Orientador: Juliane Maria Guerreiro Tanomaru / Resumo: Cimentos à base de silicatos de cálcio são desenvolvidos para obturação de canais radiculares. Publicação 1: Este estudo avaliou propriedades biológicas dos cimentos experimentais (CE-1 e CE-2) compostos por silicatos de cálcio e com adição de hipoclorito de cálcio (CE-2), em comparação ao AH Plus (AHP) e TotalFill BC Sealer (TBC). A reação tecidual provocada pelos materiais no subcutâneo de ratos foi avaliada por meio da implantação de tubos de polietileno nos períodos de 7, 15, 30 e 60 dias. Cortes foram corados com hematoxilina e eosina (HE) para análises morfológica e do número de células inflamatórias/mm2(CI) e cortes foram utilizados para detecção de interleucina-6 (IL-6) e osteocalcina. Método de von Kossa foi realizado para identificação de depósitos de cálcio. Os dados foram submetidos aos testes ANOVA e Tukey, com significância de 5%. Aos 7 dias, CE-1, CE-2 e AHP apresentaram maior número de CI. AHP apresentou maior marcação para IL-6. Após 15 dias não houve diferença entre CE-2 e o grupo controle para IL-6. Aos 30 dias, AHP exibiu maior número de CI (p<0,0001) e CE-2 e o grupo controle os menores valores de CI e IL-6. Após 60 dias CE-2, TBC e controle apresentaram os menores valores (p<0,05). Os cimentos CE-1, CE-2 e TBC apresentaram estruturas positivas ao método von Kossa em todos os períodos observados e marcação positiva para osteocalcina. CE-2 apresentou quantidade de células positivas superior em todos os períodos quando comparado aos cimentos CE-1 e TBC (p< ... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Caloric intake increased over a long period of time may induce the development of obesity, causing so-called low-grade inflammation. The organism responses to the inflammation by the activation and production of cellular components of the immune system, such as macrophages or proinflammatory cytokines. The adipose tissue itself is involved in the production of bioactive molecules, including leptin and adiponectin. Increased concentration of proinflammatory cytokines can lead to a dysfunction of important metabolic pathways and impair organ's function. For the purpose of closer knowledge of the etiology of obesity and its metabolic complications, inbred strains of mice with different genetic backround are most commonly used. We aimed to define the impact of high-fat diet (HFD) on adipose and liver tissue of C57BL/6J and A/J murine strains with a different susceptibility to diet-induced obesity. We focused on description of morphological and functional changes of adipose tissue and on the evaluation of plasma leptin and adiponectin levels of mice in the early postnatal development. Next, we measured the expression of leptin mRNA in four tissues. In this study we described how the increased caloric intake leads to increased triacylglycerides (TAG) storage in the liver and to a higher inflammatory...

Prostate cancer is the most common newly diagnosed non-skin cancer and the second leading cause of cancer death in men. It is a unique neoplasm because of the large discrepancy between its clinical incidence and the much higher incidence of latent cancer. Predicting the prognosis of prostate cancer, especially the cancers detected incidentally or by screening, remains a clinically important problem. Immunoreactivity for Onco-antigen 519 (OA- 519), a recently described fatty acid synthase (FAS), has been associated with poor prognosis in breast cancers. The authors have previously shown that its detection in prostate cancer correlated with high-grade, large volume, and advanced stage tumors. This study examines the association between OA- 519 immunoreactivity in primary prostate cancer and disease progression. The authors used immunohistochemistry with an affinity-purified anti-OA-519 antibody and examined primary prostate cancers (stages A1 to D1) from 99 men with a mean follow-up of 4 years (range= 2 to 9.3). Survival analysis was used to evaluate differences in progression-free survival. OA-519 immunoreactivity was seen in 56 (57%) of the 99 primary prostate cancers examined. OA-519-positive cancers were more likely to progress than the OA- 519-negative cancers (P < .04). Univariate survival analysis showed that OA- 519 (FAS), histological grade (Gleason score), and clinical stage were significant predictors of disease progression. Multivariate analyses of all cases showed that only histological grade was significant. However, multivariate analysis of the 85 cancers with Gleason scores 2-7 (ie, low to intermediate grade) showed OA-519 (FAS) immunoreactivity to be the only statistically significant predictor of cancer progression (P<.02). Expression of the fatty acid synthase OA-519 by prostate cancers is potentially a clinically useful predictor of disease progression. It appears to be independent of histological grade (Gleason score), at least in cancers with low to intermediate grades. Further studies are needed to evaluate the role of fatty acid synthase in malignancy and the potential therapeutic implications of enzyme blockers.

A new approach to study angiotensin receptor distribution in the brain has been taken by developing antibodies to partial sequence of the angiotensin II (AII) type-1 receptor subtype (AT1) and demonstrating the presence of receptors with immunohistochemical staining. The antibody to a portion of the 3rd cytoplasmic loop of the AT1 receptor revealed distinctive punctate immunoreactive staining on cell bodies. The cell bodies were distributed in the forebrain in paraventricular and supraoptic nuclei, the organum vasculosum lamina terminalis, median preoptic area and subfornical organ. In the brainstem, the entire locus coeruleus was stained, together with the adjacent mesencephalic and motor nuclei of the trigeminal nerve. The auditory system including the cochlear nucleus and superior olivary nuclei were stained. In the medulla, all the structures involved in blood pressure control were stained including the nucleus of the solitary tract, the 12th nerve nuclei, the rostroventral lateral area and the nucleus ambiguous. Sites where AT2 receptors are located were not stained or staining was limited to specific area such as the medial accessory nucleus of the inferior olive. Immunocytochemical staining of AT1 receptors provides a new and more precise approach to the cellular localization of AII receptors.

Intrinsic cardiac neurons are core elements of a complex neural network that serves as an important integrative center for regulation of cardiac function. Although mouse models are used frequently in cardiovascular research, very little is known about mouse intrinsic cardiac neurons. Accordingly, we have dissociated neurons from adult mouse heart, maintained these cells in culture, and defined their basic phenotypic properties. Neurons in culture were primarily unipolar, and 89% had prominent neurite outgrowth after 3 days (longest neurite length of 258 ± 20 μm, n = 140). Many neurites formed close appositions with other neurons and nonneuronal cells. Neurite outgrowth was drastically reduced when neurons were kept in culture with a majority of nonneural cells eliminated. This finding suggests that nonneuronal cells release molecules that support neurite outgrowth. All neurons in coculture showed immunoreactivity for a full complement of cholinergic markers, but about 21% also stained for tyrosine hydroxylase, as observed previously in sections of intrinsic cardiac ganglia from mice and humans. Whole cell patch-clamp recordings demonstrated that these neurons have voltage-activated sodium current that is blocked by tetrodotoxin and that neurons exhibit phasic or accommodating patterns of action potential firing during a depolarizing current pulse. Several neurons exhibited a fast inward current mediated by nicotinic ACh receptors. Collectively, this work shows that neurons from adult mouse heart can be maintained in culture and exhibit appropriate phenotypic properties. Accordingly, these cultures provide a viable model for evaluating the physiology, pharmacology, and trophic factor sensitivity of adult mouse cardiac parasympathetic neurons.

The distribution of neurons in the rostral nucleus of the solitary tract (rNST) that respond to gustatory input from the anterior tongue was visualized by Fos protein immunohistochemistry following electrical stimulation of the chorda tympani (CT) nerve in rats. Maps of Fos-immunoreactive (Fos-ir) neurons were compared with the distribution of CT afferent terminal fields labeled by transganglionic transport of rhodamine-dextran in a separate group of animals. The primary concentration of Fos-ir neurons localized in register with the major terminal fields of CT afferent fibers, in the central third of the rostral 1.0 mm of the NST ipsilateral to the stimulated nerve. A similar correspondence in location and degree of labeling of Fos-ir neurons and afferent terminals was observed in the ipsilateral dorsal spinal trigeminal complex (Sp5) pars caudalis, near the obex, and the Sp5 pars oralis near the rostral pole of the rNST. Thus, the magnitude of Fos upregulation in brainstem targets of the CT nerve having chemosensory or nociceptive function, was proportional to the relative density of the CT afferent input. This correspondence, and the absence of labeling in neurons known to be one additional synapse away from the afferent input within gustatory or oral reflex pathways, suggests that the cell map obtained represents mainly neurons that are directly activated via primary afferent synapses from CT fibers. The availability of a method to histochemically identify a population of putative second-order taste neurons will facilitate analysis of the cellular/molecular properties of these neurons and of synaptic circuitry in the rNST.

In the normal ectocervix, mitoses are rare and are usually confined to the basal layers. In contrast, they occur more frequently in cervical intraepithelial neoplasia (CIN) and are seen at higher levels, suggesting that CIN may be associated with a progressive dysfunction in proliferative activity of cervical cells. The objective of this study was to use proliferating cell nuclear antigen (PCNA) immunohistochemistry to examine the proliferative activity of cervical epithelial cells in CIN lesions. Sixty- eight cervical biopsies were examined; 20 were totally benign, 14 had CIN I, 21 CIN II, and 13 CIN III. In benign epithelia, PCNA staining was usually confined to the basal layers, whereas in CIN the staining was seen at progressively higher levels of the epithelium. There was a statistically significant correlation between the CIN grade and the highest level of PCNA staining (PCNA grade, r = 0.746, P < 0.001). In addition, the PCNA grade showed significant correlation with the highest level at which mitoses were seen (mitosis grade, r = 0.706, P < 0.001), and a strong direct correlation between the mitosis and CIN grades was also observed (r = 0.955, P < 0.001). These data demonstrate that (1) PCNA immunoreactivity in neoplastic cervical epithelium is different from that seen in the normal cervix, suggesting that CIN is associated with a dysfunctional proliferation of cervical epithelium, (2) that there is a significant correlation between the PCNA grade and CIN grades, and (3) the 'mitosis grades' have a strong correlation with the CIN grades.

Organisms are often exposed to multiple stressors in their natural habitat. While the effects of individual stressors alone are generally well-studied, their combined interactions are often unknown. One such unknown interaction is that between copper, an essential micronutrient that is toxic in high levels, and hypoxia, a commonly experienced environmental stressor. This project sought to examine the effects that copper has on the hypoxic response as well as the combined effects of copper and hypoxia exposure using freshwater-acclimated adult killifish (Fundulus heteroclitus), a model euryhaline teleost. It was found that copper blunts the acute ventilatory response to hypoxia, ammonia, and combined hypoxia and ammonia exposure. Gill morphometry was also examined and it was found that while copper alone has no effect, when combined with hypoxia exposure it eliminates the increase in filament length and cross-sectional area seen when fish were exposed to hypoxia alone. Furthermore, when neuroepithelial cell size was examined, copper decreased cell projection area with or without the combined presence of hypoxia. Taken as a whole, this thesis indicates that copper exposure can significantly and negatively impact the ability of aquatic organisms to mount appropriate physiological responses to hypoxia. / Thesis / Master of Science (MSc)

The integrins play a crucial role in cancer cell proliferation, migration, differentiation, survival and angiogenesis. It has been shown that integrin expression is positively correlated to cancer dissemination, this suggests targeting selected integrins as an anti-metastatic strategy. The aim of this study is to investigate the effect of novel antagonists of α5β1, αvβ3 and αvβ5 integrins on cancer cell migration, a key process in tumour cell dissemination. Immunohistochemistry was used to evaluate the expression of α5, αv, β3 and β5 integrin subunits in prostate cancer tissues. Furthermore the expression of these integrin subunits in tumour and normal human head and neck tissues was compared. The expression profile of these integrin subunits in established human cancer cell lines was subsequently evaluated using immunodetection methods in cells and xenograft tumour samples. The effect of integrin inhibition on cell migration was then assessed using neutralizing antibodies against αvβ3, αvβ5, and α5β1 integrins in the scratch-wound healing assay. This assay was then used to evaluate the potential of novel small molecule integrin antagonists in preventing tumour cell migration. In H & N tissues, αvβ3, αvβ5 and α5β1 integrins are extensively expressed in tumour tissues but weakly expressed in normal tissue from the same patient. Further, prostate cancer tissues expressed variable levels of αvβ3, αvβ5 and α5β1 integrins. αvβ3 and αvβ5 integrins were expressed in variable levels in OSC-19, PC-3, DU145, DLD-1, HT-29, HUVEC, MCF-7, MCF-7ADR and M14 human tumour cell lines and in OSC-19, PC-3, HT-29 and MCF-7 xenografts. α5β1 integrin was expressed in all cell lines and xenografts except in MCF-7 cell line and HT-29 cell line and xenograft. Overall, the expression was elevated in xenografts compared to the corresponding cultured cells. Based on the expression profile and ability of cells to migrate, three cell lines (DLD-1 colon, DU145 prostate and OSC-19 HNSCC) were selected as models to further evaluate the potential of novel small molecule integrin antagonists to inhibit cell migration. The cell lines were characterized by using neutralizing antibodies against αvβ3, αvβ5, and α5β1 integrins to determine which of these three integrins were primarily involved in tumour cell migration. In DLD-1 and DU145, blocking αvβ5 and αvβ3 significantly inhibited migration, whilst the migration of OSC-19 was 50% inhibited by a multi-integrin inhibitor combination. Among the antagonists, ICT9055 and ICT9072 significantly decreased DLD-1 cell migration by 70% and 60% respectively while ICT9023, ICT9024, and ICT9026 significantly decreased DU145 cell migration by 60%, 60% and 50% respectively. The findings suggest that single integrin inhibition is not sufficient to prevent cell migration whereas dual or multiple inhibition is more effective. Two novel anti-migratory agents were identified in colon cancer and three in prostate cancer which would warrant further investigation. / Princess Nora Bint Abdul Rahman University

Predicting the prognosis of patients with prostate cancer is a clinically important problem. Previous studies have indicated that the expression of haptoglobin-related protein epitopes in samples of breast cancer in early stages was associated with earlier relapses and higher risk for tumor recurrence. Oncogenic antigen 519 (OA-519) is the new marker designation for molecules expressing haptoglobin-related protein epitopes. The objective of this immunohistochemical study was to examine OA-519 expression in prostate cancer samples and its relationship to the established prognostic indicators of tumor grade, tumor volume, and clinical stage. Forty-two consecutive tissue samples of prostate adenocarcinoma were examined using an affinity- purified anti-OA-519 antibody. Twenty specimens (48%) tested positive, whereas 22 (52%) tested negative. No staining was observed in normal or hyperplastic prostate tissue. Staining occurred in 6 of 9 (67%) grade III, 14 of 23 (61%) grade II, and in none of 10 (0%) grade I cases (I vs. II and/or III: Fisher exact test, P < 0.006). Twenty-three of the 42 samples were transurethral resection specimens with cancer; 11 (48%) of these tested positive. The mean percentage of tissue chips with tumor, a measure of tumor volume, was significantly higher in the positive group (57%) than in the negative group (15%) (P = 0.004). The proportion of positively stained cases increased with advancing clinical stage, with 25% of Stage A cases expressing OA-519, and 46%, 67%, and 64% of Stages B, C, and D, respectively, expressing OA-519. OA-519 expression correlates with higher tumor grades, larger tumors, and possibly with advanced stage, and thus, it is potentially of prognostic value in prostate cancer.