Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

A temperatura é um fator abiótico que exerce profundos efeitos no funcionamento dos ecossistemas marinhos, influenciando na biologia das espécies. Os peixes são um bom modelo para se estudar este fator, pois são pecilotérmicos e possuem importância comercial e ecológica. O objetivo deste trabalho foi analisar o comportamento, a tolerância térmica e os mecânicos moleculares de juvenis de peixe pampo Trachinotus carolinus em relação à elevação da temperatura. Para isso, foram submetidos ao aumento controlado da temperatura da água (2°C/h) a partir da temperatura de coleta (22°C) até 26°C, 30°C, 32°C, 34°C e 36°C e nestas foram mantidos por 120 minutos, ou foram aquecidos até a morte no estudo de tolerância térmica. O comportamento foi analisado usando-se vídeos e as expressões de Hsp70 e p53 nas brânquias e no coração dos pampos foram obtidas por meio da técnica de imuno-histoquímica Os resultados mostraram que os pampos comportam-se de forma agitada com o aquecimento até a temperatura de 36°C, e a partir dela apresentam uma desorganização dos sistemas e órgãos. A expressão de Hsp70 e p53 está relacionada ao aumento de temperatura até 34°C e decai nas temperaturas de 36°C e TCS. Assim, conclui-se que o aumento de temperatura afeta o comportamento e a expressão de Hsp70 e p53. / Temperature is an abiotic factor that influences the biology of marine species. Fishes are a good model to study temperature because they are poikilothermic and have ecological and commercial importance. The aim of this study was to analyze the behavior and study the expression of Hsp70 and p53 using the immunohistochemistry assay. The Expression of these two protein was observed in relation to temperature rise in heart and gills tissues of juvenile pompano fish Trachinotus carolinus. The Fishes were exposed to controlled increase of the water temperature (2°C/h), from 22°C (control) to 26°C, 30°C, 32°C, 34°C and 36°C and they were maintained in these temperature for 120 minutes . After that fishes were maintained in these temperature for 120 minutes. In addition, experiments were performed to determinate the critical temperature survival (TCS). The results revealed that the expression of Hsp70 and p53 were proportional to the temperature rise until 34°C and that it declines at temperatures of 36°C and TCS. The fishes behavior was frantic with heating until a temperature of 36°C and subsequently they have showed a disorganization of systems and organs. We concluded that the temperature increase affects the behavior and expression of p53 and Hsp70 in pompanos fishes.

O fosfato inorgânico é um composto essencial por estar relacionado com diversos processos metabólicos e biossíntese de moléculas relevantes à sobrevivência celular. Em bactéria são conhecidos dois tipos principais de transportadores específicos de fosfato inorgânico, o sistema Pit, de baixa afinidade, e o sistema Pst de alta afinidade, um ABC transportador, ativado sob carência de fosfato extracelular. A interação deste sistema com a fisiologia e patogênese de Streptococcus mutans, o principal agente etiológico da cárie dental, foi estudada neste trabalho. Os genes que codificam o sistema Pst de S. mutans UA159, estão organizados em um óperon policistrônico (pstS, C1, C, B, smu.1134 e phoU). A análise das sequências de aminoácidos das proteínas do sistema Pst de S. mutans com ortólogos do gênero Streptococcus, demonstrou maior identidade com bactérias da cavidade oral. Análise de prevalência do gene pstS, que codifica a proteína ligadora, demonstrou conservação entre todas as cepas laboratoriais e clínicas avaliadas. A deleção de pstS reduziu a capacidade de incorporação de ortofosfato que se refletiu na diminuição da taxa de replicação celular em meios com diferentes concentrações de fosfato inorgânico. A mutação também reduziu a capacidade da bactéria em aderir à superfícies abióticas e diminuiu sua hidrofobicidade de superfície, além de aumentar a resistência intrínseca a ácido. No entanto, não houve alteração na eficiência de transformação bacteriana. Por fim, o gene pstS de S. mutans foi clonado, expresso e a proteína purificada utilizada para obter anticorpos que inibiram o crescimento de S. mutans in vitro. Deste modo, segere-se que o sistema Pst é relevante na fisiologia e patogênese de S. mutans. / Inorganic phosphate is an essential compound related to several metabolic process and biosynthesis of molecules relevant to cellular survival. Bacteria are known to posses two main types of specific inorganic phosphate transporters, the low-affinity Pit system, and the high affinity Pst system, an ABC transporter family constituent, activated by extracellular phosphate starvation. The aim of this work was to study the role of Pst system in physiology and pathogenesis of Streptococcus mutans, the main etiological agent of dental caries. The genes of the Pst system of S. mutans UA159, are organized in a polycistronic operon (pstS, C1, C, B, smu.1134 and phoU). The amino acid sequence analysis of Pst proteins of S. mutans related to orthologs of Streptococcus genus, showed the highest identity value when compared to oral cavity bacteria. The PstS binding protein was present in all tested clinical and laboratory strains of S. mutans. The pstS mutant showed a decreased capacity of ortophosphate uptake which leaded to growth deficiency in different phosphate concentrations. The mutation also reduced the bacteria adherence to abiotic surfaces and the hydrophobicity of the cell surface, diverging from the increase the intrinsic acid resistance. Otherwise, there was no change in the bacterial transformation eficiency. Finally, the S. mutans pstS gene was cloned, expressed and the purified protein was used to obtain antibodies that inhibited the growth of S. mutans in vitro. Taking together, these results suggesting that the Pst transport system is relevant to S. mutans physiology and pathogenesis.

Les thérapies ciblées sont des agents dont le but est d’inhiber une voie oncogénique spécifiquement activée dans une tumeur. Ces thérapies peuvent donc cibler l’angiogenèse tumorale, les voies de signalisation cellulaire, la prolifération infinie, l’anti-apoptose ou le pouvoir métastatique. Outre leur effet en monothérapie, leur intérêt repose également sur leur combinaison avec les traitements standards, chimiothérapie, radiothérapie ou même chirurgie. Dans ce contexte, l’association des thérapies ciblées et de la radiothérapie paraît séduisante. En effet, les mécanismes qui sous-tendent la sensibilité ou la résistance à une irradiation sont maintenant mieux connus et permettent d’envisager des manipulations thérapeutiques afin d’améliorer encore les résultats d’une radiothérapie ou d’une radiochimiothérapie. L’objectif de cette thèse était d’exploiter les données d’histologie moléculaires des carcinomes pulmonaires pour évaluer de nouvelles approches de radiosensibilisation basée sur l’inhibition de signaux de survie en ciblant les protéines Bcl-2 et Bcl-XL et la voie IGF-1 dans les carcinomes à petites cellules (CPC) et en ciblant la voie EGFR dans les carcinomes non à petites cellules du poumon (CPNPC). L’un des objectifs était également d’intégrer une méthodologie de combinaison plus précise compte-tenu des discordances très fréquemment observées entre les effets pré-cliniques d’une association et les résultats des études cliniques de la même combinaison. Dans une première partie, nous avons cherché à inhiber les mécanismes antitapoptotiques mis en jeu dans les CPC au moyen de deux nouvelles classes de thérapies ciblées : un oligonucléotide antisens ciblant l’ARNm du gène BCL2 (oblimersen) et un « BH3 mimetic » inhibiteur des protéines Bcl-2/Bcl-XL (S44563). Nous avons démontré dans ces deux études l’intérêt du ciblage de « l’anti-apoptose » pour radiosensibliser les lignées de CPC. En utilisant le S44563, un « BH3 mimetic » qui induit une apoptose via la voie mitochondriale dans les lignées qui surexpriment les cibles à savoir Bcl-2 et Bcl-XL,, nous avons montré que l’inhibition de Bcl-2 et Bcl-XL permettait d’induire une radiosensibilisation par induction de l’apoptose. Le mécanisme d’interaction reposait probablement par une induction de l’expression des protéines anti-apoptotiques, en particulier Bcl- XL à la suite d’une irradiation via l’activation de la voie NF-κB. Ceci est confirmé par les études de séquence montrant que l’administration pendant et après l’administration de la radiothérapie est plus efficace réalisant ainsi une chimiosensibilisation sous l’effet d’une irradiation rendant les cellules tumorales plus dépendantes (concept d’addiction oncogénique contextuelle) aux mécanismes de résistance à l’apoptose. Dans une seconde partie, nous avons également montré que l’inhibition d’une voie de signalisation cellulaire (la voie IGF-1) permettait également d’obtenir une radiosensibilisation tumorale validant ainsi l’intérêt de ces combinaisons. En particulier, L’anticorps monoclonal ciblant IGF-1R, le R1507 augmente l’efficacité du cisplatine et au final améliore l’efficacité de la radio-chimiothérapie dans plusieurs lignées de CPC via la diminution de l’expression de IGF-1R avec pour conséquence une diminution de l’activation de ses effecteurs, en particulier AKT, indiquant que le R1507 augmente la radiosensibilité en supprimant l’activation de IGF-1R secondaire à l’irradiation. Cependant, nous avons montré par différentes approches d’étude du transcriptome que les cellules tumorales traitées par le R1507 pendant 4 semaines s’adaptaient en ré-exprimant IGF-1R, en activant p-IRS1 et en activant différentes voies oncogéniques telles que l’angiogenèse ou d’autres voies de signalisation cellulaire. Ces résultats suggèrent donc les limites d’un schéma adapté chez l’homme lors d’une radio-chimiothérapie concomitante et plaident pour une association précoce à d’autres thérapies ciblées. / Targeted therapies are drugs that block a specific molecular target involved in following alterations in cell physiology: growth signal self-sufficiency, insensitivity to growth-inhibitory signals, evasion of apoptosis, an unlimited replicative potential, sustained angiogenesis, tissue invasion, and metastasis. Although these compounds showed efficacy when given alone, there is now a rationale to combine these agents with other antitumor therapies such as chemotherapy, radiation and surgery. In this context, there is compelling data supporting the association between targeted therapies and radiation. The better understanding of mechanisms of sensitivity or resistance to radiation may help to envision new strategies to improve its efficacy. The primary goal of this work was to assess new strategies of radiosensitization based on molecular characteristics of both small cell lung cancer (by targeting Bcl-2 and Bcl- XL proteins as well as IGF-I pathway) and non-small cell lung cancer (by targeting EGFR pathway). The second objective was also to assess new methods to better investigate combination of radiation with new targeted therapies. In the first part of the work, we evaluated the impact of the inhibition of BCL-2 in small cell lung cancer cell lines with oblimersen, an antisense BCL-2 oligodeoxynucleotide and with a small peptide BH3 mimetic, S44563 which targets both Bcl-2 and Bcl- XL proteins. We showed that inhibiting anti-apoptotic mechanisms could enhance radiosensitivity of SCLC cells. S44563 caused SCLC cells to acquire hallmarks of apoptosis through activation of the mitochondrial pathway in Bcl2 and Bcl- XL overexpressing cell lines. S44563 markedly enhanced the sensitivity of SCLC cells to radiation in both in vitro and in vivo assays through apoptosis induction. This positive interaction was explained by the induction of radiation-induced anti-apopototic proteins, mainly Bcl- XL by the NF-κB pathway. These data were confirmed by in vivo experiments showing that the radiosensitization was greater when S44563 was given after the completion of the radiation in the context of radiation-induced oncogenic addiction. In the second part of the work, we showed that IGF-1R targeting increases the antitumor effects of DNA-damaging agents in SCLC model. R1507 (a monoclonal antibody directed against IGF-1R), exhibited synergistic effects with both cisplatin and IR in SCLC cell lines through IGF-IR downregulation and reduced activation of downstream AKT. However, we observed a transient reduction of IGF-1R staining intensity in vivo, concomitant to the activation of multiple cell surface receptors and intracellular proteins involved in proliferation, angiogenesis, and survival. These data underscore the challenge of the combination of concomitant radiotherapy and chemotherapy and support the early use of targeted therapies to improve the antitumor efficacy.

Made available in DSpace on 2014-06-11T19:31:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-12-20Bitstream added on 2014-06-13T21:02:11Z : No. of bitstreams: 1 tanaka_fy_dr_araca.pdf: 1088210 bytes, checksum: 03e8b8b712b01bee40429585b8ec19b1 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O objetivo deste trabalho foi analisar a viabilidade celular comparando os meios de conservação para enxerto ósseo. A preservação de células viáveis em procedimentos de enxerto ósseo é de fundamental importância para que se tenha a osteogênese. Foram utilizados 43 ratos machos. Após a antissepsia do campo operatório foi realizada incisão linear na região mediana da calota craniana para obtenção do enxerto da região parietal direita e esquerda as quais foram removidas com auxílio de trefina de 5mm de diâmetro acoplada em micro-motor de baixa rotação, sob constante irrigação com solução de soro fisiológico 0,9% estéril. As peças do enxerto foram acondicionadas em tubos de ensaio estéreis os quais foram devidamente identificadas de acordo com o grupo e mantidas dentro deste tubo conforme cada condição do grupo. Como meio de conservação da viabilidade celular do enxerto foi utilizado o soro fisiológico a 0,9% (Grupo I) e a solução de Euro Collins® (Grupo II) e ainda para verificar se a temperatura tem influência direta na manutenção da viabilidade celular foi analisado o enxerto ósseo conservado em temperatura ambiente (Grupo III) e o enxerto ósseo sem nenhuma solução, porém mantido em gelo (GrupoIV). Para avaliar a viabilidade celular foi utilizada análise histológica e imunoistoquímica imediata e ainda em cada grupo analisou-se a viabilidade celular no período de 6 horas, 12 horas, 24 horas e 30 horas. Como resultado observou-se que a solução de Euro Collins® apresentou-se superior ao soro fisiológico no que se diz respeito à manutenção da viabilidade celular do enxerto ósseo onde se pode notar viabilidade celular até o período de 30 horas. / The aim of this study was to analyze cellular viability comparing storage media for skull vault bone graft. Preservation of viable cells in bone graft procedures is of paramount importance to obtain osteogenesis. Forty-three male used in this study. After antisepsis of the operative field, a linear incision was made on the middle region of the skull vault to obtain a bone graft from the right and left parietal areas. The grafts were removed with a 5-mm diameter trephine bur coupled to low-speed handpiece under continuous irrigation with sterile 0.9% saline. The graft pieces were placed in sterile 5-mL test tubes with caps, and were properly identified according to the group and maintained inside the test tubes as per each group conditions. The storage media evaluated for preservation of graft cellular viability were 0.9% saline (Group I) and Euro Collins® solution (Group II). In order to assess whether the temperature has a direct influence on the maintenance of cellular viability, the analysis was extended to bone grafts stored at room temperature (Group III) and bone grafts with no solution, but maintained in ice (Group IV). Cellular viability was evaluated by immediate histological and immunohistochemical analyses. For each group, cellular viability was analyzed at 6, 12, 24 and 30 hours after procedure. The results of this study showed that Euro Collins® solution yielded better performance than 0.9% saline as regards the maintenance of bone graft cellular viability (up to 30 hours).

A temperatura é um fator abiótico que exerce profundos efeitos no funcionamento dos ecossistemas marinhos, influenciando na biologia das espécies. Os peixes são um bom modelo para se estudar este fator, pois são pecilotérmicos e possuem importância comercial e ecológica. O objetivo deste trabalho foi analisar o comportamento, a tolerância térmica e os mecânicos moleculares de juvenis de peixe pampo Trachinotus carolinus em relação à elevação da temperatura. Para isso, foram submetidos ao aumento controlado da temperatura da água (2°C/h) a partir da temperatura de coleta (22°C) até 26°C, 30°C, 32°C, 34°C e 36°C e nestas foram mantidos por 120 minutos, ou foram aquecidos até a morte no estudo de tolerância térmica. O comportamento foi analisado usando-se vídeos e as expressões de Hsp70 e p53 nas brânquias e no coração dos pampos foram obtidas por meio da técnica de imuno-histoquímica Os resultados mostraram que os pampos comportam-se de forma agitada com o aquecimento até a temperatura de 36°C, e a partir dela apresentam uma desorganização dos sistemas e órgãos. A expressão de Hsp70 e p53 está relacionada ao aumento de temperatura até 34°C e decai nas temperaturas de 36°C e TCS. Assim, conclui-se que o aumento de temperatura afeta o comportamento e a expressão de Hsp70 e p53. / Temperature is an abiotic factor that influences the biology of marine species. Fishes are a good model to study temperature because they are poikilothermic and have ecological and commercial importance. The aim of this study was to analyze the behavior and study the expression of Hsp70 and p53 using the immunohistochemistry assay. The Expression of these two protein was observed in relation to temperature rise in heart and gills tissues of juvenile pompano fish Trachinotus carolinus. The Fishes were exposed to controlled increase of the water temperature (2°C/h), from 22°C (control) to 26°C, 30°C, 32°C, 34°C and 36°C and they were maintained in these temperature for 120 minutes . After that fishes were maintained in these temperature for 120 minutes. In addition, experiments were performed to determinate the critical temperature survival (TCS). The results revealed that the expression of Hsp70 and p53 were proportional to the temperature rise until 34°C and that it declines at temperatures of 36°C and TCS. The fishes behavior was frantic with heating until a temperature of 36°C and subsequently they have showed a disorganization of systems and organs. We concluded that the temperature increase affects the behavior and expression of p53 and Hsp70 in pompanos fishes.

In USA more than 12.5% of all infants are born preterm. Approximately 75% of all perinatal deaths occur among these preterm infants. Preterm infants are frequently very low in birth weight (VLBW) and receive multiple red blood cell (RBC) transfusions. These transfusions pose increased risk of infections and other complications. Since erythropoietin (EPO) stimulates RBC production, EPO treatment of VLBW infants has received attention as a modality for reducing transfusions in this group. The overall hypothesis of this work is that treatment optimization of EPO of anemia in preterm infants requires a comprehensive knowledge of the behavior of RBC and the pharmacokinetic/pharmacodynamics (PK/PD) relationship between EPO and erythropoiesis. Under that overall hypothesis, the specific aims were: 1) To describe erythropoiesis dynamics in preterm infants, 2) To determine and explain the variability in the response to EPO in preterm infants, 3) To evaluate newborn sheep as an experimental model for erythropoiesis in preterm infants, 4) To test the hypothesis that RBC lifespan is shortened under acute hypoxic stress conditions, 5) To test the hypothesis that EPO receptor (EPOR) pool size increases under hypoxic stress conditions and the change in EPOR pool size can be predicted using EPO clearance measurements, 6) To describe the effect of EPOR pool size changes on erythropoiesis kinetics. A model that describes erythropoiesis dynamics in preterm infants as a function of the plasma EPO concentration is presented in Chapter 2. In Chapter 3, several covariates are tested for their ability to identify infants with good EPO responsiveness. The lamb is also tested as an animal model for the erythropoiesis in preterm infants (Chapter 4). In Chapters 5-7, the effect of hypoxic stress conditions on RBC survival was explored defining the relation between the efficacy of EPO and survival of RBC produced as a result of EPO administration. RBC lifespan measurement methods are reviewed in Chapter 5. In Chapter 6, a new methodology for the measurement of RBC lifespan under stress conditions is developed. This new methodology is applied in Chapter 7 to explore the effect of hypoxic stress conditions on the survival of RBC. The study presented in Chapter 8 is undertaken to investigate changes in both EPOR pool size and EPO clearance under hypoxic conditions. An erythropoiesis model that accounts for change in the EPOR pool size under stress conditions is presented in Chapter 9. Analysis of erythropoiesis dynamics in preterm infants demonstrated that a three fold increase in the amount of RBC produced by preterm infants is possible by EPO administration. This emphasizes the potential of using EPO for the management of anemia in preterm infants. Covariate screening identified gestational age as a potential marker for the responsiveness to EPO treatment. PD analysis results in lambs demonstrated similarities between lambs and preterm infants in different erythropoietic characteristics such as sensitivity to EPO in producing RBC, Hb production rate before birth and blood volume. Survival analysis demonstrated that RBC lifespan is not shortened under acute hypoxic conditions Analysis of EPOR mRNA level demonstrated an up regulation of EPOR level under stress conditions accompanied by a parallel increase in EPO clearance. EPOR up regulation under stress conditions level was incorporated in a PD model presented in Chapter 9. The developed model provides a framework for optimizing EPO dosing. Accordingly, an optimal dosing strategy should in general maximize the interaction between EPO and EPOR. Specifically, EPO should be administered when the number of EPOR are close to maximally up-regulated.

Prostate cancer is one of the leading causes of death among males in the United States. In this study, we hypothesized that an activated PKC-iota-dependent anti-apoptotic pathway, drives the cell cycle proliferation and survival of prostate cancer cells. We investigated the role of atypical PKC-iota (PKC-ι) in androgen- independent prostate DU-145 carcinoma, androgen-dependent prostate LNCaP carcinoma compared to transformed non-malignant prostate RWPE-1 cells. Western blotting and immunoprecipitations demonstrated that PKC-ι is associated with cyclin-dependent activating kinase (CAK/Cdk7) in androgen-dependent, RWPE-1 and LNCaP cells but not in androgen-independent DU-145 cells. Treatment of prostate RWPE-1 cells with PKC-ι silencing RNA (siRNA) decreased cell proliferation, cell cycle accumulation at G2/M phase and decreased phosphorylation of Cdk7 and cdk2. In addition, PKC-ι siRNA treatment provoked a decrease in phosphorylation of Bad and increased Bad/Bcl-xL heterodimerization, leading to cell apoptosis. In DU-145 cells, PKC-ι is anti-apoptotic and still required for cell survival. Treatment with PKC-ι siRNA blocked an increase in cell number, and inhibited G1/S transition. In addition to cell cycle arrest, both RWPE-1 cells and DU-145 cells underwent apoptosis via mitochondria dysfunction and activating apoptosis cascades such as release of cytochrome c, activation of caspase-7, and poly-(ADP-ribose) polymerase (PARP) cleavage. Mechanistic pathways involving aPKCs in the NF-κB survival pathway were established using pro-inflammatory cytokine, tumor necrosis factor alpha (TNFα). Results demonstrated that RWPE-1 cells and DU-145 cells are insensitive to TNFα whereas LNCaP cells are sensitive to TNFα treatment and undergo apoptosis. In DU-145 cells, TNFα induced PKC-ι activation of IκB kinase, IKKα/ß, while in RWPE-1 cells, PKC-ζ activates IKKα/ß. Both RWPE-1 and DU-145 show degradation of IκBα allowing NF-κB/p65 translocation to the nucleus. In LNCaP cells, the upstream kinase activation IKKα/ß was not observed, although there have been reports that LNCaP cells weakly activate IKKα and have NF-κB activation. In vivo kinase assay demonstrates that PKC-ι is the substrate of IKKα/ß. A putative PKC-ι inhibitor (ICA-1) inhibited activation of IKKα/ß in vivo. Hence, PKC-ι is an antiapoptotic protein and this suggests that anti-PKC-ι therapy may be a viable option for prostate carcinoma cells.

Orientador: Wirley Gonçalves Assunção / Resumo: A utilização de biomateriais que visam devolver o volume ósseo perdido após a perda dentária tem se expandido. O osso é um tecido conjuntivo altamente especializado, possuindo dinâmica aposicional onde o equilíbrio entre neoformação e reabsorção envolve a interação de fatores endócrinos, parácrinos e autócrinos. Diversas associações de materiais diversos e substitutos ósseos têm sido estudadas, porém, o MTA (Agregado Trióxido Mineral) ainda carece de informações acerca da sua utilização como substituto ósseo ou em associação com demais substitutos. Assim, o objetivo deste estudo foi avaliar a influência da presença do MTA Angelus Branco® nas proprorções de 5%, 10% e 15% em associação com substituto ósseo de Hidroxiapatita e β- Tricálcio Fosfato na citotoxicidade, resposta tecidual e reparo ósseo, em defeito crítico em calvária de ratos. Para tanto, utilizou-se cultura celular da SAOS-2 para avaliação citológica e ensaio MTT do contato direto e eluentes. Também foram utilizados 112 ratos machos Wistar distribuídos em 7 grupos e avaliados em 2 tempos (7 e 28 dias). Após eutanasiados foram submetidos à microtomografia e por coloração de hematoxilina e eosina para análise histológica e histomorfométrica. A análise da homocedasticidade foi realizada pelo teste Shapiro-Wilk, para distinção dos dados paramétricos e não paramétricos. Para análise dos dados paramétricos, foi realizada análise de variância One-Way (ANOVA One-Way) e realizado o pós-teste de Tukey para os parâmetros micr... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The use of biomaterials that aim to return lost bone volume after tooth loss has expanded. Bone is a highly specialized connective tissue with appositional dynamics where the balance between neoformation and resorption involves the interaction of endocrine, paracrine and autocrine factors. Several associations of different materials and bone substitutes have been studied. However, the MTA (Mineral Trioxide Aggregate) still lacks information about its use as bone substitute or in association with other substitutes. Thus, the aim of this study was to evaluate the influence of presence of MTA Angelus Branco® in the 5%, 10% and 15% proportions in association with Hydroxyapatite and β-Tricalcium Phosphate bone substitute in cytotoxicity, tissue response and bone repair in critical defect in rat calvaria. For this, SAOS-2 cell culture was used for cytological evaluation and MTT assay of direct contact and eluents. We also used 112 male Wistar rats distributed into 7 groups and evaluated at 2 distinct times (7 and 28 days). After euthanasia, the specimens were submitted to microtomography and hematoxylin and eosin staining for histological and histomorphometric analysis. The analysis of homoscedasticity was performed by the Shapiro-Wilk test for distinction of parametric and non-parametric data. For analysis of parametric data, one-way analysis of variance (One-Way ANOVA) was performed and Tukey's post-test was performed for the microtomographic parameters and for the cytotoxicity t... (Complete abstract click electronic access below) / Mestre

<p>The focus of this thesis is the life and death of adult retinal ganglion cells (RGCs). RGCs are neurons that convey visual information from the retina to higher centers in the brain. If the optic nerve is transected (ONT), adult RGCs die by a form of cell death called apoptosis, and a general hypothesis is that neurotrophic factors can support the survival of injured neurons.</p><p>With the intention to gain knowledge about systems that can be used to decrease RGC death after ONT, we have studied growth factor receptors belonging to the tyrosine kinase family of receptors (RTK), known to mediate important cell survival signals. We found that the RTK Ret and its coreceptor GFRα1 were expressed by RGCs, and to test the above-mentioned hypothesis, we intraocularly administered glial cell-line derived factor, which activates a Ret-GFRα1 complex, and found transiently mediated RGC survival after ONT. </p><p>To identify new, potential neurotrophic factor receptors expressed by RGCs, with the aim to improve RGC survival after ONT, we developed a method for the molecular analysis of acutely isolated RGCs. The method involves retrograde neuronal tracing, mechanical retinal layer-separation, and isolation of individual RGCs under UV-light for RT-PCR analysis. Using this method, in combination with degenerate PCR directed towards the tyrosine kinase domain, several RTKs were identified. Axl, Sky, VEGFR-2, VEGFR-3, CSF-1R, and PDGF-βR are expressed by adult RGCs, and considered to be receptors with potential neurotrophic activity. Other results have shown that RGCs may require depolarization or increase in intracellular cAMP levels in order to fully respond to exogenously added trophic factors. We found that melanocortin receptors (MCRs) were expressed by RGCs, and MCRs can mediate elevation of intracellular AMP. We observed that α-MSH induced neurite outgrowth from embryonic retinal cells, indicating that MCR ligands have direct effects on retinal cells. RTKs and their ligands may be involved in endogenous systems for neuronal repair within the visual system. BDNF, NT-3, FGF2, and HGFR all increased in the retina after ONT and may be a part of an activated system for neuronal repair locally within the retina. </p><p>Adult axotomized RGCs die by apoptosis, therefore we examined the regulation of apoptotic genes after ONT. Bim and Bax increased in the retina after ONT, and may promote death of axotomized RGCs, whereas the increase in Bcl-2 may contribute to limit RGC apoptosis after ONT. </p><p>All in all, this thesis provides insights into the expression and regulation of molecules involved in the death and survival of RGCs. The results have revealed a number of potential neurotrophic receptors expressed by RGCs, and both identified RTKs and MCRs will serve as new targets in therapeutic approaches aiming at counteraction of RGC death after injury.</p>