Orientador: Cássio Luís Zanettini Riccetto / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T07:18:23Z (GMT). No. of bitstreams: 1 Dias_FernandoGoulartFernandes_M.pdf: 14775900 bytes, checksum: 18591a82df67b2439fbeb82e6cffca13 (MD5) Previous issue date: 2014 / Resumo: INTRODUÇÃO: Telas sintéticas representam, na atualidade, o pilar do tratamento da incontinência urinária e de prolapsos vaginais, sendo o polipropileno monofilamentar o material sintético mais utilizado. Apesar de taxas de cura de até 90%, complicações relacionadas à integração, tais como exposição ou erosão das telas, não podem ser negligenciadas. O colágeno, por ser um material biologicamente compatível, pouco imunogênico e com propriedades moduladoras do processo inflamatório, pode ser utilizado como um importante agente cicatricial melhorando a integração das telas. OBJETIVO: Avaliar, por meio de técnicas imunohistoquímicas, o efeito do recobrimento de tela de polipropileno monofilamentar, implantada no subcutâneo de ratas, com gel purificado de colágeno bovino, do ponto de vista da resposta imuno-inflamatória, do metabolismo do colágeno, angiogênese e citotoxicidade. MATERIAIS E MÉTODOS: Após aprovação no Comite de Ética em Experimentação Animal, foram utilizadas 20 ratas fêmeas da raça Wistar, tendo sido implantadas, em cada animal, de um lado da parede abdominal, uma tela de polipropileno monofilamentar (PP) e, do outro lado, uma tela semelhante recoberta com gel purificado de colágeno (PPC). Os animais foram divididos em quatro sub-grupos contendo 5 animais cada e foram eutanasiados em 7, 14, 21 e 90 dias após o implante. Foram utilizados reagentes específicos para avaliação dos aspectos de interesse: a) Imunológicos (Interleucina 1 ¿ IL-1); b) Metabolismo do colágeno (Metaloproteinases de Matriz 2 e 3 ¿ MMP-2 e 3); c) Angiogênese (Antígeno de Superfície CD-31); d) Citotoxicidade (Receptor do Fator de Necrose Tumoral alfa ¿ TNF ¿?). A análise das imunorreatividades foi realizada com auxílio do software analisador de imagens AxioVisionTM. RESULTADOS: A análise comparativa das variáveis entre os 4 períodos definidos (7,14, 21 e 90 dias) e entre os 2 grupos (PP e PPC) apontou diferença significativa para: CD-31 com maior número de vasos no grupo PPC, no subgrupo 14 dias (p=0.002) em relação ao grupo PP, e diminuição após 90 dias (p=0.002) no grupo PPC; MMP-2 com redução na densidade média no grupo PPC (p=0.046) nos subgrupos 21 e 90 dias em relação ao grupo PP ; MMP-3 com maior estabilidade ao longo do tempo no grupo PPC, de modo que houve queda significativa da área percentual reativa no grupo PP após 14 e 90 dias (p=0.017), bem como redução da densidade média logo após 21 dias, mas apenas após 90 dias no grupo PPC (p<0.001). CONCLUSÃO: O recobrimento com gel purificado de colágeno bovino determinou alterações significantes na resposta tecidual das ratas às telas de polipropileno, do ponto de vista imunohistoquímico, quanto à angiogênese e atividade das metaloproteinases na área do implante, sem influência significativa sobre a resposta imuno-inflamatória local (expressas por meio da IL-1 e TNF¿?) / Abstract: INTRODUCTION: The use of synthetic meshes, specially the polypropylene mesh, has become the standart treatment of urinary incontinence and vaginal prolapses. Even though presenting high cure rates, complications related to integration issues, such as exposure or erosion of the mesh, cannot be neglected. The collagen, well known as an important immunoinflammatory modulator, has been speculated to be a usefull tool in the healing process and possibly improving integration of meshes. OBJECTIVE The aim of this study is to evaluate, using immunohistochemical techniques, the effect of the use of a new purified collagen gel covering the monofilament polypropylene mesh implanted subcutaneously in rats, regarding immune-inflammatory response, collagen metabolism, angiogenesis and cytotoxicity. METHODS: After Ethics Committee on Animal Use¿s approval, in 20 female Wistar rats were implanted, at one side of abdominal wall, a monofilament polypropylene mesh (PP) and on the other side, the same mesh covered with a new developed purified collagen gel (PPC). The animals were divided into four sub-groups containing 5 animals each and were euthanized at 7, 14, 21 and 90 days after implantation. The immunohistochemical assessment of the samples was done by using specific reagents for the evaluation of points of interest: a) Immunologic (Interleukin 1 (IL-1)), b) Collagen metabolism (Metalloproteinases 2 and 3 (MMP-2 and 3)), c) Angiogenesis (surface antigen CD-31), d) Cytotoxicity (Tumor Necrosis Factor-alpha Receptor - TNF- ?). The objective analysis was performed using the image analysis software AxioVision TM. RESULTS: Comparative analysis of variables between the four periods defined (7, 14, 21 and 90 days) and between the 2 groups (PP and PPC) showed: higher vessel density in PPC group after 14 days (p=0.002) and decrease after 90 days (p=0.002); decrease of MMP-2 average density in PPC group after 21 and 90 days (p=0.046); more stability in MMP-3¿s behavior in PPC group along the periods with MMP-3 percent reactive area showing significant decrease just in PP group after 14 and 90 days (p=0.017) and also for MMP-3 average density, in which reduction was significant after 21 days in PP group, but just after 90 days in PPC (p<0.001).CONCLUSION: Highly purified collagen coating causes significant changes in angiogenesis and in metalloproteinase's immunohystochemical expression in meshes implants in rats, without significant influence on the local immuno-inflammatory response (expressed by IL-1 and TNF-?) / Mestrado / Fisiopatologia Cirúrgica / Mestre em Ciências

Cigarette smoking during pregnancy contributes to the development of neurological health problems in offspring. As a result, public health organizations are recommending NRT to pregnant women to wean them off tobacco. If nicotine itself is injurious to the developing brain, then nicotine substitution may not eliminate the deleterious health outcomes of maternal smoking. In studies of cognitive decline, estradiol elicits a neuroprotective effect through ER activation. However, the underlying mechanism remains unclear. Evidence suggests that estrogen-mediated neuroprotection is activated through glial cell interaction, mitigating inflammation and protecting neurons critical for learning and memory. If NRT antagonizes these cellular targets, it may put individuals at risk for future cognitive impairments. Randomly assigned nulliparous female Wistar rats were injected subcutaneously with 1 mg/kg/day of nicotine bitartrate or saline for 2 weeks before mating until weaning (PND 21). Pups (saline n=6 and nicotine, n=6) were sacrificed at 26 weeks of age and the hippocampal formation was processed for Nissl and immunohistochemical staining for GFAP and ERα. Gestational exposure to nicotine only produced a significant increase in the expression of ERα in the DG of the hippocampus. While additional research is needed, these findings suggest that NRT might indeed interfere with proper brain development, making offspring increasingly susceptible to long-term adverse health effects. Le tabac fumé pendant la grossesse affecte de manière importante le développement neurologique de la progéniture, y compris à long terme. C’est pourquoi, les autorités de la santé publique recommandent les substituts nicotiniques comme soutien au sevrage tabagique chez les femmes enceintes. Si le danger se situe dans la nicotine de la cigarette, alors les produits de substitution nicotiniques risquent également d’interférer avec le développement cérébral. De nombreuses données expérimentales convergent pour attribuer un rôle protecteur à l’oestradiol sur le fonctionnement cognitif. Par contre, le mécanisme sous-jacent est inconnu. Il se peut que l’oestradiol arrive à neutraliser la réaction inflammatoire provoquée par les cellules gliales, amoindrissant la détérioration des neurones impliqués au niveau de la mémoire. Ainsi, une perturbation de ce mécanisme par la nicotine pourrait engendrer une détérioration progressive des fonctions cognitives. Des rats femelles Wistars nullipares assignées de façon alléatoire à un groupe ont reçu soit une injection sous-cutanée de 1mg/kg/jour de nicotine bitartrate ou de saline, 2 semaines avant l’accouplement jusqu'au sevrage au jour 21 postnatal. A 26 semaines, les ratons furent sacrifiés (saline n=6 et nicotine, n=6) et une analyse du Nissl et immunohistochimique de GFAP et ERα furent réalisées sur les formations hippocampiques. L’exposition prénatale à la nicotine a seulement augmenté significativement l’expression de ERα dans le GD de l’hippocampe. Alors que des études plus approfondies sont nécessaires, ces résultats suggèrent que les substituts nicotiniques affectent le développement neurologique périnatal, ce qui risque d’entrainer des répercussions a long terme sur la santé.

Orientador: Oslei Paes de Almeida / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-26T10:32:36Z (GMT). No. of bitstreams: 1 Martinez_MarisolMartinez_D.pdf: 11593706 bytes, checksum: 9204e3ab03e2563ba5f0fa74b21ca2e2 (MD5) Previous issue date: 2014 / Resumo: Os tumores odontogênicos (TO) são um grupo heterogêneo de lesões derivadas de tecidos dentários, que ocorrem nos ossos gnáticos e tecidos adjacentes. Os TO são raros, na grande maioria benignos, representando menos de 4% de todos os espécimes recebidos em laboratórios de patologia bucal. Os TO malignos frequentemente apresentam dificuldades para diagnóstico e correta classificação, com vários pontos ainda não bem esclarecidos. O objetivo desse trabalho foi comparar as características clínicas, histológicas e imunohistoquímicas do carcinoma ameloblástico (CA) e fibrosarcoma ameloblástico (FSA) e dos seus respectivos correspondentes benignos ou seja ameloblastoma (AM) e fibroma ameloblástico (FA). O carcinoma escamocelular intra-ósseo primário (CEIP) também foi comparado com carcinoma escamocelular de mucosa bucal (CEMB). Foi avaliada a expressão imunohistoquímica de citoqueratinas 5, 7, 8, 14, 19, marcadores de proliferação celular Ki-67, p53, p63 e moléculas de adesão celular CD138 (Syndecan), E-cadherin e ?-catenin. Os resultados mostraram que o CA expressaram mais altos níveis Ki-67, p53 e p63 do que AM por tanto, poderiam ser usados como marcadores para diferenciar essas duas lesões. A expressão de CKs 5 e 19 foram alteradas com relação ao padrão observado em AM, pelo que alterações genéticas de essas proteínas poderiam estar presentes nas células malignas. CK19 pode ser um bom marcador para tumores odontogênicos benignos como AM mas não em malignos pois a expressão nestes é variável. Nas moléculas de adesão celular particularmente CD138 não foi observada diminuição da expressão em AC comparada com AM. As características histológicas assim como o perfil imunohistoquímico do CEMB e CEIP foram similares, por tanto, sugere-se que as características clínicas e radiográficas sejam os principais parâmetros para ser considerados no diagnóstico. Nos casos de fibroma ameloblástico (FA) e fibrosarcoma ameloblástico (AFS) a expressão de CKs foi similar em intensidade e localização e Ki-67 em FSA foi mais alto comparado com FA enquanto que p53 foi negative e ambas lesões. A expressão das moléculas de adesão foi similar em ambas lesões sendo mais intensa com CD138. Finalmente, foram ilustradas as características clínicas e histopatológicas de um caso de fibroodontoma ameloblástico pigmentado e um caso de CEIP originado a partir da transformação maligna de um queratocisto / Abstract: Odontogenic tumors correspond to a heterogenous group of lesions derived from dental tissues, involving the jaws and adjacent tissues. OT are rare, the majority benign, representing less than 4% of all specimens of an oral pathology laboratory. OTs frequently present difficulties for the correct diagnosis and classification, with various points yet to be clarified. The objective of this study was to analyze the clinical, histological and the expression of immunohistochemical markers in malignant odontogenic tumors and the benign counterparts. We assessed CK5, 7, 8, 14, 19, Ki-67, p53, p63, CD138 (Syndecan), E-cadherin and ?-catenin expression by immunohistochemistry in 15 and 9 cases of ameloblastoma (AM) and ameloblastic carcinoma (AC) respectively. Dez cases of primary intraosseous squamous cell carcinoma (PIOSCC), 9 cases of squamous cell carcinoma of mouth (OSCC), 8 cases of ameloblastic fibroma and ameloblastic fibrosarcoma (AFS). Also, these were compared expression patterns between these groups. In summary, Ki-67, p53 and p63 expression were higher in AC than in AM and could be used as markers of malignant transformation of AM. CKs 5 and 19 expression in AC are altered in relation to the pattern found in AM, also CK19 is a good marker for benign odontogenic tumors as AM. Our results do not permit to confirm that expression of CD138, is decreased in AC in relation to AM. The histological features as well as the immunohistochemical profile of OSCC and PIOSCC were similar, therefore, suggests that the clinical and radiographic features are the main parameters to be considered for diagnosis. In both AF and AFS the CKs expression in odontogenic islands was similar in intensity and localization, except CK7 that in most cases of AF was focally positive, while in AFS most cases were negative. Ki-67 proliferative index was higher in AFS that in AF, and p53 was negativo in both lesions. Expression of adhesion molecules was similar in AF and AFS, being most intense for CD138 / Doutorado / Patologia / Doutora em Estomatopatologia

Dysregulation of the chemokine receptor 4 (CXCR4) and its primary ligand CXCL12 (SDF-1, stromal cell-derived factor-1), has been implicated in the progression of melanoma and the mechanisms by which the CXCR4/CXCL12 axis has been shown to activate cell cycle progression is via stimulation of the mitogen-activated protein kinase (MAPK) pathway. Given this, we sought to ascertain the potential cooperativity of CXCR4 with established histopathologic prognosticators including the BRAF status in primary cutaneous melanoma. In this IRB approved study, archived tissue samples with diagnosis of primary cutaneous melanoma were retrieved from the Skin Pathology Laboratory at BUSM, Boston, MA and a total of 107 cases identified as meeting criteria for inclusion. Protein expression of CXCR4 and CXCL12 were assessed using commercially available rabbit polyclonal antibodies (Ab2074 and, ab9797 respectively, Abcam, Cambridge, MA, USA). CXCR4 gene expression (mRNA) was measured by semiquantitative RT-PCR with appropriate controls. For IHC, a semi-quantitative scoring (ranging from 0-3) was used and cases with a score of ≥2 (>10%) were considered positive. Molecular analysis for CXCR4 gene expression and BRAF exon 15 mutation status was performed using mRNA semi-quantitative RT-PCR and DNA Sanger sequencing respectively. Univariate analyses of CXCR4 mRNA expression revealed a statistically significant correlation between elevated CXCR4 expression (low ΔCt value) and presence of the BRAF mutation and absence of a host response (p=0.03 and p=0.0003 respectively). Univariate analyses revealed a significant correlation between elevated CXCR4 mRNA (low ΔCt value) and the following: presence of BRAF mutation and absence of a host response (p=0.03 and 0.0003 respectively). CXCR4 mRNA was significantly higher among both AJCC stage 1 and stage 3 compared to stage 2 (p=0.01). Compared with CXCR4 negative samples, univariate analyses of CXCR4 protein showed that the proportion of CXCR4 positives was significantly greater in melanomas with absence of mitoses (p<0.0001), ulceration (p=0.0008) and regression (p=0.02). Patients presenting at shallower stages (AJCC 1-2) exhibited a larger proportion of CXCR4 positives (76.9%, p<0.0001 and 69.0%, p=0.004), while those at deeper stages (AJCC 3-4) exhibited a larger proportion of negatives (75.0%, p=0.002 and 66.7%, p=0.10). In a multivariable analysis, lower odds of CXCR4 protein expression were associated with AJCC stage-3 (OR=0.16, p=0.01), stage-4 (OR=0.17, p=0.04), and mitoses (OR=0.21, p=0.01). Lack of correlation between CXCR4 mRNA and protein expression suggests that further study is required for a more precise understanding of mRNA-protein interaction for CXCR4 in order to identify factors contributing to the lack of concordance. CXCR4 protein appears to be associated with established prognosticators of good clinical outcome as its expression is less frequently observed in melanomas with mitoses, ulceration and depth >2 mm. The association between CXCR4 mRNA and a brisk host response suggests that it may serve as a biomarker for recruiting melanoma patients for immunotherapy. Higher CXCR4 mRNA in patients with a BRAF mutation suggests its utility as a putative therapeutic target.

Electrophysiological studies of myenteric neurons in the guinea-pig antrum suggest that different neuroactive compounds are involved in synaptic transmission. It is not known what neurotransmitters and neuropeptides are present and to what extent they colocalize. Immunohistochemical stainings were performed on whole-mount preparations of the guinea-pig antrum. Immunoreactivity for neuron-specific enolase was used as a general marker and was set at 100%. There was no overlap between cholinergic and nitrergic neurons, resulting in two separate subpopulations. The presence of choline acetyltransferase immunoreactivity was used to identify the cholinergic subset, which accounted for 56% of the cells. Immunoreactivity for nitric oxide synthase, on the other hand, was displayed in 40.7% of the neurons. Substance-P immunoreactivity was present in 37.4% of the cells and vasoactive intestinal peptide and neuropeptide Y in 21.7% and 28.6%, respectively. Small subsets of neurons had immunoreactivity for serotonin (3.9%), calretinin (6.8%) and calbindin (0.5%). Colocalization studies revealed several subgroups of neurons, containing one or more of the screened markers. Though some similarity is found in the chemical coding of the antrum compared to that of the small intestine and the corpus, remarkable differences can be seen in the occurrence of some subpopulations. Cholinergic neurons are not as predominant as in other parts of the gut, serotonin presence is doubled and some vasointestinal-peptide-positive neurons express substance P. These differences might reflect the highly specialized function of the antrum; however, the exact role of these classes remains to be established.

Although it is fairly well accepted that Helicobacter pylori infection plays a significant role in causing gastric cancer, the exact mechanisms involved in its pathogenesis are unclear. We have examined the relationship between H. pylori infection and oncogene expression in different stages of disease progression from precursor lesions to gastric carcinoma. We used Diff-Quik stain to diagnose H. pylori infection and immunohistochemical stains against c-erbB-2, p53, ras, c-myc, and bcl-2 to determine expression of oncogenes. H. pylori infection was found in all cases of chronic gastritis, atrophic gastritis, intestinal metaplasia, and early gastric carcinoma, and in 16 of 30 (53%) cases of advanced gastric carcinoma. Overexpression of c-erbB-2 was found in 2 (7%) cases of advanced gastric carcinoma, which were H. pylori negative. Suppressor gene, p53, was overexpressed in 3 (30%) cases of intestinal metaplasia, 2 (33%) cases of early gastric carcinoma, and 18 (60%) cases of advanced gastric carcinoma. Of these 18 p53-positive advanced gastric cancer cases, 11 (61%) were H. pylori positive. Expression of ras p21 was found in 4 (40%) cases of H. pylori-negative normal mucosa, 10 (100%) cases of chronic gastritis, 1 (10%) case of atrophic mucosa, 6 (60%) cases of intestinal metaplasia, 2 (33%) cases of nonneoplastic mucosa adjacent to early gastric carcinoma, and 7 (23%) nonneoplastic mucosa adjacent to advanced gastric carcinoma, all of which showed H. pylori. No evidence of expression of either c-myc or bcl-2 was detected in any of the above-mentioned samples. The data suggest that H. pylori infection may increase expression of ras p21 proteins and induce p53 suppressor gene mutation early in the process of gastric carcinogenesis.

Perineuronal nets (PNNs) decrease neuroplasticity, increase stability of neural systems, and are key to ending the neurodevelopmental critical period. PNNs are found throughout the brain, including the mediodorsal (MD) thalamus, a key region in cortico-thalamo-cortical communication with the prefrontal cortex (PFC). I examined the structure and location of PNN elements relative to axons in the MD thalamus from post-mortem human brain tissue in three normal individuals. Using electron microscopy and two-dimensional analysis, my results showed that about a third of axons have a consistent distribution of the PNN element brevican. A vital component of PNNs, the chondroitin sulfate proteoglycan (CSPG) brevican was found within the cytoplasm, within the myelin, and outside the myelin along the length of axons. Three-dimensional analyses and axon reconstructions showed that the quantity of brevican varied periodically along axons in a wave-like manner. These findings suggest a model for the arrangement of brevican in a weaving pattern through myelin, cytoplasm, and external surface of axons. This model of PNN elements has various functional implications, including influence on the growth and function of axons, ion homeostasis along the axon, and the ability of neurons to produce and transmit action potentials. These structures likely have a significant impact on the function of MD. The combined influence of PNNs and connections of the MD thalamus with the PFC may play powerful roles in various cognitive disorders, including schizophrenia.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25006号 / 医博第5040号 / 新制||医||1070(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 高橋 淳, 教授 荒川 芳輝, 教授 林 康紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

During an early developmental period, some glycinergic synapses in the brainstem and spinal cord release predominately GABA, which activates GABAA receptors on the postsynaptic membrane. The function of this early GABAergic transmission is unknown but presumed to contribute to synapse maturation. Classically, the enzyme glutamic acid decarboxylase (GAD), which synthesizes GABA from glutamate, has been considered the sole source of GABA in neurons. GABAergic neurons typically express one or both of the two known isoforms of this enzyme, GAD65 and GAD67. However, co-transmitting synapses in the midbrain were recently reported to acquire GABA through other means – GABA transporters (GAT1 and GAT3) and/or aldehyde dehydrogenase (ALDH1A1). To determine the source of GABA in immature glycinergic neurons of the auditory brainstem, we immunostained for GADs, GATs, and ALDH1A1, co-staining with markers for glial cell and synaptic terminals to verify cellular and subcellular location. GAD65 was expressed in synaptic terminals whereas GAD67 was localized to neuronal cell bodies, proximal dendrites, and presumabed synaptic terminals. However, during the peak period of GABA transmission in the first postnatal week, expression levels of both GAD65 and GAD67 were surprisingly low. Although GAT1 and GAT3 expression levels coincided with the peak period of GABA transmission, neither GAT was localized to neuronal cell bodies. In contrast, ALDH1A1 was expressed during the first postnatal week and was localized to neuronal cell bodies. These results suggest that immature glycinergic neurons of the auditory brainstem may not acquire GABA through classical GABA synthesis or GABA reuptake, but perhaps are able to synthesis GABA through the putrescine degradation pathway mediated by ALDH1A1. / Thesis / Master of Science (MSc) / Evolutionarily older parts of the mammalian brain, such as the brainstem, typically play little role in higher-order functions, but contain regulatory centers that are critically important for keeping the organism alive. As conventional wisdom has been that brainstem centers require fast inhibitory communication (mediated by the neurotransmitter glycine) to carry out their critical functions, an ongoing mystery lies in why many immature inhibitory neurons in the developing brainstem use the relatively slow inhibitory neurotransmitter, GABA. We and others have speculated that inhibitory neural circuits of the brainstem require GABA for maturation and/or refinement. As a first step in addressing this question in the auditory brainstem, we looked for the cellular and molecular sources of GABA by performing antibody stains for various proteins known to be involved in GABA synthesis and transport. Our results suggest, somewhat surprisingly, that GABA in the immature brainstem likely arises from non-classical sources.