Orientador: Kellen Cristine Tjioe / Resumo: Objetivo: Avaliar a expressão do p16Ink4a por imunohistoquímica e a presença do HPV16 pela Real time PCR em tecido fresco, plasma e saliva de pacientes com leucoplasia bucal (LB) e hiperplasia fibrosa inflamatória (HFI). Material e métodos: Foram incluídos 67 pacientes com o diagnóstico de LB e 44 pacientes com diagnóstico de HFI no estudo. Foram coletados dados sociodemográficos, clinicopatológicos, amostras de tecido fresco, sangue e saliva, que foram armazenados em um freezer a -80o C para realização de análise molecular. Também foi utilizado o tecido parafinizado para realização da imunohistoquímica para a p16Ink4a. As amostras de materiais biológicos obtidos dos pacientes foram submetidas à detecção do DNA do HPV-16 pela Real time PCR. O tecido parafinizado dos mesmos pacientes foram utilizados para avaliar a expressão da p16Ink4a pela imunohistoquímica. Resultados: Dos 67 pacientes incluídos de LB no estudo, 55,2% eram do sexo masculino com uma média de idade de 57,1 anos. Os pacientes idosos (>45 anos) compuseram 86,6% da amostra. As localizações anatômicas mais acometidas por LB foram a mucosa jugal (35,8%) e língua (20,9%). Sobre o tabagismo, 71,8% dos pacientes eram fumantes, onde a maioria (47,8%) foi classificada como tabagista leve. Em relação ao consumo de álcool, 47,4% eram alcoolistas, sendo a sua maioria classificada como alcoolista leve (59,3%). O grupo HFI foi composto por 54,5% pacientes do sexo masculino com uma média de idade de 57,3 anos. 90,9% dos paci... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Orientador: Mariza Akemi Matsumoto / Coorientador: Mariza Akemi Matsumoto / Coorientador: Idelmo Rangel Garcia Júnior / Banca: Roberta Okamoto / Banca: André Luís da Silva Fabris / Resumo: Os materiais vítreos podem ser processados por diferentes rotas, dentre elas a convencional por fusão e solidificação ou por rota sol-gel, o que melhora sua bioatividade. O objetivo do presente estudo foi o de avaliar o comportamento biológico da vitrocerâmica Biosilicato® sintetizada pelo processamento sol-gel durante o processo de reparo ósseo em modelo animal. Para tanto, foram utilizados 30 ratos Albinus Wistar, machos, com cerca de três meses de idade e pesando em média 450 gramas. Os mesmos foram submetidos a procedimento cirúrgico para confecção de um defeito de 5mm de diâmetro no osso parietal direito e divididos em 2 grupos, de acordo com o biomaterial estudado: Grupo BS - defeitos preenchidos com Biosilicato® particulado convencional (180-212 µm), e Grupo BG - defeitos preenchidos com Biosilicato® particulado produzido via rota sol-gel (180-212 µm). Após os períodos de 7, 21 e 45 dias, os animais foram submetidos à eutanásia para remoção dos espécimes para serem preparados e submetidos para análise microscópica morfológica e imuno-histoquímica. Aos 7 dias do grupo BS observaram-se partículas do biomaterial circundadas por tecido de granulação, no centro do defeito próximo e tecido ósseo neoformado próximo as suas paredes. Aos 21 dias, presença marcante de CGMs em contato com o biomaterial e aos 45 dias, biomaterial ora substituído por leucócitos MNs e CGMs ora circundado por tecido conjuntivo. Já no grupo BG aos 7 dias observaram-se numerosas partículas arredondadas... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The vitreous materials can be processed by different routes, among them the conventional one by fusion and solidification or by route sol-gel, which improves their bioactivity. The objective of the present study was to evaluate the biological behavior of the glass-ceramic Biosilicato® synthesized by sol-gel processing during the bone repair process in animal model. For this, 30 male Albinus Wistar rats were used, about three months old and weighing on average 450 grams. They were submitted to a surgical procedure to make a 5mm diameter defect in the right parietal bone and divided into 2 groups according to the biomaterial studied: BS group - defects filled with conventional particulate biosilicate (180-212 μm), and Group BG - defects filled with particulate Biosilicate® produced via sol-gel route (180-212 μm). After the 7, 21 and 45 day periods, the animals were submitted to euthanasia to remove the specimens to be prepared and submitted for microscopic morphological and immunohistochemical analysis. At 7 days of the BS group biomaterial particles were observed surrounded by granulation tissue, close to the defect wall and noticed if neoformed bone tissue at 21 days, the presence of CGMs in contact with the biomaterial and at 45 days, biomaterial ora replaced by leukocytes MNs and CGMs or surrounded by connective tissue, already in the BG group at 7 days there are numerous rounded biomaterial particles surrounded by granulation tissue at 21 days, presence of CGMs in contact ... (Complete abstract click electronic access below) / Mestre

Le profil d'activité des neurones dopaminergiques semble fortement évoluer au cours des premières semaines post-natales, faisant intervenir des modifications des propriétés intrinsèques et synaptiques, mais la connaissance des mécanismes à l'origine de ces changements et de leur cinétique est encore parcellaire.Dans un premier article, nous avons caractérisé le profil d'expression des sous-unités des principaux canaux ioniques sensibles au potentiel somato-dendritiques des neurones dopaminergiques au cours des premières semaines de développement post-natal. Nous avons pu décrire les principaux changements d'expression de ces canaux entre P6, P21 et P40. Dans un second article, nous avons défini l'évolution du comportement électrophysiologique des neurones dopaminergiques lors des 4 premières semaines postnatales. Nous avons pu montrer que l'acquisition du phénotype électrique "mature" des neurones dopaminergiques implique essentiellement deux transitions développementales, intervenant respectivement entre P3 et P5 puis entre P9 et P11.Enfin dans une troisième étude, nous avons tenté de déterminer les principaux changements morphologiques intervenant au cours des premières semaines post-natales et de définir leur impact sur le profil électrophysiologique des neurones dopaminergiques. Nos résultats suggèrent que la morphologie de l'axone et du segment initial de l'axone changent fortement au cours des trois premières semaines post-natales.Nous avons ainsi pu caractériser les principales transitions développementales intervenant dans l'acquisition du phénotype électrique des neurones dopaminergiques ainsi que les changements morphologiques et biophysiques associés. / The firing pattern of dopaminergic neurons seems to strongly evolve during the first postnatal weeks, involving changes in intrinsic and synaptic properties, but our knowledge of the mechanisms underlying these changes and their precise timecourse is still fragmented.In a first study, we characterized the expression profile of several somato-dendritic voltage-gated ion channels in dopaminergic neurons during postnatal development. Our results describe the major changes in expression of these ion channels occurring between P6, P21 and P40.In a second study, we described the modifications in electrophysiological behavior of dopaminergic neurons across the first four postnatal weeks. We show that the acquisition of the mature electrical phenotype of dopaminergic neurons mainly involves two developmental transitions occurring between P3 and P5 and then between P9 and P11, respectively.Finally, in a third study, we attempted to define the major morphological changes occurring during early postnatal development and to measure their impact on the electrophysiological profile of dopaminergic neurons. Our results suggest that the morphology of the axon and the axon initial segment strongly change during the first three postnatal weeks, even though these changes do not seem to significantly influence the excitability of dopaminergic neurons or the shape of their action potential.We were able to characterize the main developmental transitions leading to the acquisition of the mature electrical phenotype of dopaminergic neurons, and to describe some of the biophysical and morphological changes associated with this electrophysiological maturation.

The management of significant bone defects, delayed and non-union of fractures can be extremely challenging. Development of specific treatment is hindered by an absence of information regarding the molecular events which regulate these processes. In this thesis, a bilateral cancellous bone defect model of the femur and tibia was developed in a rodent and the spatiotemporal profile of TGF-β, BMP 2 and 7, Smads 1, 4 and 5 characterised. Next, the capability of acid solution to augment healing was tested in both a bone defect and in a closed femoral fracture model. Finally, a long term oestrogen deficiency (OVX) rat model of postmenopausal osteoporosis was characterised and the spatiotemporal profiles of IGF-1, IGFR-1, MMP-1, MMP-3, MMP-9, MMP-13, TIMP-1, TIMP-2, BMP-2, BMP-4, BMP-7, TGF-β, Smad4, Smad7, VEGF, Flt-1, Ihh and FGF-2 were compared in femoral osteotomies between OVX and Sham groups. The bilateral cancellous defect model was successfully created with a number of advantages with which to recommend its use in future studies. TGF-β, BMP 2 and 7, Smads 1, 4 and 5 had characteristic spatiotemporal profiles during cancellous bone defect healing suggesting that they have a regulatory role. The results of the acid study were inconclusive and problems with substance delivery and maintenance at the desired site need to be addressed in the future to fully test this hypothesis. No significant differences were detected on histology or three-point mechanical testing between the fracture calluses of acid and control groups. In the final study, OVX rats after six months had significantly increased weight and decreased bone mineral density compared to their sham counterparts. A histological delay in osteotomy healing was observed in the OVX group but no significant differences on tensile testing were seen between OVX and Sham groups up to six weeks. Immunohistochemistry revealed that delayed healing may be due to the down-regulation of IGF-1, BMP-2, 4, and 7 and the up-regulation of MMP-3 in OVX compared to Sham groups. In conclusion, the results of this thesis give some insight into the molecular biology of bone defects and osteoporotic fractures. This information may also be useful in the development of specific treatments aimed at augmenting healing in bone defects and osteoporotic fractures.

Due to the costly and technically challenging nature of genetic testing, methods have been developed to target more specifically those who are at increased risk of carrying the Hereditary Non-Polyposis Colorectal Cancer (HNPCC) mutation. HNPCC is an inherited colorectal cancer syndrome. Testing of tumour material (which has previously been removed during surgery) for features of HNPCC has been found to be an effective and economic method of identifying those at higher risk of having a mutation. Only those at higher risk of having a mutation will undergo genetic testing. This practice of “tumour testing” has become widespread. / There is currently no clarity about requirements for consent prior to testing of stored tumour tissue. The person giving consent to tumour testing does not always have an appointment with a genetics service prior to giving consent. This can be contrasted to genetic testing on blood samples where laws and guidelines state that informed consent is required prior to genetic testing and that comprehensive genetic counselling and support should be provided as part of this process. Protocols for genetic testing have been developed as a result of extensive research around the impact and implications of genetic testing. / Consumer opinion and participation through research is an important aspect of health policy and guideline development. Accordingly the purpose of this study was to contribute to such development by gaining insight into the experiences, understandings, decision making processes and opinions of those who had given consent to have their own or their relatives tumour tested. Seventeen people who had given consent for tumour testing either for themselves, or on behalf of a deceased relative were recruited through a Familial Cancer Centre and in-depth interviews conducted. The interviews were transcribed and analysed using thematic analysis. / Some participants had no memory of consenting to tumour testing. Others remembered basic concepts. Negative implications of testing were unknown or viewed as unimportant. Participants did not understand the difference between tumour testing and germline testing. Despite lack of memory or understanding participants did not want additional or more detailed pre-test information although they did want more follow-up and support after receipt of results. The decision to consent to testing was made as soon as participants were informed of the availability of tumour testing - the major reason being to provide information for the family that would aid in cancer prevention. Participants were more concerned with accessibility to testing than pre test information and counselling. / Findings in this study indicated participants made decisions heuristically rather than systematically and this as well as participants’ opinions and other decision-making research has implications for the traditional view of informed consent around genetic related decisions. This in turn has implications for policy and guidelines in the area. Implications for current practise as a result of findings from this study include ensuring participants understand negative implications of testing and follow up and support of those with negative as well as positive results to tumour testing.

<p>Bronchial carcinois are subdivided into typical and atypical. Atypical carcinoids are more malignant, but typical carcinoids may also influence survival. In the present study immunohistochemistry was performed to identify prognostic markets in patients with typical bronchial carcinoids. The diagnostic efficacy of octreoscan was evaluated, in comparison with CT and bone scan, and finally our experience of treating patients with metastatic bronchial carcinoids is reported. In an unselected material of 43 patients with typical bronchial carcinoids, metastatic disease was found in 12 patients (28%). Five patients (12%) developed distant metastases and died from their disease. High Ki-67 index, as well as positive staining for bcl-2 or p53 was associated with de- creased survival time. Positive staining for CD44s, v7-8 and v9, as well as positive nuclear staining for nm23 correlated to decreased mortality. Staining for CD44 and Ki-67 should be performed routinely for prognostic evaluation in these patients. </p><p>Octreoscan positive tumors were found in altogether 20/28 patients (71%). The primary tumor was detectable in 81% and intrathoracic metastases in 78% of the patients on octreoscan; the corresponding figures for CT were 94% and 89% respectively. Liver metastases, as shown by CT, were demonstable by octreoscan in 64% of patients. Octreoscan showed 70% and bone scan 90% sensitivity for identification of bone metastases. </p><p>Plasma chromogranin A was elevated in 28/30 patients (94%) with metastatic bronchial carcinoids and was the most sensitive tumor marker. Increased urinary 5'HIAA was found in 68%. </p><p>Biotherapy with α-interferon and Octreotide relieved carcinoid syndrome in 7/16 patients. However, only 4/27 patients showed stable disease during median 15 months, while 23 patients progressed. Treatment with cisplatinum + etoposide resulted in an objective response or stable disease for 6-8 months in 3/8 patients with widespread tumors. Doxorubicin combined with streptozotocin or paclitaxel was associated with stable disease for 9 months in 2/2 patients each. All 7 patients treated with streptozotocin+5-FU progressed. </p><p>Among the 43 unselected typical bronchial carcinoid patients, 5-year and 10-year survival was 95% and 91%, respectively. The prognosis in patients with bronchial carcinoids showing distant metastases was poor: 5-year survival was 70% from diagnosis and 22% from treatment start. </p>

<p>The amyloidoses are biochemically heterogeneous diseases with patholophysiologic deposits of various proteins. Amyloid deposits can occur either localized to one organ or tissue or as part of a systemic disease with deposits in many different tissue. The clinical course, prognosis and therapy are different for each type of amyloidosis and therefore a type specific diagnosis is demanded as early as possible. We describe a method for typing of the most common systemic amyloidoses based on Western blot analysis combined with specific</p><p>in- house antibodies, using subcutaneous fat biopsies. We found that the method is reliable and easy to perform and the tissue sample needed is obtained by minor surgery.</p><p>In the aortic intima amyloid deposits are often associated with atherosclerosis plaques. In our study we also investigated the prevalence of intimal amyloid from 10 patients age 58-94, amyloid deposits were present in 50% of the cases.</p> / <p>Amyloidos är ett sjukdomstillstånd där proteiner som normalt är lösliga i kroppen felveckas och formar långa olösliga fibriller som ansamlas i vävnader och organ såsom t.ex. hjärta, hjärna och lever. Det finns cirka 25 proteiner som kan ge upphov till amyloidos. Man kan skilja på två huvudgrupper av amyloidos, systemisk och lokaliserad. Vid lokal amyloidos kan inlagringar förekomma i specifika vävnader vid framför allt vissa åldersberoende sjukdomar som t.ex. Alzheimers sjukdom. Vid systemisk amyloidos förekommer inlagringar i praktiskt taget alla vävnader. Symtomatologin vid systemisk amyloidos är variabel och sjukdomsbilden kan vara svårtolkad men tidig och specifik diagnostik ger möjlighet till riktad terapi mot den bakomliggande sjukdomen. Syftet med denna studie var att utvärdera en Western blot metod som använts för typning av vanligaste formerna av systemisk amyloidos. De slutsatser som nåtts är att denna metod är snabbt, pålitligt och enkel att utföra. Diagnos erhölls med finnålsbiopsi av bukfettvävnad som är enkel, snabb och billig metod med liten risk för patienternas hälsa. Vi lyckades också med hjälp av immunhistokemisk infärgning titta på prevalens av amyloid i aortas intima.</p>

It is well established that air pollution has detrimental effects on both human health as well as the environment. Exposure to ozone and particulate matter pollution, is associated with an increase in cardiopulmonary mortality and morbidity. Asthmatics, elderly and children have been indicated as especially sensitive groups. With a global increase in use of vehicles and industry, ambient air pollution represents a crucial health concern as well as a political, economical and environmental dilemma. Both ozone (O3) and diesel exhaust (DE) trigger oxidative stress and inflammation in the airways, causing symptoms such as wheezing, coughing and reduced lung function. The aim of this thesis was to further examine which pro-inflammatory signaling pathways that are initiated in the airways by ozone, as compared to diesel exhaust. Furthermore, to study the effects of these two ambient air pollutants in a sequential exposure, thus mimicking an urban profile. In order to investigate this in healthy as well as asthmatic subjects, walk-in exposure chambers were utilized and various airway compartments were studied by obtaining induced sputum, endobronchial biopsies, or airway lavage fluids. In asthmatic subjects, exposure to 0.2 ppm of O3 induced an increase in the cytokines IL 5, GM-CSF and ENA-78 in the bronchial epithelium six hours post-exposure. The healthy subjects, however, displayed no elevations of bronchial epithelial cytokine expression in response to the ozone exposure. The heightened levels of neutrophil chemoattractants and Th2 cytokines in the asthmatic airway epithelium may contribute to symptom exacerbations following air pollution exposure. When examining an earlier time point post O3 exposure (1½ hours), healthy subjects exhibited a suppression of IL-8 as well as of the transcription factors NFκB and c-jun in the bronchial epithelium, as opposed to after filtered air exposure. This inhibition of early signal transduction in the bronchial epithelium after O3 differs from the response detected after exposure to DE. Since both O3 and DE are associated with generating airway neutrophilia as well as causing direct oxidative damage, it raises the query of whether daily exposure to these two air pollutants creates a synergistic or additive effect. Induced sputum attained from healthy subjects exposed in sequence to 0.2 ppm of O3 five hours following DE at a PM concentration of 300 µg/m3, demonstrated significantly increased neutrophils, and elevated MPO levels, as compared to the sequential DE and filtered air exposure. O3 and DE interactions were further investigated by analyses of bronchoalveolar lavage and bronchial wash. It was demonstrated that pre-exposure to DE, as compared to filtered air, enhances the O3-induced airway inflammation, in terms of an increase in neutrophil and macrophage numbers in BW and higher EPX expression in BAL. In conclusion, this thesis has aspired to expand the knowledge of O3-induced inflammatory pathways in humans, observing a divergence to the previously described DE initiated responses. Moreover, a potentially adverse airway inflammation augmentation has been revealed after exposure to a relevant ambient combination of these air pollutants. This provides a foundation towards an understanding of the cumulative airway effects when exposed to a combination of ambient air pollutants and may have implications regarding future regulations of exposure limits.

Bronchial carcinois are subdivided into typical and atypical. Atypical carcinoids are more malignant, but typical carcinoids may also influence survival. In the present study immunohistochemistry was performed to identify prognostic markets in patients with typical bronchial carcinoids. The diagnostic efficacy of octreoscan was evaluated, in comparison with CT and bone scan, and finally our experience of treating patients with metastatic bronchial carcinoids is reported. In an unselected material of 43 patients with typical bronchial carcinoids, metastatic disease was found in 12 patients (28%). Five patients (12%) developed distant metastases and died from their disease. High Ki-67 index, as well as positive staining for bcl-2 or p53 was associated with de- creased survival time. Positive staining for CD44s, v7-8 and v9, as well as positive nuclear staining for nm23 correlated to decreased mortality. Staining for CD44 and Ki-67 should be performed routinely for prognostic evaluation in these patients. Octreoscan positive tumors were found in altogether 20/28 patients (71%). The primary tumor was detectable in 81% and intrathoracic metastases in 78% of the patients on octreoscan; the corresponding figures for CT were 94% and 89% respectively. Liver metastases, as shown by CT, were demonstable by octreoscan in 64% of patients. Octreoscan showed 70% and bone scan 90% sensitivity for identification of bone metastases. Plasma chromogranin A was elevated in 28/30 patients (94%) with metastatic bronchial carcinoids and was the most sensitive tumor marker. Increased urinary 5'HIAA was found in 68%. Biotherapy with α-interferon and Octreotide relieved carcinoid syndrome in 7/16 patients. However, only 4/27 patients showed stable disease during median 15 months, while 23 patients progressed. Treatment with cisplatinum + etoposide resulted in an objective response or stable disease for 6-8 months in 3/8 patients with widespread tumors. Doxorubicin combined with streptozotocin or paclitaxel was associated with stable disease for 9 months in 2/2 patients each. All 7 patients treated with streptozotocin+5-FU progressed. Among the 43 unselected typical bronchial carcinoid patients, 5-year and 10-year survival was 95% and 91%, respectively. The prognosis in patients with bronchial carcinoids showing distant metastases was poor: 5-year survival was 70% from diagnosis and 22% from treatment start.

In this thesis, methods to study gene and protein expression in cells and tissues were developed and utilized in combination with protein-specific antibodies, with the overall objective to attain greater understanding of protein function. To analyze protein expression in in vitro cultured cell lines, a cell microarray (CMA) was developed, facilitating antibody-based protein profiling of cell lines using immunohistochemistry (IHC). Staining patterns in cell lines were analyzed using image analysis, developed to automatically identify cells and immunohistochemical staining, providing qualitative and quantitative measurements of protein expression. Quantitative IHC data from CMAs stained with nearly 3000 antibodies was used to evaluate the adequacy of using cell lines as models for cancer tissue. We found that cell lines are homogenous with respect to protein expression profiles, and generally more alike each other, than corresponding cancer cells in vivo. However, we found variability between cell lines in regards to the level of retained tumor phenotypic traits, and identified cell lines with a preserved link to corresponding cancer, suggesting that some cell lines are appropriate model systems for specific tumor types. Specific gene expression patterns were analyzed in vitiligo vulgaris and malignant melanoma. Transcriptional profiling of vitiligo melanocytes revealed dysregulation of genes involved in melanin biosynthesis and melanosome function, thus highlighting some mechanisms possibly involved in the pathogenesis of vitiligo. Two new potential markers for infiltrating malignant melanoma, Syntaxin-7 and Discs large homolog 5, were identified using antibody-based protein profiling of melanoma in a tissue microarray format. Both proteins were expressed with high specificity in melanocytic lesions, and loss of Syntaxin-7 expression was associated with more high-grade malignant melanomas. In conclusion, the combination of antibody-based proteomics and microarray technology provided valuable information of expression patterns in cells and tissues, which can be used to better understand associations between protein signatures and disease.