This body of work is concerned with the genetics of craniofacial morphology and specifically with that of the cranial sutures which form fibrous articulations between the calvarial bones. The premature fusion of these sutures, known as craniosynostosis, is a common developmental abnormality and has been extensively utilised here as a tool through which to study the genetics of suture morphogenesis and craniofacial diversity. Investigations began with a search for polymorphisms associated with normal variation in human craniofacial characteristics. Denaturing High-Performance Liquid chromatography was used to identify polymorphisms in two genes causative for craniosynostosis by analysing DNA from a large cohort of individuals from four ethnogeographic populations. A single nucleotide polymorphism in fibroblast growth factor receptor 1 was identified as being associated with variation in the cephalic index, a common measure of cranial shape. To further, and specifically, investigate the molecular processes of suture morphogenesis gene expression was compared between unfused and prematurely fusing/fused suture tissues isolated from patients with craniosynostosis. Two approaches, both utilising Affymetrix gene expression microarrays, were used to identify genes differentially expressed during premature suture fusion. The first was a novel method which utilised the observation that explant cells from both fused and unfused suture tissue, cultured in minimal medium, produce a gene expression profile characteristic of minimally differentiated osteoblastic cells. Consequently, gene expression was compared between prematurely fused suture tissues and their corresponding in vitro de-differentiated cells. In addition to those genes known to be involved in suture morphogenesis, a large number of novel genes were identified which were up-regulated in the differentiated in vivo state and are thus implicated in premature suture fusion and in vivo osteoblast differentiation. The second microarray study involved an extensive analysis of 16 suture tissues and compared gene expression between unfused (n=9) and fusing/fused sutures (n=7). Again, both known genes and a substantially large number of novel genes were identified as being differentially expressed. Some of these novel genes included retinol binding protein 4 (RBP4), glypican 3 (GPC3), C1q tumour necrosis factor 3 (C1QTNF3), and WNT inhibitory factor 1 (WIF1). The known functions of these genes are suggestive of potential roles in suture morphogenesis. Realtime quantitative RT PCR (QRT-PCR) was used to verify the differential expression patterns observed for 11 genes and Western blot analysis and confocal microscopy was used to investigate the protein expression for 3 genes of interest. RBP4 was found to be localised on the ectocranial surface of unfused sutures and in cells lining the osteogenic fronts while GPC3 was localised to suture mesenchyme of unfused sutures. A comparison between each unfused suture (coronal, sagittal, metopic, and lambdoid) demonstrated that gene expression profiles are suture-specific which, based on the identification of differentially expressed genes, suggests possible molecular bases for the differential timing of normal fusion and the response of each suture to different craniosynostosis mutations. One observation of particular interest was the presence of cartilage in unfused lambdoid sutures, suggesting a role for chondrogenesis in posterior skull sutures which have generally been thought to develop by intramembranous ossification without a cartilage precursor. Finally, the effects of common media supplements used in in vitro experiments to stimulate differentiation of calvarial suture-derived cells were investigated with respect to their ability to induce in vivo-like gene expression. The response to standard differentiation medium (ascorbic acid + β-glycerophosphate) with and without dexamethasone was measured by both mineralisation and matrix formation assays and QRT-PCR of genes identified in the above described microarray studies. Both media induced collagen matrix and bone nodule formation indicative of differentiating osteoblasts. However, the genes expression profiles induced by both media differed and neither recapitulated the levels and profiles of gene expression observed in vivo for cells isolated from both fused and unfused suture tissues. This study has implications for translating results from in vitro work to the in vivo situation. Significantly, the dedifferentiation microarray study identified differentially expressed genes whose products may be considered candidates as more appropriate osteogenic supplements that may be used during in vitro experiments to better induce in vivo-like osteoblast differentiation. This study has made a substantial contribution to the identification of novel genes and pathways involved in controlling human suture morphogenesis and craniofacial diversity. The results from this research will stimulate new areas of inquiry which will one day aid in the development of better diagnostics and therapeutics for craniosynostosis, and other craniofacial and more general skeletal abnormalities.

Dissertation (Ph.D.)--University of South Florida, 2009. / Includes vita. Includes bibliographical references. Also available online.

Dissertation (Ph.D.)--University of South Florida, 2009. / Title from PDF of title page. Document formatted into pages; contains 103 pages. Includes vita. Includes bibliographical references.

La polyarthrite rhumatoïde (PR) est la maladie auto-immune la plus fréquente d’une prévalence de 1%. Les cellules résidentes de la cavité synoviale, les fibroblast-like synoviocytes (FLS), sont des acteurs majeurs de la PR. Leur activation par des récepteurs de l’immunité innée participe à l’acquisition d’un phénotype agressif menant à la destruction ostéo-articulaire. Dans cette étude, nous avons évalué le rôle régulateur de miARN sur les voies de signalisation des Toll-like receptors (TLR). L’activation de TLR2 et de TLR4 dans les FLS induit la diminution de l’expression de plusieurs miARN, dont miR-19a et b (miR-19), alors que TLR2 est surexprimé. Nous avons pu ainsi montrer que miR-19 régule Tlr2 et que la transfection de mir-19 dans les FLS activés induit une diminution de l’expression de TLR2 et de la synthèse d’IL-6 et de MMP-3. Mir-19 appartient au cluster miR-17~92, dont l’expression est abaissée dans les FLS. Il code pour 6 miARN dont miR-20a. miR-20a est également sous-régulé après activation de TLR2 et TLR4 dans les FLS et les THP-1. Nous avons montré que miR-20a régule directement l’expression d’Ask1, impliquée et surexprimée après activation de TLR4. La transfection de miR-20a in vitro nous a permis de montrer que miR-20a contrôle l’expression d’ASK1 et induit une inhibition de la synthèse de cytokines majeures de la PR dans les FLS et les THP-1. Des résultats équivalents ont été obtenus ex vivo chez la souris. Ces travaux ont permis d’identifier dans les FLS rhumatoïdes des miARN anti-inflammatoires dont la baisse d’expression permet une augmentation de l’expression de TLR2 et d’ASK1. Ces miARN pourraient donc constituer de nouvelles cibles thérapeutiques. / Rheumatoid arthritis (RA) is the most frequently autoimmune disease with a prevalence of 1%. Resident cells of joints, the fibroblast-like synoviocytes (FLS), act as key players in RA. Their activation through Pattern-recognition receptors leads to an aggressive phenotype, leading in the osteo-articular destruction of the joints. In this study, we aimed to discuss the link between Toll-like receptors (TLR) and miRNA pathway. We established the down-regulation of a few miRNA when FLS were activated through TLR2 and TLR4, including miR-19a and miR-19b (miR-19). We showed that miR-19 regulates directly Tlr2 and that transfection of miR-19 mimics leads to a decrease of IL-6 and MMP-3 synthesis in FLS. miR-19 belongs to the cluster miR-17~92, which is also down-regulated in activated FLS. This primary transcript encodes for 6 miRNA, including miR-20a, which is also down regulated upon TLR2 and TLR4 activation in FLS and further in THP-1, a monocyte cell-line. Then, we validated the predicted regulation of miR-20a on Ask1, an important kinase involved in TLR4 pathway. The transfection of miR-20a mimics in vitro represses ASK1 expression and inhibits several major cytokines in RA both in FLS and THP-1. Further, we confirmed these results on ex vivo experiments on peritoneal macrophages. These works allowed us to identify new anti-inflammatory miRNA that are downregulated and allow overexpression of TLR2 and ASK1 in RA FLS. These results open new experiments on in vivo models. All together, these data give new insights for identify new therapeutics in RA.

O propósito do presente estudo foi avaliar o comportamento de células pulpares humanas expostas ao TGFβ1 e ao aFGF, em cultura, nas seguintes concentrações: TGFβ1 a 1ng/mL, TGFβ1 a 5ng/mL, TGFβ1 a 1ng/mL + aFGF a 5ng/mL, TGFβ1 a 5ng/mL + aFGF a 5ng/mL e aFGF a 5ng/mL. Foi avaliada a morfologia celular, a atividade da fosfatase alcalina, através de ensaio com pNPP como substrato e a expressão das proteínas osteocalcina, sialoproteína óssea e sialofosfoproteína de dentina, através de RT-PCR. Após quatro dias, verificou-se que a média do número de nucléolos no grupo tratado com TGFβ1 a 1ng/mL foi significativamente maior que no grupo tratado com aFGF a 5ng/mL. A média da atividade da fosfatase alcalina no grupo tratado com TGFβ1 a 1ng/mL foi significativamente maior que no grupo tratado com TGFβ1 a 5ng/mL + aFGF a 5ng/mL. Foi observada a expressão de osteocalcina em todas as células pulpares humanas que proliferaram em cultura. Entretanto, no grupo em que foi utilizado o aFGF a 5ng/mL houve diminuição da expressão da osteocalcina. A exposição dos fatores não induziu a expressão de componentes da matriz de dentina tais como BSP e DSPP. Sugere-se que as células expostas ao TGFβ1 1ng/mL foram estimuladas, apresentando uma maior atividade celular e as células expostas ao aFGF 5ng/mL foram inibidas, apresentando uma menor atividade celular. / The aim of the present work was to evaluate the behavior of human dental pulp cells exposed to TGFβ1 and aFGF in culture, at the following concentrations: TGFβ1 1ng/mL, TGFβ1 5ng/mL, TGFβ1 1ng/mL + aFGF 5ng/mL, TGFβ1 5ng/mL + aFGF 5ng/mL e aFGF 5ng/mL. We assessed the cellular morphology, alkaline phosphatase activity, using pNPP as substrate, and expression of osteocalcin, bone sialoprotein, dentin sialophosphoprotein proteins by RT-PCR. After four days, the nucleolus media in the group treated with TGFβ1 1ng/mL was significantly higher than the group treated with aFGF 5ng/mL The alkaline phosphatase activity in the TGFβ1 1ng/mL treated group was significantly higher than the media observed in TGFβ1 5ng/mL + aFGF 5ng/m treated group. Osteocalcin expression was observed in all human dental pulp cell cultures. However, in the aFGF 5ng/mL treated group the osteocalcin expression decreased. The exposure to growth factors did not induced the expression of dentin matrix components such as BSP or DSPP. Our data suggest that the cells exposed to TGFβ1 1ng/mL were stimulated and had a higher cell activity, and that cells exposed to aFGF 5ng/mL were inhibited having a cell activity decrease.

No description available.

O propósito do presente estudo foi avaliar o comportamento de células pulpares humanas expostas ao TGFβ1 e ao aFGF, em cultura, nas seguintes concentrações: TGFβ1 a 1ng/mL, TGFβ1 a 5ng/mL, TGFβ1 a 1ng/mL + aFGF a 5ng/mL, TGFβ1 a 5ng/mL + aFGF a 5ng/mL e aFGF a 5ng/mL. Foi avaliada a morfologia celular, a atividade da fosfatase alcalina, através de ensaio com pNPP como substrato e a expressão das proteínas osteocalcina, sialoproteína óssea e sialofosfoproteína de dentina, através de RT-PCR. Após quatro dias, verificou-se que a média do número de nucléolos no grupo tratado com TGFβ1 a 1ng/mL foi significativamente maior que no grupo tratado com aFGF a 5ng/mL. A média da atividade da fosfatase alcalina no grupo tratado com TGFβ1 a 1ng/mL foi significativamente maior que no grupo tratado com TGFβ1 a 5ng/mL + aFGF a 5ng/mL. Foi observada a expressão de osteocalcina em todas as células pulpares humanas que proliferaram em cultura. Entretanto, no grupo em que foi utilizado o aFGF a 5ng/mL houve diminuição da expressão da osteocalcina. A exposição dos fatores não induziu a expressão de componentes da matriz de dentina tais como BSP e DSPP. Sugere-se que as células expostas ao TGFβ1 1ng/mL foram estimuladas, apresentando uma maior atividade celular e as células expostas ao aFGF 5ng/mL foram inibidas, apresentando uma menor atividade celular. / The aim of the present work was to evaluate the behavior of human dental pulp cells exposed to TGFβ1 and aFGF in culture, at the following concentrations: TGFβ1 1ng/mL, TGFβ1 5ng/mL, TGFβ1 1ng/mL + aFGF 5ng/mL, TGFβ1 5ng/mL + aFGF 5ng/mL e aFGF 5ng/mL. We assessed the cellular morphology, alkaline phosphatase activity, using pNPP as substrate, and expression of osteocalcin, bone sialoprotein, dentin sialophosphoprotein proteins by RT-PCR. After four days, the nucleolus media in the group treated with TGFβ1 1ng/mL was significantly higher than the group treated with aFGF 5ng/mL The alkaline phosphatase activity in the TGFβ1 1ng/mL treated group was significantly higher than the media observed in TGFβ1 5ng/mL + aFGF 5ng/m treated group. Osteocalcin expression was observed in all human dental pulp cell cultures. However, in the aFGF 5ng/mL treated group the osteocalcin expression decreased. The exposure to growth factors did not induced the expression of dentin matrix components such as BSP or DSPP. Our data suggest that the cells exposed to TGFβ1 1ng/mL were stimulated and had a higher cell activity, and that cells exposed to aFGF 5ng/mL were inhibited having a cell activity decrease.

Made available in DSpace on 2014-12-17T15:36:44Z (GMT). No. of bitstreams: 1 EudesESL_TESE.pdf: 7375820 bytes, checksum: c195e08c1d621b363793dbcfb6727636 (MD5) Previous issue date: 2014-06-06 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / A number of evidences show the influence of the growth of injured nerve fibers in Peripheral Nervous System (PNS) as well as potential implant stem cells (SCs) to make it more suitable for nerve regeneration medium. In this perspective, this study aimed to evaluate the plasticity of mesenchymal stem cells from bone marrow of mice in the presence of culture medium conditioned with facial nerve explants (D-10) and fibroblast growth factor-2 (FGF-2). In this perspective, the cells were cultivated only with DMEM (group 1), only with D-10(group 2), only with FGF-2(group 3) or with D-10 and FGF-2(group 4). The growth and morphology were assessed over 72 hours. Quantitative phenotypic analysis was taken from the immunocytochemistry for GFAP, OX-42, MAP-2, β-tubulin III, NeuN and NF-200 on the fourth day of cultivation. Cells cultured with conditioned medium alone or combined with FGF-2 showed distinct morphological features similar apparent at certain times with neurons and glial cells and a significant proliferative activity in groups 2 and 4 throughout the days. Cells cultived only with conditioned medium acquired a glial phenotype. Cells cultured with FGF-2 and conditioned medium expressed GFAP, OX-42, MAP-2, β-tubulin III, NeuN and NF-200. On average, area and perimeter fo the group of cells positive for GFAP and the ?rea of the cells immunostained for OX-42 were higher than those of the group 4. This study enabled the plasticity of mesenchymal cells (MCs) in neuronal and glial nineage and opened prospects for the search with cell therapy and transdifferentiation / Uma s?rie de evid?ncias mostra a influ?ncia do meio no crescimento de fibras nervosas lesadas no Sistema Nervoso Perif?rico (SNP), assim como o potencial do implante de C?lulas Tronco (CTs) em tornar esse meio mais prop?cio ? regenera??o nervosa. Nessa perspectiva, esse estudo teve como objetivo avaliar a plasticidade de c?lulas tronco mesenquimais (CTMs) da medula ?ssea de ratos diante da presen?a de meio de cultura condicionado com explantes de nervo facial (D-10) e fator de crescimento fibrobl?stico-2 (FGF-2). Dessa maneira as c?lulas mesenquimais foram cultivadadas com meio DMEM (grupo 1), s? com meio D-10(grupo 2), s? com FGF-2(grupo 3) ou com meio D-10 e FGF-2(grupo 4). O crescimento e a morfologia celular foram avaliados ao longo de 72 horas. Al?m disso, a avalia??o fenot?pica foi feita a partir da imunocitoqu?mica para GFAP, OX-42, MAP-2, β-tubulina III, NeuN e NF-200 no quarto dia de cultivo. As c?lulas cultivadas com meio condicionado sozinho ou combinado com FGF-2 demonstraram caracter?sticas morfol?gicas semelhantes a neur?nios e c?lulas gliais e uma significativa atividade proliferativa nos grupos 2 e 4 ao longo dos dias. As c?lulas cultivadas com meio condicionado desprovido de tratamento com FGF-2 adquiriram fen?tipo glial demostrando imunorreatividade para GFAP e OX-42. As c?lulas cultivadas com meio condicionado com adi??o de FGF-2 expressaram GFAP, OX-42, MAP-2, β-tubulina III, NeuN e NF-200. Em m?dia, a ?rea e per?metro das c?lulas do grupo 2 positivas para GFAP e a ?rea das c?lulas imunomarcadas para OX-42 foram maiores do que as do grupo 4. O estudo possibilitou a plasticidade de c?lulas mesenquimais (CMs) numa linhagem neuronal e glial e abriu perspectivas para busca de novas t?cnicas com terapia e transdiferencia??o celular

The pathogenesis of insulin resistance is a complex and still intensively studied issue. Endocrine and paracrine activity of the adipose tissue together with mi- tochondrial dysfunction are the most discussed potential factors included in the development of insulin resistance. In the first part of our study we examined the involvement of the adipose tissue and its secretory products in the etiopathogenesis of insulin resistance in patients with Cushing's syndrome, acromegaly and simple obesity. We focused on three important regulators of metabolic homeostasis - fibroblast growth factors 21 and 19 (FGF-21 and FGF-19) and adipocyte fatty acid binding protein (FABP-4). We found significantly elevated circulating levels of FGF-21 and FABP-4 ac- companying insulin resistance in both patients with simple obesity and patients with obesity connected to Cushing's syndrome, as compared to healthy controls. The concentrations of both substances were comparable between hypercortisolic and obese patients. This finding together with the absence of correlation be- tween the levels of FGF-21 resp. FABP-4 and cortisol suggest that the reason for elevation of their concentrations is obesity and its metabolic consequences themselves rather then the effect of hypercortisolism on FGF-21 and FABP-4 production. We found no...

Hyperglycemia in critically ill patients was considered for many years an adaptive response to stress conditions being present in both patients with and without previous history of diabetes. Hyperglycemia is caused mainly by peripheral insulin resistance induced by the factors acting counteracting insulin signalling at the postreceptor level. Furthermore, hyperglycemia itself can then increase serum levels of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-6 (Il-6) and interleukin-8 (Il- 8) and others. On the contrary, peripheral insulin resistance induced by pro- inflammatory cytokines may further potentiate hyperglycemia. White adipose tissue represents in addition to its energy storage function also a very active endocrine active organ. In addition to regulation of a number of metabolic processes it also significantly modulates the inflammatory response. In critically ill patients, adipose tissue changes its morphology, i.e. the adipocytes are shrinking and adipose tissue is abundantly infiltrated by macrophages. Paradoxically, overweight and obese critically ill patients have lower mortality than underweight, lean and morbidly obese subjects. In our studies, we selected population of the patients undergoing elective major cardiac surgery with extracorporeal...