ABSTRACT Vascular permeability and angiogenesis are crucial events in the rodent and human uterus in early pregnancy and are regulated by vascular endothelial growth factor (VEGF) and prostaglandins liberated from arachidonic acid by cyclooxygenase 2 (COX2). These events coincide with the typical morphological features of the receptive uterus and are regulated by synchronized release of ovarian hormones (oestrogen and progesterone). However, administration of follicle stimulating hormone (FSH) and human chorionic gonadotropin (hCG), commonly used in assisted reproduction, affect the synchrony of the hormonal milieu, particularly by increasing oestrogen levels. This causes detrimental changes to the uterine morphology and affects vascular permeability at the site of implantation. In the present study, the expression of COX2 and VEGF was compared between control and hyperstimulated rat uteri during the peri-implantation period using immunohistochemistry and Western blot analysis. While in control pregnant rats COX2 and VEGF immunolocalization occurred in the luminal epithelial cells and stroma on consecutive days, strong immunolocalization of COX2 and VEGF occurred in the luminal epithelial cells but was inhibited in the stroma of the hyperstimulated rats. This appears to have resulted in the suppression of stromal decidualization and vascular permeability. Western blot analysis did not show any results. This may be due to low concentrations of the protein in the sample. Since vascular permeability and angiogenesis are critical to the process of implantation and are influenced by VEGF and COX2, disturbance of the pattern of these two proteins by hyperstimulation may contribute to the low implantation rate in IVF programes. immunohistochemistry and Western blot analysis. While in control pregnant rats COX2 and VEGF immunolocalization occurred in the luminal epithelial cells and stroma on consecutive days, strong immunolocalization of COX2 and VEGF occurred in the luminal epithelial cells but was inhibited in the stroma of the hyperstimulated rats. This appears to have resulted in the suppression of stromal decidualization and vascular permeability. Western blot analysis did not show any results. This may be due to low concentrations of the protein in the sample. Since vascular permeability and angiogenesis are critical to the process of implantation and are influenced by VEGF and COX2, disturbance of the pattern of these two proteins by hyperstimulation may contribute to the low implantation rate in IVF programes.

In this work, we examine the structure and function of two important human proteins. The first is human kallikrein 6 (hK6), which is a newly identified enzyme in the serine proteinase family that is expressed in the central nervous system. In chapter 2, the X-ray crystal structure of mature, active recombinant human kallikrein 6 at 1.75 Å is presented. This high resolution model provides the first three-dimensional view of one of the human kallikreins and one of only a few structures of serine proteinases predominantly expressed in the central nervous system. Enzymatic and X-ray data provide support for the characterization of human kallikrein 6 as a degradative proteinase with structural features more similar to trypsin than the regulatory kallikreins. In chapter 3, we have re-solved the structure of hK6 to a resolution of 1.56 Å. In addition, a detailed analysis of the preferred substrate specificity of hK6 at the positions P3, P2, P1′, P2′, and P3′ is undertaken using internally quenched fluorescent substrates based on a peptide background sequence of the identified autolysis region. Furthermore, the identified optimized substrate sequence is modeled into the 1.56 Å structure of human kallikrein 6 using docking in order to identify structural aspects of the protein responsible for this preference. The substrate specificity data show that human kallikrein 6 displays little discrimination for particular amino acids at the tested positions with the exception of P2′, where there is a pronounced preference for proline. The second protein studied in this work is human fibroblast growth factor-1 which is a member of the β-trefoil superfold. In chapter 4, a 1.10 Å atomic-resolution x-ray structure of human fibroblast growth factor 1, a member of the β-trefoil superfold, is reported. The FGF-1 structure exhibits numerous core packing defects detectable using a 1.0Å radius probe. In addition to contributing to the relatively low thermal stability of FGF-1, these defects may also permit domain motions within the structure. The availability of refined ADP's permits a translation/libration/ screw (TLS) analysis of putative rigid body domains. The observed rigid body motion in FGF-1 appears related to the ligand-binding functionalities. / Dissertation / PhD

No indivíduo adulto, a angiogênese ocorre particularmente em situações patológicas como nos tumores em crescimento, no desenvolvimento de metástases e no processo de cicatrização, sendo o fator de crescimento do endotélio vascular (VEGF) o principal fator envolvido na angiogênese tumoral. Por essa razão, o presente estudo teve como objetivo avaliar os níveis circulantes de VEGF no soro de cães com sarcoma de tecidos moles e sua relação com as células sanguineas, comparados com um grupo de animais sem câncer (controle), a expressão do VEGF e a densidade de microvasos nos espécimes tumorais dos cães com sarcoma de tecidos moles. O grupo controle foi composto de 30 cães machos e o grupo de animais com sarcoma de tecidos moles foi de 25 cães (18 machos e 7 fêmeas castradas) os quais foram avaliados prospectivamente. A coleta sangüínea foi realizada apenas uma vez no grupo controle e em três tempos nos animais com sarcoma (pré-operatório, duas semanas e três meses de pós-operatório) da mesma maneira. Após a colheita o sangue foi processado, para extração do soro e determinação dos níveis de VEGF a partir de um método quantitativo ELISA (enzime-linked immunosorbent assay). A expressão do VEGF e a densidade de microvasos foi investigada por meio da prova de imunoistoquiímica utilizando-se anticorpos anti-VEGF e anti-fator VIII respectivemente. Não houve diferença entre o nível sérico de VEGF dos animais controles e portadores de sarcoma de tecidos moles no tempo pré-operatório. O nível de VEGF sérico no pré-operatório mostrou-se discretamente aumentado em relação a duas semanas e três meses de pós-operatório. Houve correlação positiva entre VEGF sérico e contagem neutrófilos e correlação negativa entre o VEGF e quantidade de hemoglobina nos animais com sarcoma. Houve uma tendência dos animais com hemangiopericitoma apresentarem níveis maiores de VEGF sérico em relação aos portadores de tumor maligno da bainha neural periférica. Houve expressão do VEGF em 65% dos casos, sendo o hemangiopericitoma aquele que expressou maior quantidade de VEGF intratumoral. Não houve diferença na densidade de microvasos entre os tumores negativos e positivos ao VEGF. Os resultados encontrados sugerem contribuição das células do sangue circulante para os níveis de VEGF do soro de cães portadores de sarcomas de tecidos moles, células tumorais e outros tipos celulares parecem ser responsáveis pela angiogênese tumoral, mas não contribuem para elevação da concentração sérica desse fator. / In adult individual, angiogenesis occurs particularly in pathological situations as tumors growth, development of metastasis and in the wound healing process. The vascular endothelial growth factor (VEGF) is the main agent involved in tumor angiogenesis. Therefore, the aim of the present study was to evaluate the circulating levels of VEGF in the serum of dogs suffering from soft tissue sarcoma and its relation with the blood cells, and also the expression of VEGF and the micro vascular density in tumors specimens. A group of health animals was used as control. The control group and treatment group were composed of 30 male dogs 25 dogs (18 males and 7 castrated females) respectively. The blood collection was carried once in the control group and three times in the animals with sarcoma (timely, pre-surgery, two weeks and three months post-surgery) following the same protocol. The blood was processed and a quantitative method ELISA (enzime-linked immunosorbent assay) was used to determinate of the levels of VEGF. The expression of the VEGF and the microvascular density were investigated by means of the immunohistochemical test using anti-VEGF and anti-factor VIII antibodies respectively. There was no difference between serum level of VEGF of the controls animals and serum level of VEGF in the animal with soft tissue sarcoma in pre-surgical time. Serum level of VEGF in pre-surgical time revealed discrete increased in relation the two weeks and three months of post-surgery. There was a positive correlation between serum VEGF and neutrophils counting and negative correlation between the VEGF and amount of haemoglobin in the animals with sarcoma. The animals with hemangiopericytoma showed a trend to higher levels of VEGF in relation to the malignant peripheral nerve sheath tumor. The expression of the VEGF was detected in 65% of the cases; the hemangiopericytoma is the one that expressed greater intratumoral amount of VEGF. There was no difference in the microvascular density between the negative and positive tumors to the VEGF. The results suggest contribution of the circulating blood cells for the levels of serum VEGF of the of the dogs with of soft tissue sarcomas, tumors cells and other cells types seem to be responsible for tumor angiogenesis, but they don\'t contribute for rise serum level of VEGF.

O objetivo deste trabalho foi avaliar, in vitro, os efeitos de diferentes concentrações do fator de crescimento transformador beta 1 (TGF-β1) em células-tronco derivadas da polpa de dentes decíduos esfoliados humanos (SHED), com relação à viabilidade, proliferação, migração e diferenciação celular. As SHED foram mantidas em meio de cultura MEMα + soro fetal bovino (FBS) 10% + penicilina e estreptomicina 1% e tratadas com TGF-β1 na concentração de 1,0; 5,0 e 10,0 ng/mL. Após 1, 3, 5 e 7 dias, foram avaliadas a viabilidade celular pelo método MTT e a proliferação pelo método SRB. Após 24 h de tratamento com TGF-β1, foi realizado um ensaio de migração celular por meio de insertos com poros de 8 μm. Para a avaliação da diferenciação celular de SHED em odontoblastos foram analisados por meio da RT-PCR os marcadores DSPP e DMP-1, após tratamento com TGF-β1 nas diferentes concentrações por 14 dias. Os resultados foram submetidos à ANOVA seguido do teste de Tukey. Em relação à viabilidade celular, as diferentes concentrações de TGF-β1 não tiveram efeito citotóxico sobre SHED. As células tratadas com diferentes concentrações de TGF-β1 apresentaram maiores taxas de proliferação que as do controle negativo (MEMα + 10% de FBS) a partir do 3o dia (p=0,000). Observou-se maiores taxas de migração em direção aos meios contendo TGF-β1, mas sem diferença estatisticamente significativa entre as diferentes concentrações utilizadas, entretanto, houve diferença estatisticamente significativa entre as diferentes concentrações de TGF-β1 com o controle positivo (p=0,000), controle negativo (p=0,000) e entre o controle positivo e negativo (p=0,002). A expressão de DMP-1 foi observada de forma crescente nas doses de 1,0 e 5,0 ng/mL de TGF-β1 ao longo do período (1, 7 e 14 dias) e na dose de 10,0 ng/mL a marcação foi mais intensa desde o primeiro dia do estímulo. Em relação à expressão de DSPP, o grupo tratado com 10,0 ng/mL apresentou marcação após 14 dias de tratamento. Sendo assim, este estudo permite concluir que as diferentes concentrações de TGF-β1 estimularam a proliferação e migração celular, sem efeito citotóxico sobre as células ao longo do período do estudo. Em relação à diferenciação celular a concentração 10,0 ng/mL de TGF-β1 estimulou à expressão de DMP-1 e DSPP. / The aim of this study was to evaluate, in vitro, the effect of transforming growth factor beta 1 (TGF-β1) in stem cells derived from the pulp of human exfoliated deciduous teeth (SHED) regarding to cell viability, proliferation, migration and differentiation. SHED were maintained in MEMα culture medium + 10% fetal bovine serum (FBS) + 1% penicillin and streptomycin, and treated with TGF-β1 at the following concentrations of 1.0; 5.0 and 10.0 ng/mL. After 1, 3, 5 and 7 days, cell viability was assessed by MTT assay and proliferation by the SRB method. After 24h of TGF-β1 treatment, cell migration assay was carried out using inserts of 8 μm pore size. To evaluate SHED differentiation into odontoblasts, DMP-1 and DSPP markers were analyzed by RT-PCR, after treatment at different concentrations of TGF-β1 for 14 days. The results were submitted by ANOVA and Tukey test. With respect to cell viability, the different TGF-β1 concentrations did not have cytotoxic effect on SHED. The cells treated by different TGF-β1 concentrations showed higher proliferation rates than those of the negative control (MEMα + 10% FBS) after the third day (p = 0.000). Higher rates of migration towards the media containing TGF-β1 were observed, but there were no statistically significant differences among the concentrations. All different TGF-β1 concentrations showed statistically significant differences with the positive control (p=0.000) and negative control (p=0.000). Statistically significant differences were observed between positive and negative control (p=0.002). DMP-1 expression was observed incrementally at TGF-β1 concentrations of 1.0 and 5.0 ng/mL at 1, 7, and 14 days and the concentration of 10.0 ng/mL was more intense from day one of the stimulus. DSPP expression was more intense after 14 days of treatment with the concentration of 10.0 ng/mL. Thus, this study concluded that different TGF-β1 concentrations stimulated cell proliferation and migration, without cytotoxic effect on the cells throughout the study period. From the perspective of cell differentiation, TGF-β1 concentration of 10.0 ng/mL was capable of stimulating DMP-1 and DSPP expression.

Os hemoderivados têm sido utilizados com frequência cada vez maior na medicina equina, sendo caracterizados como um produto autólogo, com maior quantidade de fatores de crescimento e que melhora a capacidade de cicatrização de tecidos com pouco aporte sanguíneo, como tendões e articulações, diminuindo o tempo de recuperação do animal. Sabe-se que os fatores de crescimento são derivados das plaquetas, porém a correlação positiva entre o aumento na contagem plaquetária e a maior concentração de fatores de crescimento ainda é motivo de discussão entre os autores. Com o intuito de se obter um produto final com maior contagem plaquetária, é frequente o aumento da velocidade ou número de centrifugações na metodologia empregada, aumentando o risco de agregação plaquetária precoce. O objetivo do presente trabalho é estudar o efeito da dupla centrifugação no preparo de PRP, por meio da comparação entre contagens plaquetárias, concentrações de fator de crescimento TGF-β1, e grau de ativação plaquetária por meio da porcentagem de agregação. Foram utilizados 12 equinos, machos, de 3 a 5 anos, clinicamente sadios. Para tanto foram realizados dois protocolos distintos: um com centrifugação única e o outro com dupla centrifugação. No primeiro, o sangue com anticoagulante foi centrifugado a 141G/12 minutos; enquanto no segundo a primeira centrifugação foi de 300G/5 minutos seguida de 700G/15 minutos, com repouso entre as mesmas e após. Os produtos obtidos após cada centrifugação foram submetidos à contagem plaquetária, teste de agregação e quantificação de TGF--β1 por meio de kit ELISA. Os resultados obtidos demonstraram maior concentração plaquetária quando utilizado protocolo de dupla centrifugação. Agregometria evidenciou maior ativação das plaquetas durante o preparo do PRP quando submetidas a maiores velocidades de centrifugação (força gravitacional) e não ao fato das amostras serem centrifugadas duas vezes. A quantificação do TGF--β1 não mostrou diferença quando realizado em amostras com apenas uma centrifugação, mas demonstrou valores maiores no produto final da segunda centrifugação. A avaliação por meio de coeficiente de determinação e coeficiente de correlação de Pearson evidenciou correlação positiva entre contagem plaquetária e de TGF--β1. O protocolo com dupla centrifugação se mostrou mais eficaz em concentrar plaquetas e TGF--β1, não sendo prejudicado pela ativação precoce dessas plaquetas durante o preparo. / Blood derived products have been used in equine medicine with increasing frequency, being characterized as an autologous product, with greater amount of growth factors and be capable of improvement the healing capacity in tissues with poor blood supply, such as tendons and joints, reducing the time of recovery of the animal. It is known that the growth factors are derived from platelets, but the positive correlation between the increase in platelet count and the higher concentration of growth factors is still a reason for discussion among the authors. In order to obtain a final product with a higher platelet count, it is frequent to increase the speed or number of centrifugations in the methodology employed, increasing the risk of early platelet aggregation. The aim of the present study is to verify the effect of double centrifugation in PRP preparation by comparing platelet counts, TGF-β1 growth factor concentrations, and degree of platelet activation through percentage of aggregation. Twelve horses, male, aged 3 to 5 years-old, clinically healthy were subjected. Two different protocols were performed: one with single centrifugation and the other with double centrifugation. In the first one, the anticoagulated blood was centrifugated at 141G/12 minutes; while in the second one the first centrifugation was 300G/5 minutes followed by 700G/15 minutes, with rest between them and after. The products obtained after each centrifugation were submitted to platelet counting, aggregation test and measurement of TGF-β1 by ELISA kit. The results showed a higher platelet concentration when double centrifugation protocol was used. The aggregometry test evidenced a greater activation of the platelets during the preparation of PRP when submitted to higher centrifugation velocities (times g), and not to double centrifugation. Quantification of TGF-β1 showed no difference when performed on samples with only one centrifugation, but was higher values in the final product of the second centrifugation. The determination coefficient and Pearsons correlation coefficient showed a positive correlation between the platelet count and TGF-β1 concentration. The double centrifugation protocol proved to be more effective at concentrating platelets and consequently higher amounts of TGF-β1, not being impaired by early activation during obtainment.

Um dos grupos de doenças mais importante que acomete o desenvolvimento da caixa craniana humana é o das craniossinostoses, caracterizado pelo fechamento prematuro de uma ou mais suturas cranianas. Entre as formas mendelianas das craniossinostoses sindrômicas, mutações dominantes em FGFR2 são uma das causas mais frequentes e estão associadas às síndromes de Apert, de Crouzon e de Pfeiffer. A sinalização intracelular subseqüente à ativação de FGFR2, tanto selvagem quanto mutante, é bastante intrincada e pode sofrer inúmeras bifurcações. As porções iniciais destas vias, imediatamente subsequentes à ativação do receptor, são relativamente bem compreendidas. Grande parte, porém, do controle dessas vias, principalmente no que tange a regulação transcricional e sua associação com alterações em comportamentos celulares, não é entendido. Assim sendo, os objetivos gerais deste trabalho foram: 1) estudar o potencial de diferenciação e o perfil diferencial de transcrição gênica de culturas primárias de células fibroblastóides isoladas a partir do periósteo das suturas coronais de pacientes acometidos por síndrome de Apert (heterozigotos para a mutação de ganho de função p.Ser252Trp em FGFR2, a mutação mais comum em pacientes com esta síndrome) e 2) estudar o potencial de diferenciação osteogênico e o perfil transcricional respectivamente de células mesenquimais e de tecido provenientes de sutura coronal de um modelo murino para Síndromes de Crouzon/Pfeiffer (heterozigotos para a mutação p.Cys342Tyr em Fgfr2, a mutação mais comum associada a estas síndromes). Certificamo-nos da expressão gênica e proteica de FGFR2 nas células fibroblastóides humanas e de Fgfr2 nas células mesenquimais murinas. Em seguida, testamos o potencial osteogênico (in vitro e in vivo ) e adipogênico (in vitro ) das células de pacientes com Síndrome de Apert, comparadas a células do mesmo tecido mas de indivíduos sem esta mutação e o potencial osteogênico (in vitro ) das células mesenquimais de camundongos portadores da mutação p.Cys342Tyr em Fgfr2, comparadas a células também das suturas coronais mas de animais selvagens. O potencial de diferenciação das células mutantes, nos dois grupos de experimentos, foi muito aumentado em relação ao potencial das células livres destas mutações. Conduzimos experimentos de microarrays de expressão gênica (sistema CodeLink) com 7 amostras de culturas primárias de células de pacientes com S. de Apert e as comparamos com 7 amostras de culturas primárias controles. Identificamos 263 genes com valores de expressão estatisticamente diferentes (SNR ≥ |0.4|, P ≤ 0,05) nas amostras de pacientes com S. de Apert quando comparadas às controles (118 superexpressos, 145 subexpressos). Categorias funcionais enriquecidas foram regulação de proliferação celular, metabolismo de nucleotídeos, regulação de expressão gênica, adesão celular, organização de matriz extracelular e cascata PI3K MAPK. Para a validação deste experimento constatamos superexpressão, por PCR em tempo real, de genes identificados como superexpressos na assinatura de expressão associada às células mutadas, além de verificarmos o mesmo comportamento destes genes em células controles tratadas com FGF2 exógeno para superativação do receptor. Os experimentos de expressão gênica com os tecidos de suturas coronais do modelo murino foram feitos com 15 amostras de tecidos de animais mutantes em 3 grupos de 5 e comparadas a amostras de mesmo tecido de animais selvagens agrupadas da mesma forma. Identificamos três listas de genes diferencialmente expressos: a primeira contendo 188 transcritos (P ≤0,05, FC ≥ 1,5,sendo 91 superexpressos e 97 subexpressos), e as outras duas filtradas previamente para coeficiente de variação < 50% dentro de cada grupo, contendo 488 transcritos (P ≤0,05, FC ≥ 1,2, sendo 183 superexpressos e 305 subexpressos) e 31 transcritos (P ≤0,05, FC ≥ 1,5, sendo 11 superexpressos e 20 subexpressos). Categorias funcionais mais enriquecidas foram crescimento, proliferação e ciclo celular, diferenciação celular, sinalização célula-célula, resposta imune mediada por células e sinalização por receptor Wnt. Estes resultados nos permitiram: a) demonstrar que células fibroblastóides de periósteo craniano de paciente portadores de S. de Apert (mutação p.Ser252Trp em FGFR2) e células mesenquimais do modelo murino para S. de Crouzon e Pfeiffer, portador da mutação p.Cys342Tyr em Fgfr2, apresentam potencial osteogênico aumentado, agregando evidências que sugerem que esta alteração de comportamento celular tem função fundamental no desencadeamento das craniossinostoses nestas síndromes; b) revelar assinaturas de expressão gênicas associadas a estas mutações nas condições estudadas, que podem reger este comportamento celular anormal; c) identificar um novo grupo de genes associados à patofisiologia da Síndrome de Apert ou às características fenotípicas do modelo murino investigado, podendo também ser genes candidatos a outras craniossinostoses de causa desconhecida. / Craniosynostosis is one of the most important group of diseases linked to the development of the human skull and is characterized by the premature fusion of one or more cranial sutures. Dominant mutations in FGFR2 are frequent molecular causes amongst the mendelian inherited forms of the syndromic craniosynostosis and are associated to Apert, Crouzon and Pfeiffer syndromes. The intracellular signaling pathways following the activation of wild type or mutant FGFR2 are very complex due to several possible bifurcations. The initial portions of these pathways, immediately following the receptor activation, are relatively well delineated. However the great majority of the events related to the control of these pathways is still not well understood, mainly concerning its transcriptional regulation and its association to other cell behavior anomalies. Therefore the key scopes of this work were: 1) to study the differentiation potential and the differential gene expression profile of primary fibroblastoid cell cultures isolated from the periosteum of the coronal sutures of Apert Syndrome patients (heterozygous for the mutation p.Ser252Trp in FGFR2, the most common cause of the Apert Syndrome condition) and 2) to study the osteogenic differentiation potential and the transcriptional profile of mesenchymal cells and tissue isolated from the coronal sutures of a mouse model for the Crouzon and Pfeiffer Syndromes (heterozygous for the p.Cys342Tyr mutation in Fgfr2, the mutation most commonly associated to these syndromes). We assured the FGFR2 /FGFR2 gene and the protein expression in human fibroblastoid cells and Fgfr2 /Fgfr2 expression in the mesenchymal murine cells. We tested the (in vitro and in vivo ) osteogenic and the (in vitro ) adipogenic potentials of the Apert Syndrome patients cells compared to cells from the same tissue but from subjects without this mutation and the (in vitro ) osteogenic potential of mesenchymal cells from mice bearing the p.Cys342Tyr mutation in Fgfr2 compared to coronal suture cells but from wild type mice. On both experiments the differentiation potential of the mutant cells were very increased when compared to the potential of the wild type cells. We conducted gene expression microarray experiments (CodeLink system) using 7 samples from primary cultures of cells from Apert Syndrome patients compared to 7 samples from primary control cultures. We identified 263 genes with significantly different expression (SNR ≥ |0.4|, P ≤ 0,05) associated to the Apert Syndrome profile (118 upregulated, 145 downregulated). Enriched functional cathegories were regulation of cell proliferation, nucleotide metabolism, gene expression regulation, cell adhesion, extracellular matrix organization and PI3K MAPK cascades. In order to validate this gene expression signature we confirmed through Real-Time PCR the upregulation of genes identified as upregulated in the Apert cell profile in samples from the microarray experiment and in control cells treated with exogenous overactivate the receptor. The gene expression experiments with the coronal suture tissues from the mouse model were performed with 15 samples of mutant animal tissue in 3 groups of 5 and compared to samples from the same tissue of wild type animals, with identical grouping. We identified three sets of differentially expressed genes: the first set containing 188 transcripts (P ≤0,05, FC ≥ 1,5, 91 upregulated e 97 downregulated), and the other two filtered for coeficient of variation < 50% in each group, containing 488 transcripts (P ≤0,05, FC ≥ 1,2, sendo 183 upregulated and 305downregulated) e 31 transcripts (P ≤0,05, FC ≥ 1,5, 11 upregulated and 20 downregulated). The most enriched functional categories were growth, proliferation and cell cycle, cell differentiation, cell-to-cell signaling, cell mediated immune response and Wnt receptor signaling. These results allowed us: a) to demonstrate that fibroblastoid cells from coronal periosteum PF Apert Syndrome patients (p.Ser252Trp mutation in FGFR2) and mesenchymal cells from the coronal tissue of the mouse model for Crouzon and Pfeiffer syndromes (bearing the p.Cys342Tyr in Fgfr2) have enhanced osteogenic potential, summoning evidences suggesting that this cell behavior alteration have a fundamental role to the craniosynostotic process in these syndromes; b) to unravel gene expression signatures linked to these mutations in the studied conditions, that could orchestrate this abnormal cell behavior; c) to identify a ser of genes associated to the pathophysiology of Apert Syndrome and to the phenotypic characteristics of the animal model investigated, which might be candidate genes to other craniosynostosis of unknown cause.

The primary purpose of this study was to investigate the effects of aerobic and resistance training on BDNF and IGF-I expression in a 3xTg-AD mouse model of Alzheimer’s disease. Twenty-four 3xTg-AD mice were randomly assigned to either an aerobic (AT, n=8), resistance (RT, n=8), or control (CNT, n=8) group. Intervention groups underwent 9 weeks of exercise training. Motor behavior and grip strength were measured pre- and post- intervention. Our results showed a significant increase in hippocampal BDNF expression in AT mice after a 9-week intervention. Further, AT mice were found to have higher concentrations of IGF-I, and improved motor behavior when compared to RT and CNT. No significant differences were observed in IGF-I concentration between RT and other groups. RT improved grip strength after nine weeks of training. These findings support the use of AT and RT as a tool to improve comorbidities found in Alzheimer’s disease. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

Obesity is associated with elevated levels of the pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), contributing to systemic insulin resistance. Fibroblast growth factor 21 (FGF21) is a vital metabolic and inflammatory regulator, however circulating FGF21 concentrations are elevated in obese individuals. Acute aerobic exercise increases systemic FGF21 in normal-weight individuals, however the effect of acute aerobic exercise on plasma FGF21 response and the relationships with inflammation (IL-6 and TNF-α), insulin resistance, and energy expenditure in obese individuals is unknown. Following 30 minutes of treadmill running at 75% VO2max, plasma FGF21 response, as indicated by area-under-the-curve “with respect to increase” (AUCi) analyses, was attenuated in 12 obese compared to 12 normalweight subjects. Additionally, FGF21 AUCi positively correlated with glucose AUCi, total relative energy expenditure, and relative VO2max, suggesting that cardiorespiratory fitness levels may predict FGF21 response, contributing to the enhanced regulation of glucose and energy metabolism. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2014. / FAU Electronic Theses and Dissertations Collection

Pang Yee Man Flora. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 111-128). / Abstracts in English and Chinese. / Abstract (in English) --- p.ii / Abstract (in Chinese) --- p.iv / Acknowledgement --- p.v / Table of Contents --- p.vii / List of Figures --- p.xii / Symbols and Abbreviations --- p.xv / Scientific Names --- p.xvii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Vitellogenesis --- p.2 / Chapter 1.2 --- Vitellogenin --- p.3 / Chapter 1.2.1 --- Structure --- p.3 / Chapter 1.2.2 --- Vitellogenin synthesis in the liver --- p.4 / Chapter 1.3 --- Regulation of vitellogenin synthesis --- p.5 / Chapter 1.3.1 --- Estradiol --- p.5 / Chapter 1.3.1.1 --- Mechanism of action --- p.6 / Chapter 1.3.1.2 --- Estradiol-stimulated vitellogenin expression --- p.7 / Chapter 1.3.1.3 --- Memory effects --- p.9 / Chapter 1.3.2 --- Testosterone --- p.10 / Chapter 1.3.3 --- Cortisol --- p.13 / Chapter 1.3.4 --- Progesterone --- p.14 / Chapter 1.3.5 --- Growth Hormone --- p.14 / Chapter 1.3.6 --- Prolactin --- p.15 / Chapter 1.3.7 --- Thyroid hormone --- p.15 / Chapter 1.4 --- Growth factors --- p.16 / Chapter 1.4.1 --- Activin --- p.16 / Chapter 1.4.1.1 --- Structure --- p.16 / Chapter 1.4.1.2 --- Functions --- p.17 / Chapter 1.4.2 --- Epidermal growth factors (EGF) --- p.18 / Chapter 1.4.2.1 --- Structure --- p.18 / Chapter 1.4.2.2 --- Functions --- p.19 / Chapter 1.5 --- Objectives of the present study --- p.20 / Chapter Chapter 2 --- Expression of Goldfish Vitellogenin in vivo and in vitro --- p.25 / Chapter 2.1 --- Introduction --- p.25 / Chapter 2.2 --- Materials and Methods --- p.26 / Chapter 2.2.1 --- Materials --- p.26 / Chapter 2.2.2 --- Sequencing --- p.27 / Chapter 2.2.3 --- Cell culture --- p.28 / Chapter 2.2.4 --- RNA extraction --- p.29 / Chapter 2.2.5 --- Northern hybridization --- p.31 / Chapter 2.2.6 --- Slot blot hybridization --- p.32 / Chapter 2.2.7 --- Data analysis --- p.33 / Chapter 2.2.8 --- SDS-PAGE analysis --- p.33 / Chapter 2.2.9 --- in situ hybridization --- p.34 / Chapter 2.3 --- Results --- p.37 / Chapter 2.3.1 --- Validation of vitellogenin mRNA detection --- p.37 / Chapter 2.3.2 --- Basal and estradiol-stimulated vitellogenin expression and production invivo --- p.38 / Chapter 2.3.3 --- Localization of vitellogenin expression in the liver --- p.39 / Chapter 2.3.4 --- Expression of vitellogenin in vitro --- p.40 / Chapter 2.4 --- Discussion --- p.54 / Chapter Chapter 3 --- Effects of Steroids on the Expression of Goldfish Vitellogenin in vitro --- p.60 / Chapter 3.1 --- Introduction --- p.60 / Chapter 3.2 --- Materials and Methods --- p.62 / Chapter 3.2.1 --- Materials --- p.62 / Chapter 3.2.2 --- Animal --- p.62 / Chapter 3.2.3 --- Primary culture of dispersed hepatic cells --- p.62 / Chapter 3.2.4 --- Drug treatment --- p.64 / Chapter 3.2.5 --- Total RNA isolation --- p.64 / Chapter 3.2.6 --- Messenger RNA isolation --- p.65 / Chapter 3.2.7 --- Slot blot analysis --- p.66 / Chapter 3.2.8 --- Data analysis --- p.68 / Chapter 3.2.9 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.68 / Chapter 3.2.10 --- Cloning of aromatase cDNA --- p.69 / Chapter 3.2.11 --- Sequencing --- p.70 / Chapter 3.3 --- Results --- p.71 / Chapter 3.3.1 --- Effect of 17-β estradiol on vitellogenin mRNA expression --- p.71 / Chapter 3.3.2 --- Effect of testosterone on vitellogenin mRNA expression --- p.71 / Chapter 3.3.3 --- Detection of aromatase mRNA expression in the liver by RT-PCR --- p.72 / Chapter 3.3.4 --- Effect of aromatase inhibitors on testosterone-stimulated vitellogenin expression --- p.73 / Chapter 3.4 --- Discussion --- p.81 / Chapter Chapter 4 --- Effects of Epidermal Growth Factor (EGF) and Activin on the Expression of Vitellogenin in the Goldfish Hepatic Cells in vitro --- p.86 / Chapter 4.1 --- Introduction --- p.86 / Chapter 4.2 --- Materials and Methods --- p.88 / Chapter 4.2.1 --- Materials --- p.88 / Chapter 4.2.2 --- Primary culture of dispersed hepatic cells --- p.89 / Chapter 4.2.3 --- Slot blot analysis --- p.91 / Chapter 4.2.4 --- Data analysis --- p.91 / Chapter 4.3 --- Results --- p.92 / Chapter 4.3.1 --- Effect of activin on vitellogenin mRNA expression --- p.92 / Chapter 4.3.2 --- Effect of EGF and TGF-α on vitellogenin mRNA expression --- p.93 / Chapter 4.4 --- Discussion --- p.99 / Chapter Chapter 5 --- General Discussion --- p.104 / Chapter 5.1 --- Overview --- p.104 / Chapter 5.2 --- Contribution of the present study --- p.106 / Chapter 5.2.1 --- Expression of goldfish vitellogenin in vivo and in vitro --- p.106 / Chapter 5.2.2 --- Effects of steroids on the expression of goldfish vitellogenin in vitro --- p.106 / Chapter 5.2.3 --- Effects of EGF and activin on the expression of vitellogenin in the goldfish hepatic cells in vitro --- p.107 / Chapter 5.3 --- Future prospects --- p.108

by Chung Lung Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 149-157). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abbreviations --- p.ii / Abstracts --- p.v / List of figures --- p.ix / List of tables --- p.xii / Contents --- p.xiii / Contents / General Introduction --- p.1 / Chapter CHAPTER ONE --- CISPLATIN RESISTANCE MECHANISMS / Chapter 1.1 --- INTRODUCTION --- p.3 / Chapter 1.1.1 --- History of Cisplatin as An Anticancer Drug --- p.3 / Chapter 1.1.2 --- Active Mechanisms of Cisplatin --- p.8 / Chapter 1.1.3 --- Formation of DNA Adducts --- p.8 / Chapter 1.1.4 --- Cisplatin Resistance Mechanisms --- p.9 / Chapter 1.1.4.1 --- Intracellular Accumulation of Cisplatin --- p.11 / Chapter 1.1.4.2 --- Glutathione-S-transferase and Glutathion --- p.12 / Chapter 1.1.4.3 --- Metallothionein --- p.16 / Chapter 1.1.4.4 --- Cell Cycle Perturbation --- p.16 / Chapter 1.1.4.5 --- P-glycoprotein --- p.17 / Chapter 1.1.4.6 --- Multidrug Resistant Protein --- p.19 / Chapter 1.1.4.7 --- Topoisomerase II --- p.20 / Chapter 1.1.4.8 --- DNA Repair --- p.22 / Chapter 1.1.4.9 --- Induction of Programme Cell Death --- p.23 / Chapter 1.2 --- OBJECTIVES --- p.27 / Chapter 1.3 --- MATERIALS AND METHODS / Chapter 1.3.1 --- Materials --- p.28 / Chapter 1.3.2 --- Methods --- p.31 / Chapter 1.3.2.1 --- Cell Lines --- p.31 / Chapter 1.3.2.2 --- Drug Sensitivity Assay --- p.31 / Chapter 1.3.2.3 --- Platinum Uptake --- p.32 / Chapter 1.3.2.4 --- Cell Cycle Analysis --- p.32 / Chapter 1.3.2.5 --- Western Blot Analysis --- p.33 / Chapter 1.3.2.6 --- Glutathione Content Determination --- p.36 / Chapter 1.3.2.7 --- DNA Fragmentation --- p.36 / Chapter 1.3.2.8 --- JC-1 Staining --- p.37 / Chapter 1.3.2.9 --- HE and DCF Staining --- p.38 / Chapter 1.3.2.10 --- Quantitative RT-PCR --- p.38 / Chapter 1.4 --- RESULTS / Chapter 1.4.1 --- Cisplatin Sensitivity of A431 Cells by MTT Assay --- p.40 / Chapter 1.4.2 --- Cross-resistance to Anti-cancer Drugs --- p.40 / Chapter 1.4.3 --- Quantitation of Cisplatin Accumulation in A431 Cells by AAS --- p.44 / Chapter 1.4.4 --- Drug Detoxification Agent --- p.45 / Chapter 1.4.5 --- Detection of Cell Cycle Arrest by Flow Cytometer --- p.47 / Chapter 1.4.6 --- Expression of Drug Resistance Related Genes --- p.48 / Chapter 1.4.7 --- Detection of Apoptosis by DNA Fragmentation --- p.50 / Chapter 1.4.8 --- Role of Mitochondria and Reactive Oxygen Species by Flow Cytometer --- p.52 / Chapter 1.4.9 --- Detection of Apoptotic mRNA Level by Quantitative RT-PCR --- p.57 / Chapter 1.4.10 --- Detection of Apoptotic Protein Level by Western Blot Analysis --- p.57 / Chapter 1.5 --- DISCUSSIONS --- p.59 / Chapter CHAPTER TWO: --- THE INTERACTION BETWEEN DRUG RESISTANCE MECHANISMS AND GROWTH FACTOR SIGNALLING PATHWAY / Chapter 2.1 --- INTRODUCTION --- p.63 / Chapter 2.1.1 --- Structure of EGF and EGFR --- p.64 / Chapter 2.1.2 --- Growth Factor Signal Transduction Pathway --- p.69 / Chapter 2.1.3 --- Biological Effect of EGF --- p.69 / Chapter 2.1.3.1 --- Modification of Drug Sensitivity by EGF --- p.71 / Chapter 2.2 --- OBJECTIVES --- p.74 / Chapter 2.3 --- MATERIALS AND METHODS / Chapter 2.3.1 --- Materials --- p.75 / Chapter 2.3.2 --- Methods / Chapter 2.3.2.1 --- Cell Lines --- p.76 / Chapter 2.3.2.2 --- Drug Sensitivity Assay --- p.77 / Chapter 2.3.2.3 --- Northern Blot Analysis --- p.77 / Chapter 2.3.2.4 --- Southern Blot Analysis --- p.78 / Chapter 2.3.2.5 --- Others --- p.78 / Chapter 2.4 --- RESULTS / Chapter 2.4.1 --- Sensitivity to EGF --- p.79 / Chapter 2.4.2 --- EGFR Expression Levels --- p.80 / Chapter 2.4.3 --- EGF Induced Protein Phosphorylation Pattern --- p.84 / Chapter 2.4.4 --- Effect of EGF on A431 Cells --- p.86 / Chapter 2.4.5 --- Response of Cells to Agents Targeting on EGF Signalling Pathway --- p.91 / Chapter 2.4.6 --- Response of Cells to Other Growth Factors --- p.97 / Chapter 2.4.7 --- Sensitivity of Cells to Different Anti-cancer Drugs --- p.99 / Chapter 2.4.8 --- Drug Resistance Mechanisms --- p.103 / Chapter 2.4.9 --- 5-Fluorouracil Sensitivity in A431 Cells --- p.108 / Chapter 2.4.10 --- Cisplatin Sensitivity in A431 Cells --- p.113 / Chapter 2.5 --- DISCUSSIONS --- p.117 / Chapter CHAPTER THREE: --- IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENE IN A431 CELLS BY DIFFERENTIAL DISPLAY / Chapter 3.1 --- INTRODUCTION --- p.122 / Chapter 3.2 --- MATERIALS AND METHODS / Chapter 3.2.1 --- Materials --- p.128 / Chapter 3.2.2 --- Methods / Chapter 3.2.2.1 --- Identification of Differentially Expressed Genes by RT-PCR / Chapter 3.2.2.2 --- Cloning of a Differentially Expressed cDNAs --- p.129 / Chapter 3.2.2.3 --- Screening and Sequencing of cDNA Inserts --- p.130 / Chapter 3.2.2.4 --- Rapid Amplification of cDNA Ends (RACE) --- p.131 / Chapter 3.2.2.5 --- Amplifcation Reaction --- p.131 / Chapter 3.2.2.6 --- Cloning and Sequencing of the RACE Fragment --- p.132 / Chapter 3.3 --- RESULTS / Chapter 3.3.1 --- Identification of novel cDNA by mRNA differential display --- p.133 / Chapter 3.4 --- DISCUSSIONS --- p.145 / General Conclusion --- p.147 / References --- p.149