The global demographic shift towards population aging will result in a dramatic increase in the numbers of elderly in the population. In order to cope with these changing demographics, it is imperative that we better understand aging and age-related pathologies. The reproductive tract provides an excellent system in which to study aging in that it is affected by aging, without compromising the overall health of the individual. In the Brown Norway rat model, the male reproductive tract (testis and epididymis) shows numerous signs of aging when other systems remain relatively unaffected by age, thus making this system ideal for studies on underlying causes of aging. One of the many theories proposed to account for the aging process is oxidative stress. Moreover, some of the changes that take place in the aging epididymis are suggestive of oxidative stress. In order to understand the contribution of oxidative stress to aging of the epididymis, we undertook global gene expression studies of the tissue in the young animal and then assessed how this gene expression was affected by age. We manipulated oxidative stress by caloric restriction and antioxidant (vitamin E) supplementation and deficiency. In characterizing the longitudinal gene expression in the young epididymis, we identified many genes never before reported in this tissue. We found that age profoundly affects gene expression in the epididymis and that the expression of oxidative stress related transcripts decreased, most dramatically in the distal (corpus and cauda epididymidis) regions of the tissue. Caloric restriction attenuated or reversed many of the gene expression changes that took place with age. The effect of caloric restriction was greatest for transcripts associated with protein synthesis and mitochondrial function. Finally, we found that long term antioxidant (vitamin E) deficiency resulted in increased expression of oxidative stress transcripts and exacerbated the effect

We hypothesized that if normal cells carry a regulator which is inactivated in cancer cells, the transfer of this regulator from a normal cell to a cancerous one should inactivate the telomerase. Available in the laboratory was a panel of mouse/human microcell hybrids, each containing a tagged normal human chromosome fragment integrated into a mouse melanoma (telomerase-positive) background. We screened these hybrids for telomerase activity using both the polymerase chain reaction (PCR)-based telomeric repeat amplification protocol (TRAP) assay and mTERT mRNA amplification. / We were able to find a telomerase negative microcell hybrid, B78MC27. Following multiple passages in culture most of the cells undergo senescence; however a small population reactivates telomerase to ensure their survival. The revertant B78MC27-R4, where the human tagged chromosome has been removed, retains telomerase inhibition and also behaves like B78MC27 at later passages. In spite of our efforts, PCR mapping and fluorescence in situ hybridization (FISH) analysis on these hybrids failed to identify the exact region containing the putative repressor. Transfection of a reporter gene (luciferase) under the control of the hTERT promoter into these hybrids confirmed that the telomerase regulation is affecting the hTERT transcription level. / Finally, upon chromosome transfer containing the telomerase repressor into the PC-3M-Pro4GP1 human prostate cancer cell line, we observed hTERT gene transcriptional down-regulation and inhibition of telomerase activity. Thus, telomerase regulation is subject to a dominant negative inhibition likely involving the direct binding of a repressor on the hTERT promoter. The discovery of the natural telomerase regulator could eventually lead to potential cancer therapy, particularly for tumors having short telomeres, as it is seen in prostate cancer.

In this era of modern medicine, impaired wound healing is still a major medical problem with limited treatment options, especially for the elderly and for diabetic patients. One of the molecules that are recognized to be critical in wound healing is transforming growth factor (TGF-beta). Exogenous direct application of TGF-beta to the wound promotes healing in animal models. Since this property of TGF-beta is cell context-dependent, the present study investigated the role of ischemia/reperfusion using a pig-skin flap model (in vivo). In addition, we studied the effects of: (i) hypoxia/reperfusion, (ii) steroids, and (iii) the combined effect of both hypoxia and steroids on the TGF-beta signaling pathway (TGF-beta, its receptors and its downstream mediators, Smad2 and Smad3) in vitro; all of these were studied using human primary skin fibroblasts and a human keratinocyte cell line (HaCat cells). / Our in vivo results showed that the TGF-beta receptors (RI, RII, and RIII) and TGF-beta1 are markedly increased under acute ischemic conditions in dermal blood vessels and fibroblasts. Our in vitro experiments showed that short-term (2 hours) and long-term (24 hours) exposure to hypoxia differentially regulates TGF-beta components, namely, active TGF-beta, RII mRNA, phosphorylated (P) and total Smad2 and Smad3. Our studies have also shown that steroids are able to strongly modulate TGF-beta signaling and that these effects are critically dependent on the type of steroid used and on treatment duration. Analysis of the combined effect of hypoxia and steroids revealed that steroid effects on the TGF-beta signaling machinery is potentially modulated by oxygen tension, with hypoxia acting synergistically or antagonistically in a steroid-specific manner. In summary, we have identified oxygen tension and steroids as regulators of the action of TGF-beta in skin cells. We demonstrated that their effects are critically dependent on the duration of treatment and that they may act synergistically or antagonistically on the components of the TGF-beta pathway, emphasizing the complexity of TGF-beta signaling. Identification of these agents as modulators of TGF-beta signaling provides a basis for the development of strategies for the manipulation of TGF-beta action in the skin. This may contribute towards the development of agents that promote wound healing.

The pituitary gland is a very convenient model to study mechanisms implicated in cellular differentiation. This endocrine gland is composed of six cell lineages, each committed to the production of a different hormone: thyrotrophs (TSH), somatotrophs (GH), lactotrophs (PRL), gonadotrops (LH, FSH), melanotrophs (alpha-MSH) and corticotrophs (ACTH). ACTH and alpha-MSH are both processed from the same precursor, proopiomelanocortin (POMC). Pituitary hormone-producing cells differentiate sequentially from a common epithelial primordium, Rathke's pouch, under the combinatorial action of a subset of tissue- and cell-restricted transcription factors. In corticotrophs, Tpit and NeuroD1 are important for POMC transcription. Their expression closely precedes that of POMC, suggesting a role in the differentiation of this lineage. In order to better define the role of Tpit in POMC cell differentiation, we generated Tpit-null mice. These mice are deficient in POMC-expressing cells while other pituitary lineages are normal. Analysis of other corticotroph markers, such as NeuroD1, showed that lineage commitment was preserved in absence of Tpit. We next tested if Tpit and -NeuroD1 could be jointly required for corticotroph determination by producing Tpit-/- NeuroD1-/- mice. This experiment revealed that these two factors are not essential for early determination of corticotroph cells, suggesting different levels of control for commitment of POMC lineages compared to cell survival or to cell-specific transcription of POMC. Further investigation of Tpit-/- mice has revealed that Tpit is a negative regulator of the gonadotroph fate. These studies suggest a binary model of cell fate decisions for pituitary cell differentiation, suggesting the presence of a pituitary pluripotent precursor. Finally we have shown that mutations in the coding sequence of human TPIT are associated with isolated ACTH deficiency, a pathology very similar to that found in Tpit-null m

Tumor necrosis factor alpha (TNF), a proinflammatory cytokine produced primarily by macrophages and crucial in immune responses, is regulated both transcriptionally and posttranscriptionally upon exposure to various pathogens. Posttranscriptional regulation of TNF expression depends on interaction of RNA-binding proteins with an AU-rich element (ARE) within the 3' untranslated region of TNF mRNA that modulates translational efficacy and mRNA stability. We screened a macrophage protein expression library with a TNF-ARE cRNA probe and identified the RNA-binding protein FXR1P, closely related to fragile X mental retardation syndrome protein FMRP. Macrophage cell lines were generated from FXR1 knockout mice and their littermate controls. The FXR1 knockout macrophages had enhanced TNF protein production following activation compared to control macrophages. Expression of several other proteins encoded by ARE-containing transcripts was also affected by FXR1P deficiency. A GFP-ARE reporter that has green fluorescent protein (GFP) expression under control of the 3'-UTR of TNF mRNA had enhanced expression in transfected macrophages deficient in FXR1P. The ablation of FXR1P led to a dramatically enhanced association of the TNF mRNA with polyribosomes, demonstrating the important role of FXR1P in the posttranscriptional regulation of TNF expression. Our data suggests that release of this repression by FXR1P occurs during LPS-induced macrophage activation. Complementation of the knockout macrophages with recombinant FXR1P resulted in decreased TNF protein production, supporting our findings that FXR1P operates as a repressor of TNF translation. Using 2D electrophoresis gel coupled with mass spectrometry to explore the functional role of FXR1P in macrophages, we showed that FXR1P deficiency had an impact on several proteins, including G3BP1 and CBF-A that have been linked to posttranscriptional regulation. Finally, phosphorylation analysis of FXR1P indi

In most species, highly sophisticated global regulatory networks regulate the expression of genes in response to environmental and physiological demands. The mechanisms devised control virtually every event involved in transcription and translation, as well as influencing mRNA degradation, protein stability, protein localization, protein-protein interactions, and protein function. In order to understand the general mechanism used by several organisms to control gene expression, three regulatory proteins were investigated using nuclear magnetic resonance (NMR) techniques. The systems studied were proteins involved in the control of gene expression at the transcriptional (YaeO, Chapter 2), posttranscriptional (CsrA, Chapter 3) and translational levels (aIF2Pbeta Chapter 4). / YaeO is a protein involved in the regulation of Rho-dependent transcription termination in bacteria. The solution structure of YaeO was solved, the first for a Rho inhibitor, and it was demonstrated that that YaeO binds to the primary RNA binding domain of Rho. This study suggests that YaeO inhibits transcription termination by blocking the primary RNA binding site in Rho. / The carbon storage regulator A from E. coli is a protein involved in the post-transcriptional control of numerous genes involved in carbohydrate metabolism in bacteria. It has been shown that CsrA prevents translation of its target mRNA by blocking access to the ribosome binding site. The binding studies, together with the structure of CsrA, reveal the potential RNA binding region. A model for CsrA recognition of its target mRNAs is also proposed. / The last section focuses on the archaeal translation initiation factor IF2beta. This protein is a key regulator of overall protein synthesis. The structure of aIF2beta was solved and bound zinc was proved to be necessary for the structural integrity of this translation factor. The function of aIF2beta in translation initiation was rationalized in terms of its structure and available structural data for translation factors aIF2alpha and aIF2gamma.

T cell protein tyrosine phosphatase (TC-PTP / PTPN2) is an enzyme that is essential for the proper functioning of the immune system and that participates in the control of cell proliferation, and inflammation. We previously observed that TC-PTP-/- mice display various immunodeficiencies, hypersensitivity to lipopolysaccharide (LPS) and die within three weeks of birth due to anemia and widespread inflammation. A recent analysis of the Wellcome Trust Case Control Consortium (WTCC) genome wide scan data, reported in 2007, indicated a potential role for TC-PTP in inflammatory bowel disease (IBD). To further investigate the potential role of TC-PTP in IBD, we studied heterozygous TC-PTP mutant mice challenged with dextran sulfate sodium (DSS) in their drinking water. In comparison to control animals, we observed significant changes in the colon mucosa of DSS-treated TC-PTP+/- mice, in the ratio of colon to body weight, as well as an up-regulation of mRNA transcripts for IL-6, IL-23, IL-12β, IFN-γ, TNF-α. Moreover, up-regulation of serum IL-6 levels in DSS-treated TC-PTP+/- mice confirms that mice with a single copy of the TC-PTP gene display increased susceptibility to systemic inflammation due to bowel epithelial erosion resulting from DSS challenge. Pathological and molecular analysis reveal that the deficiency of TC-PTP results in pro-inflammatory condition in the bowel of heterozygous TC-PTP+/- mice. These findings support the hypothesis that TC-PTP is an important regulator of inflammatory cytokine signaling and that it may be implicated in the pathophysiology of IBD. / La protéïne tyrosine phosphatase sécifique aux cellules T (TC-PTP) est une enzyme essentielle au fonctionnement du système immunitaire participant au contrôle de la prolifération cellulaire et de l'inflammation. Nous avons précédemment observé que des souris TC-PTP-/- sont immunodédificientes, hypersensibles à l'effect des lipopolysaccharides (LPS) et survivent jusqu'à un maximum de trois semaines d'âge en raison d'une anémie et d'inflammation disséminée. Une récente analyse génomique par le Wellcome Trust Case Control Consortium (WTCC), publié en 2007, indique un rôle potentiel pour TC-PTP (PTPN2) dans la maladie inflammatoire de l'intestin. Pour analyser de façon plus détaillée ce phénomène, nous avons soumis les souris mutantes TC-PTP hétérozygotes à un traitement de sel de dextran sulfate (DSS) dilué dans leur eau d'alimentation. En comparaison avec les animaux contrôles, nous avons observé chex les souris TC-PTP+/- soumises au traitement ave DSS des changements significatifs dans la muqueuse du colon, dans le ratio du poids du colon par rapport au poids corporel, ainsi qu'une augmentation de l'expression des ARN messagers de l'IL-6, l'IL-23, l'IL-12β, l'INF-γ et du TNF-α. De plus, une augmentation des taux sériques d'IL-6 chez les souris TC-PTP+/- soumises au DSS a confirmé que les souris possédant une seule copie du gène de TC-PTP montrent une susceptibilité accrue à une inflammation systémique causée par l'érosion de l'épithélium intestinal suivant ce traitement. Ces résultats appuient l'hypothèse selon laquelle le produit du gène TC-PTP est un régulateur important dans la voie de signalisation des cytokines pro-inflammatoires et qu'il pourrait être important dans la pathophysiologie des maladies inflammatoire de l'intestin.

Cardiovascular diseases account for more than half of all deaths in North America. Although several effective medications exist for the treatment of high blood pressure, reduction of secondary events following a heart attack remains a serious challenge. Angiotensin-(1-7) [Ang-(1-7)] has been demonstrated in multiple studies to exhibit cardio-protective effects against pathological hypertensive remodeling. Nevertheless, there is some debate as to the effect of Ang-(1-7) on kidney function. We have generated a transgenic mouse model to test for the therapeutic potential of Ang-(1-7) in long-term cardiovascular therapy. We demonstrated that low and moderate expression levels of an Ang-(1-7)-transgene in the liver had no discernable effect on viability, reproductive efficiency, blood pressure or gross histology of the heart and kidney. The highest level of transgene expression was associated with a trend toward an increase in blood pressure and a reduction in litter size. Notably, all levels of transgene expression were associated with a significant diuresis with compensated fluid and salt balance. As with our previous results obtained with over-expression of Ang-(1-7) in the heart, expression of the Ang-(1-7)-releasing transgene in the liver significantly reduced remodeling (cardiac fibrosis) in response to a hypertensive challenge. While the actual levels of Ang-(1-7) resulting from the transgene remain to be determined in our mice, the current study identifies Ang-(1-7) or its related analogs as interesting new targets in the prevention or clinical treatment of cardiovascular diseases. / Les maladies cardiovasculaires sont responsables pour plus que la moitié de la mortalité en Amérique du Nord. Malgré qu'il existe plusieurs médicaments capables de traiter l'hypertension artérielle, la réduction de risque d'un deuxième événement suite à une crise cardiaque demeure un défi important. L'angiotensine 1-7 ((Ang-(1-7)) a été démontré dans plusieurs études comme étant efficace contre le remodelage vasculaire associé à une hypertension. Malgré ceci, une incertitude quant à son effet sur la fonction rénale demeure. Nos avons généré un nouveau modèle de souris transgénique (TG) pour évaluer le potentiel thérapeutique de l'angiotensine-(1-7) [ANG-(1-7)]. Notre étude démontre qu'une expression basse ou moyenne d'un transgène pour l'Ang-(1-7) n'a aucun effet sur la viabilité, fécondité, pression artérielle ou l'histologie des reins ou du coeur des souris transgéniques. Cependant, une augmentation de la pression artérielle systolique et une réduction de la taille des portées ont été observé chez les souris TG ayant l'expression la plus élevée. Tous les animaux transgéniques présentaient une augmentation significative de la prise d'eau et de la production d'urine par rapport aux animaux témoins mais la quantité de sodium excrété sur une période de 24h était similaire chez les deux groupes. Comme nous avons remarqué précédemment avec une surproduction de l'Ang-(1-7) dans le coeur, le transgène protège le coeur contre le remodelage (fibrose du coeur) en réponse à un traitement hypertensif. Malgré que nous n'ayons pas pu déterminer la concentration actuelle de l'Ang-(1-7) dans la circulation de nos souris transgéniques, nos résultats suggèrent que le développement d'une approche pharmacologique pour mimer les effets de l'Ang-(1-7) pourrait constituer une approche valable dans le traitement des maladies cardiovasculaires.

The manipulation of the molecular mechanisms that govern distraction osteogenesis (DO) in order to increase the biomechanical strength of new bone and to accelerate its synthesis has been the topic of intense research during the past decades. / Bone morphogenetic proteins (BMPs) play an important role in bone formation. In this study, using a rabbit model of DO, the expressions of BMP's major intracellular signalling molecules, Smad proteins, was analyzed and correlated with the expression of BMP ligands and receptors. Based on these results, which confirmed post-receptor activity for the BMP signalling pathway during DO, we hypothesized that exogenous BMPs injected early in the distraction phase will accelerate bone formation in cases of DO. The cellular changes induced by local application of rhBMP-7 (OP-1) on bone formation were therefore investigated, as well as the possible pathways through which OP-1 was responsible for these effects. / The present study reveals that acceleration of bone formation can be attained after injection of OP-1 early during the distraction protocol. The enhanced bone formation, which occurs through the activation of numerous pathways, most likely depends on a non-vascular mechanism.

Micro satellite instability (MSI) is a form of genomic instability that has recently been implicated in the pathogenesis of thyroid cancer. The purpose of this study is to further define the distribution of microsatellite instability in both normal and neoplastic thyroid follicular epithelium. / Using laser capture microdissection, cells from both normal and tumor tissue were individually collected. PCR amplification of the DNA was then performed using six dinucleotide and two mononucleotide microsatellite markers. / Forty benign and malignant thyroid tumors were compared with their adjacent normal thyroid follicular tissue and were analyzed for MSI. 9/14 papillary thyroid carcinomas and 10/16 of follicular thyroid carcinomas demonstrated MSI at >30--40% of loci tested. For benign follicular adenomas, 9/10 demonstrated microsatellite stability or low-frequency MSI. / Microsatellite instability appears to play a role in thyroid pathogenesis as evidenced by the high frequency of MSI in malignant thyroid neoplasms. In addition our study showed a significant difference in MSI frequency between follicular adenomas and follicular carcinomas. More importantly, the technique of laser capture microdissection allows for more accurate selection of benign, malignant and normal DNA.