abrogé à l'aide d'un recombinant decoy receptor comme un inhibiteur pharmacologique. / Les données présentées dans cette thèse démontre que MBD2 est directement impliqué dans la réaction de déméthylation de l'ADN et que MBD2, par la déméthylation de l'ADN et l'activation de gènes pro-métastatiques, peut transformer des cellules normales en cellules cancéreuses hautement métastatique. Cette thèse soutient l'enquête inhibitrice pharmacologique de déméthylation de l'ADN en tant que médicaments anti-métastatique.
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Investigating the effects of chemotherapeutic agents for testicular cancer on the male reproductive system, progeny outcome and spermatogonial stem cells in the ratMarcon, Ludovic January 2011 (has links)
Testicular cancer (TC) is the most common type of cancer affecting men between the ages of 15 and 35 years old. The standard chemotherapy for testicular cancer is a combination of bleomycin, etoposide and cisplatin (called BEP regimen) that results in cure rates over 90%. In order to determine whether the BEP regimen induces adverse effects on the male reproductive system and progeny outcomes, in chapter 2, male Sprague-Dawley rats were treated with a combination of bleomycin, etoposide and cisplatin for 9 consecutive weeks. BEP treatment resulted in decreased testis and epididymal weights, and sperm counts, but despite the dramatic effects on spermatogenesis, paternal exposure to BEP did not affect fertility, nor were there adverse effects on litter size, pre-and post-implantation losses, fetal weight and death rate or sex ratio observed among progeny sired by treated males; however, increased mortality was observed in pups during the perinatal phase. In chapter 3, we evaluated the reversibility of a subchronic BEP treatment on the male reproductive system and progeny outcome. Male rats were treated with a 9 week subchronic BEP regimen; progeny outcome parameters were determined every 3 weeks for a total of 9 weeks during recovery. Subchronic BEP caused transient defects on spermatogenesis; however, a prolonged increase in pre-implantation loss was observed up to 9 weeks after completion of the treatment, suggesting that spermatogonial stem cells may be affected. Thus, in chapter 4, the impact of BEP chemotherapy on spermatogonial stem cells (SSCs) was investigated. Testicular cell suspension from BEP-treated GCS-EGFP transgenic rats were transplanted into busulfan-treated recipient nude mice. The number and length of colonies-derived from transplanted stem cells was assessed as a function of stem cell activity after exposure to BEP. Finally, in chapter 5, we assessed the in vitro effects of bleomycin, etoposide and cisplatin alone and in combination on cultured rat stem/progenitor spermatogonia. These results indicated that BEP has deleterious effects on male reproductive functions including SSCs and progeny outcomes. / Le cancer testiculaire (ou cancer du testicule) est le type de cancer le plus commun chez les hommes âgés de 15 à 35 ans. La chimiothérapie de première ligne pour le cancer du testicule est la combinaison de bléomycine, étoposide et cisplatine (appelé protocole BEP) qui permet un taux de guérison de plus de 90%. Afin de déterminer si le protocole BEP induit des effets néfastes sur le système reproducteur mâle ainsi que sur la progéniture, dans le second chapitre, nous avons traités des rats mâles (Sprague-Dawley) avec une combinaison de bléomycine, étoposide et cisplatine pendant 9 semaines consécutives. Le protocole BEP entraîna la diminution du poids des testicules et des épididymes, du nombre de spermatozoïdes, mais en dépit des effets spectaculaires sur la spermatogénèse, l'exposition du père au protocole BEP n'affecta pas la fertilité et aucun effet ne fut observé sur la taille de la portée, les pertes aux stades pré- et post- implantatoires, le poids et la mortalité fœtale ou encore sur le ratio mâle/femelle des fœtus issus de pères traités. Cependant, une augmentation de mortalité peu de temps après la naissance fut observée. Dans le chapitre 3, nous avons évalué la réversibilité des effets du protocole BEP sub-chronique sur le système reproducteur mâle et la progéniture. Des rats mâles furent traités avec un protocole BEP sub-chronique de 9 semaines ; les paramètres de la progéniture furent déterminés toutes les 3 semaines pour un total de 9 semaines pendant la période de rétablissement. Le protocole BEP sub-chronique entraîna des effets transitoires sur la spermatogénèse, cependant une augmentation prolongée des pertes pré-implantatoires fut observé jusqu'à 9 semaines après la fin du traitement, suggérant que les cellules souches germinales puissent être affectées. Ainsi, dans le chapitre 4, l'impact de la chimiothérapie BEP sur les cellules souches germinales (CSGs) fut étudié. Des suspensions de cellules testiculaires provenant de rats transgéniques GCS-EGFP traités avec le protocole BEP furent transplantées dans des testicules récipiendaires traités au busulfan de souris immunodéficientes. Le nombre ainsi que la longueur des colonies provenant des cellules souches transplantées fut déterminé pour évaluer la capacité des cellules souches germinales exposées au BEP à rétablir la spermatogénèse. Enfin, dans le chapitre 5, nous avons évalué les effets in vitro de la bléomycine, de l'étoposide et du cisplatine seul ou en combinaison sur des cultures de spermatogonies. Ces résultats indiquent que le protocole BEP produit des effets néfastes sur les fonctions reproductives incluant les cellules souches germinales et la progéniture.
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Effect of Islet Neogenesis Associated Protein (INGAP) peptide on axonal regrowth in the peripheral sensory nervous system and its therapeutic implications for diabetic peripheral neuropathyTam, Joseph. January 2006 (has links)
The majority of individuals with .long-term diabetes become afflicted with diabetic peripheral neuropathy (DPN), an often painful and debilitating secondary complication of the disease. Symptomatic treatments, which do not address the underlying nerve damage in diabetes, are currently the only therapeutic options for DPN, aside from rigorous control of blood glucose, which only occasionally reduces the incidence and severity of DPN. Given the progressive nature of disorder, and the failure of the classical neurotrophins in clinical trials for DPN, it is important to develop new therapeutics that can counteract the nerve damage in diabetes. / Pancreas-derived peptides that stimulate islet regeneration have gained increasing interest for use in DPN, in part due to the unique similarities that exist between pancreatic and neural tissues. We studied the effects of one such peptide, the Islet Neogenesis Associated Protein (INGAP) peptide, on axonal regrowth in the peripheral nervous system (PNS) in adult C57BLJ6 mice. Using dorsal root ganglia (DRG) explant cultures as in vitro model of axotomy, we found that INGAP peptide enhances axonal regrowth in a time- and concentration-dependent manner that involves cyclic AMP-dependent activation of protein kinase A (PKA) and stimulation of phosphatidylinositol-3 kinase (PI3K). The neuritogenic effects of INGAP peptide were reduced by blocking antibodies against a number of growth factors that are secreted by Schwann cells including nerve growth factor (NGF), insulin-like growth factor-I (IGF-I) and transforming growth factor-beta (TGF-(3), suggesting an indirect action of INGAP peptide on outgrowth from DRG neurons, achieved via a primary action on Schwann cells. / To further assess the potential usefulness of INGAP peptide in DPN, we used streptozotocin-induced type 1 diabetic mice and found that after two weeks of INGAP peptide administration, begun twelve weeks after the STZ treatment, sensory dysfunction (reduced sensitivity to heat stimulation) was corrected without inducing the hyperalgesia associated with direct administration of NGF. These effects were accompanied by increases in the levels of a number of structural proteins and transcription factors associated with nerve growth. Significantly, the beneficial effects of INGAP peptide on the PNS in vivo occurred independently of its normalizing effects on hyperglycemia. Finally, we found that INGAP peptide enhanced the mitochondrial inner transmembrane potential (DeltaPsim), and that the mitochondrial effects were largely mediated by PKA. / Taken together, these studies demonstrate that INGAP peptide enhances sensory axonal regrowth independently of islet neogenesis and the consequent secretion of insulin, and that it may be of benefit in the treatment of peripheral neuropathy associated with diabetes and possibly other clinical conditions.
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Mechanisms of FOXL2 regulated gene expression: A structural perspectiveLibasci, Vanessa January 2012 (has links)
Follicle-stimulating hormone (FSH) is a key regulator of mammalian reproduction. Activins, members of the TGFβ superfamily, selectively stimulate FSH synthesis via regulation of FSH β-subunit (Fshb) transcription. Forkhead box L2 (FOXL2) mediates activin induction of Fshb transcription in gonadotrope cells. We identified a conserved cis-element in the proximal Fshb promoter that binds FOXL2 with high affinity, which matches the consensus forkhead binding element characterized in other genes. In contrast, another study identified a different FOXL2 cis-element through screening of a random oligonucleotide library. Moreover, FOXL2 has been characterized as an important transcription factor in the regulation of other genes, including steroidogenic acute response (StAR), aromatase (CYP19), gonadotropin-releasing hormone receptor (Gnrhr), and follistatin (Fst). These data suggest that FOXL2 may exhibit flexibility in its DNA binding activity. To provide a clearer understanding of the mechanisms mediating FOXL2 binding to DNA, I aimed to solve the X-ray crystal structure of the murine FOXL2 forkhead domain in complex with its high affinity cis-element in the murine Fshb promoter. I expressed GST-tagged murine FOXL2 forkhead domain (amino acids 48-144), purified it from E.coli and examined its DNA binding activities using gel-shift assays and isothermal titration calorimetry (ITC). I also generated a homology model of FOXL2 bound to DNA, allowing me to identify crucial amino acids and nucleotides mediating binding. I optimized the conditions for protein purification using affinity, ion exchange, and size exclusion chromatography. We have obtained protein-DNA crystals that diffract X-rays to low-resolution and are currently screening to obtain better quality crystals. Ultimately, the results of our analysis will provide a structural description of FOXL2 binding to the Fshb promoter, and may provide insight into the basis for the apparent FOXL2 binding site flexibility. / L'hormone folliculo-stimulante (FSH) est un régulateur clé de la reproduction chez les mammifères. Les activines, membres de la superfamille TGFß, stimulent de manière sélective la synthèse de FSH en contrôlant la transcription de sa sous-unité β (Fshb). Dans les cellules gonadotropes de l'hypophyse, la protéine forkhead box L2 (FOXL2) est médiatrice de l'induction de la transcription de Fshb par les activines. Nous avons identifié un élément cis sur le promoteur proximal du gène Fshb qui lie FOXL2 avec haute affinité ; celui-ci correspond à l'élément consensus de liaison forkhead typique caractérisé dans d'autres gènes de la même famille. En revanche, une autre étude a identifié un élément cis différent pour FOXL2 suite à un dépistage d'une bibliothèque oligonucléotides aléatoires. De plus, FOXL2 a été caractérisée comme un facteur de transcription important dans la régulation d'autres gènes, y compris la steroidogenenic acute regulatory factor (StAR), l'aromatase (CYP19), le récepteur de l'hormone libérant la gonadotropine (Gnrhr), et follistatine (Fst). Ces données suggèrent que FOXL2 peut présenter une flexibilité dans la compostion de l'élément de liaison à l'ADN. Afin d'obtenir une meilleure compréhension des mécanismes qui explique la liaison de FOXL2 à l'ADN, j'ai cherché à résoudre la structure cristalline aux rayons X de du domaine forkhead de FOXL2 en complexe avec son élément cis de haute affinité sur le promoteur murin de Fshb. J'ai exprimé le domaine forkhead de la protéine murine de FOXL2 en fusion avec GST (acides aminés 48-144). Je l'ai ensuite purifiée à partir de bactérie E. coli et j'ai examiné sa capacité de lier d'ADN en utilisant des essais de décalage de mobilité électrophorétique (EMSA) ainsi que la calorimétrie isotherme de titration (ITC). J'ai aussi créé un modèle par homologie de FOXL2 lié à l'ADN, ce qui nous a permis d'identifier les acides aminés et les nucléotides critiques impliqués dans cette liaison entre domaine forkhead et ADN. J'ai optimisé les conditions de purification de la protéine recombinante de FOXL2 à l'aide de colonnes basées sur l'affinité, l'échange d'ions ainsi que de la chromatographie d'exclusion stérique. Nous avons obtenu des cristaux protéine-ADN qui ont effectivement diffracté les rayons X à basse résolution et somme actuellement à l'étape d'optimisation afin d'obtenir des cristaux de meilleure qualité. Finalement, les résultats de notre analyse constitue une base solide dans la quête de la structure de FOXL2 lié au promoteur Fshb et nous éclairera dans notre compréhension de l'apparente flexibilité de FOXL2 envers son site de liaison sur l'ADN.
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A novel model of Quinidine induced torsade de pointes arrhythmias in the isolated rabbit heart /Weerapura, Manjula. January 1997 (has links)
Accumulating evidence suggest the underlying danger of Class IA and Class III antiarrhythmic agents in inducing potentially lethal torsade de pointes arrhythmias (TdP) if associated with hypokalemia and slow heart rates. The primary aim of the following study was to examine the proarrhythmic activity of the Class IA agent quinidine in isolated perfused rabbit hearts in an effort towards developing a reliable model of TdP. / Thirty five rabbit hearts were perfused with therapeutic quinidine concentrations (2.5 or 5$ mu$M) in the presence of hypokalemia (K$ sp+$ - 2.7mM) and stimulated at cycle lengths (CLs) ranging from 0.5s-5.5s. Monophasic action potentials (MAPs) were recorded from left endocardial and right epicardial sites along with an ECG. / We suggest that the described model of acquired TdP in isolated rabbit hearts can be adapted for the quantitative and qualitative assessment of the proarrhythmic potential of existing and novel antiarrhythmic agents, to test the efficacy of novel therapeutic strategies for the acquired long QT syndrome and/or to clarify the specific mechanism(s) of drug induced TdP. (Abstract shortened by UMI.)
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Epigenetic crosstalk between DNA demethylation and histone acetylationOu, Jing Ni January 2009 (has links)
Abnormal methylation patterns such as regional hypermethylation and genomic hypomethylation often result in transcriptional changes of critical genes that are central to the progression of human cancers. It is therefore important to identify the mechanisms that are responsible for the alterations in order to identify proper pharmacological targets. This thesis examines whether specific cellular factors are involved in establishing the state of DNA hypomethylation in cancer cells and whether changes in chromatin structure could affect DNA methylation. MBD2 is a protein that has been previously characterized to possess distinct transcription activities; it can either function as a methylation-dependent transcription repressor, a methylation-independent transcription activator, as well as an inducer of DNA demethylation. Chapters 3 and 4 demonstrate that MBD2 induces gene-specific DNA demethylation in pancreatic and bladder cancer cells by recruiting transcriptional activator AP-2, Sp1 and the histone acetyltransferase CBP to the associated promoters. These results substantiate the idea that demethylation induced by MBD2 might facilitate the recruitment of transcription factors to the gene to activate its expression. Histone deacetylase (HDAC) inhibitors are drugs designed to target chromatin modification. In chapter 5, we showed that increasing histone acetylation by HDAC inhibitor TSA was associated with a significant decrease in global methylation. TSA also induces histone acetylation, DNA demethylation and expression of specific methylated tumor suppressor genes, such as E-CADHERIN and RARβ2 in different human cancer cell lines. Our findings provide evidence for a / Un patron de méthylation anormal, tel que l'hyperméthylation régionale ou l'hypométhylation génomique, modifie la transcription de gènes critiques jouant ainsi un rôle central dans la progression de nombreux cancers chez l'humain. Il est donc devenu essentiel d'identifier les mécanismes responsables de ces altérations afin de développer des traitements pharmacologiques ciblés. Le but principal de cette thèse est d'examiner si certains facteurs cellulaires sont impliqués dans l'établissement de l'ADN hypométhylé des cellules cancéreuses, ainsi que l'effet des changements dans la structure de la chromatine sur la méthylation de l'ADN. Il a été préalablement démontré que la protéine MBD2 possède plusieurs rôles distincts lors de la transcription, elle peut agir à la fois comme un répresseur de la transcription dépendant de la méthylation, comme un inducteur de la déméthylation ainsi qu'un activateur de la transcription indépendant de la méthylation. Les chapitres 3 et 4 présentés dans cet ouvrage démontrent que MBD2 induit la déméthylation de gènes spécifiques dans les cellules cancéreuses pancréatiques et urinaires grâce au recrutement des activateurs transcriptionnels AP-2, Sp1 ainsi que de l'histone acétyltransférase CBP au promoteur impliqué. Ces résultats supportent l'hypothèse selon laquelle la déméthylation induite par MBD2 faciliterait le recrutement de facteurs de transcription au sein du gène afin d'activer son expression. Les inhibiteurs de l'Histone déacétylase (HDAC) sont des drogues pharmaceutiques développées afin de cibler les modifications de la chromatine. Nous sommes parvenus à démontrer, dans le
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Mechanisms of leukotriene C4 synthase regulationGupta, Namrata. January 1999 (has links)
Leukotrienes are potent lipid mediators of inflammation. A member of the supergene family of Membrane Associated Proteins in Eicosanoid and Glutathione metabolism (MAPEG), LTC4 synthase catalyzes the first committed step in the biosynthesis of the biologically active cysteinyl leukotrienes. Pharmacological cross-reactivity between LTC4 synthase and other proteins in the five-lipoxygenase pathway was assessed, leading to the identification of L-699,333 as the most potent LTC4 synthase inhibitor amongst the compounds available, previously reported as a nanomolar five-lipoxygenase inhibitor and a weak LTC4 synthase inhibitor. Compounds that bind competitively to the arachidonic acid binding sites on five-lipoxygenase and five-lipoxygenase-activating protein, were found to recognize motifs that are weakly conserved on the binding site of LTC 4 synthase. L-699,333 was used to study LTC4 synthase catalysis revealing a random, rapid equilibrium mechanism, with strong substrate inhibition imparted by LTA4. Further studies showed that inhibition of LTC 4 synthase activity induced by cellular activation by phorbol esters, was due to direct phosphorylation of the enzyme. The dose-dependent phosphorylation event was found to be specific to TBP-1 cells, time-dependent with implications of negative feedback, influenced by the cellular density, and mediated by protein kinase C. These novel insights into LTC4 synthase biology further our understanding of this attractive therapeutic target against bronchial asthma.
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