Orientadores: Maria Elvira Pizzigatti Corrêa, Cecília Amélia Fazzio Escanhoela / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-20T05:39:33Z (GMT). No. of bitstreams: 1 Soares_TaniaCristinaBenetti_D.pdf: 3640740 bytes, checksum: 3cc935c816cada9809557953ff7cae3e (MD5) Previous issue date: 2012 / Resumo: A doença do enxerto contra o hospedeiro crônica (DECHc) é um processo inflamatório aloimune o qual resulta da resposta celular (células T) do doador contra o receptor em pacientes tratados pelo transplante de células progenitoras hematopoiéticas (TCTH). Os órgãos mais afetados são pele, mucosa oral, glândulas salivares e fígado. O objetivo deste trabalho foi avaliar os achados histopatológicos das glândulas salivares menores (GSM) e fígado dos pacientes afetados pela DECHc, comparando-os entre si, com as amostras destes órgãos de pacientes que não desenvolveram DECHc pós TCTH e com pacientes que não foram tratados pelo TCTH. Amostras de GSM e fígado de pacientes tratados por TCTH mieloablativo, de doadores aparentados com HLA idêntico, entre 1994 e 2006, foram analisadas. Cinquenta e sete pacientes foram selecionados, sendo 36 com DECHc oral e hepática e 21 sem DECH. O diagnóstico de DECHc foi definido por critérios clínicos (extensa/localizada), laboratoriais e de seguimento. Amostras sem alterações de pacientes não transplantados também foram avaliadas (19 de GSM e 20 de fígado). Os espécimes foram corados em hematoxilina e eosina, ácido periódico de Schiff (PAS), Tricrômio de Masson, Reticulina e Perls e tratados pela técnica de imuno-histoquímica para CD45, CD45RO, CD68, CD4, CD8, CD138 e AE1/AE3. Os critérios definidos pela classificação proposta pelo grupo de trabalho em histopatologia da reunião de consenso do National Institutes of Health (NIH) foram empregados para a avaliação de todas as amostras de ambos os órgãos. Nos espécimes corados pelo PAS, foi tomada a medida da área acinar das GSM, em imagens digitalizadas. No fígado de pacientes que desenvolveram DECH, foi observado aumento estatisticamente significante do número de células imunomarcadas para CD8 e CD45RO, quando comparado ao dos outros 2 grupos...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: Chronic graft-versus-host disease (cGVHD) is an alloimmune inflammatory process, which results from a donor-origin cellular response (T cells) against host tissues. The organs mostly affected are skin, oral mucosa, salivary glands and liver. The aim of this study was to evaluate the histopathological findings of minor salivary glands (MSG) and liver of patients affected by cGVHD, comparing the findings each other and with those of the patients who did not develop cGVHD after HSCT and patients who were not underwent HSCT. MSG and liver samples from patients who underwent myeloablative HLA-matched HSCT from sibling donors, between 1994 and 2006, were analyzed. Fifty-seven patients were selected, being 36 with oral and hepatic cGVHD and 21 without GVHD. The diagnosis of cGVHD was defined through clinical/ laboratory criteria and follow-up. Samples from non-transplanted patients were also evaluated (19 MSG and 20 liver specimens). The specimens were stained with haematoxylin & eosin, periodic acid-Schiff (PAS), Masson's trichrome, reticulin and Perls, and immunolabeled for CD45, CD45RO, CD68, CD4, CD8, CD138, and AE1/AE3. The criteria defined in the classification proposed by the consensus meeting of the National Institutes of Health were used for the evaluation of all samples of both organs. Digital images of the PAS stained sections were used to estimate the MSG acinar area. In the liver of patients who developed GVHD it was observed a statistically significant increase in the number of CD45RO and CD8 immunostained cells, comparatively with the other 2 groups...Note: The complete abstract is available with the full electronic document / Doutorado / Patologia / Doutor em Estomatopatologia

O conhecimento dos estágios iniciais do desenvolvimento embrionário de peixes é de extrema importância para o estudo de espécies nativas com potencial para a piscicultura, uma vez que permite o estabelecimento de diretrizes para a criação destes animais. Este projeto estudou o desenvolvimento embrionário da piapara (Leporinus elongatus), um peixe de grande importância na pesca esportiva e profissional na bacia dos rios Pardo e Jequitinhonha, visando compreender as fases desse animal em diversos estágios de desenvolvimento, utilizando marcadores ósseos que possibilitaram visualizar o desenvolvimento ósseo da espécie. As Proteínas Ósseas Morfogenéticas (BMP-2 e BMP-4) são consideradas moléculas essenciais reguladoras no desenvolvimento embrionário e na formação óssea, sendo ainda pouco estudadas em peixes; tais proteínas puderam ser observadas apenas no estádio larval até o período juvenil, não sendo evidenciadas nos estágios anteriores. Foram utilizadas também técnicas de Microscopia Eletrônica de Varredura e histológicas, onde foi possível visualizar as fases principais do desenvolvimento embrionário, entre elas, clivagens, diferenciação do embrião, formação dos órgãos principais, abertura de boca, pigmentação dos olhos, surgimento das nadadeiras e sistema branquial, dados estes que facilitam a compreensão sobre a ontogenia; além de criar dados embriológicos e anatômicos dessa espécie ainda pouco explorada, conhecimentos estes, imprescindíveis à biologia pesqueira e cultivo das mesmas, sendo também um auxiliar a novas pesquisas. / The knowledge of the early stages of embryonic development in fish is of great importance for the study of native species with potential for aquaculture, since it allows the establishment of guidelines for the breeding of these animals. This project studied the embryonic development of piapara (Leporinus elongatus), a fish of great importance in professional and amateur fishing from the basin of the Pardo and Jequitinhonha rivers, aiming to understand the various stages of development, using bone markers that allowed observation of the bone development in this species. The Bone Morphogenetic Proteins (BMP-2 and BMP-4) are known as key regulatory molecules in embryonic development and bone formation, and information on this subject is scarce in fish. Results shown these proteins could be observed only between the larval to the juvenile stage, not being seen at earlier stages. Scanning electron microscopy and histological techniques, where it was possible to observe the main stages of embryonic development , including , cleavage , embryo differentiation , development of major organs , mouth opening , eye pigmentation , appearance of fins and gills system. This data contributed for the understanding of ontogeny, and provided embryological and anatomical data that may help other studies like reproductive biology of this species that surely will improve reproductive techniques, important goal for raising the piapara.

This study was carried out to determine the phenotype of special dog mammary gland tumours that were grown in nude mice. 26 tumours were examined by the immunohistochemical ABC-Elite protocol. The tumour tissues were labelled with following anti-human antibodies: - AE1/AE3 (pankeratin antibody) labelled epithelial and myoepithelial cells - CD 31 labelled endothelial cells - desmin labelled cross-striated and smooth muscle cells - myosin labelled cross striated muscle cells - neurofilament (NF) labelled nerve cells - osteopontin labelled preosteoblasts, osteoblasts and osteocytes - p63 labelled nuclei of the myoepithelial cells - smooth muscle actin (SMA) labelled the cytoplasm of myoepithelial cells - type I collagen labelled the extracellular matrix in connective tissue and bone - type II collagen labelled the extracellular matrix in cartilage - vimentin labelled fibroblasts, fibrocytes, lipocytes, smooth muscle cells, endothelial cells, nerve cells, macrophages and myoepithelial cells The tumours were also submitted to a double immunolabelling study using p63 and SMA. The study could not give a final conclusion about the origin the tumours. There was still need for more research to answer that question. However, the immunohistochemical technique was analysed in detail, in order to obtain perfect labelings. Initially, all the antibodies were tested on normal dog tissue, to acquire the best working dilutions with the lowest background problems. In the tumours, good results were obtained with these dilutions for the antibodies p63, SMA, vimentin, desmin, NF, AE1/AE3 and CD 31. Except for type I collagen, type II collagen and osteopontin that gave too much unspecific labelling of the mouse connective tissue. Even, when using the Vector® M.O.M. blocking kit, the results were still very difficult to interpretate. The antigen retrieval methods were evaluated for all the antibodies. The antibodies p63, SMA, vimentin, desmin, AE1/AE3, myosin, neurofilament and CD 31 needed the antigen retrieval treatment. The antibodies type I collagen and type II collagen needed the treatment with the enzyme pepsin, while osteopontin did not need any pretreatment at all. The double immunolabelling with p63 and SMA gave excellent results. Different combinations were tried out with different substrates, namely Vector® Nova RED, Vector® DAB and Vector® SG. Vector® methyl green was used as counterstaining, but it interfered with the other substrates, and better results were obtained without this counterstaining.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Athymic nude mouse models have been extensively used to study biological behaviour of normal and diseased human tissues. In such models, immune-deficient mice act as hosts for cysts constructed from human material. A unique biocyst model that entails transplantation of human vaginal cysts into athymic nude mice has been implemented to study diseases of oral mucosa. To date, only one immunohistochemical study of this biocyst model has been reported. Nevertheless, conclusions made in that study were only based on the observed expression patterns of human and murine markers. Statistical assessment of immunohistochemical data had been omitted by the investigator. Therefore, the objective of this study was to further delineate the immunohistochemical profile of normal human vaginal tissue and human vaginal tissue that had been xenografted into nude mice.Experimental cysts constructed from human vaginal mucosa were xenografted into athymic nude mice and harvested 9-weeks post transplantation. Immunohistochemical analysis of normal human vaginal tissue and human vaginal tissue that had been xenografted into nude mice was performed using a panel of human and murine markers. Expression patterns of human and murine markers were assessed. Human markers included cytokeratin 1,cytokeratin 5, cytokeratin 13, cytokeratin 14, collagen type IV, laminin, elastin, fibronectin,Langerhans cells and VEGFR-3. Murine markers included collagen type IV, laminin,fibronectin, Langerhans cells and VEGFR-2. Staining intensities were quantified and statistically analysed using one-way ANOVA with subsequent Friedman’s test for multiple comparisons. Since the sample size was small, the power of the test statistic was enhanced by including Dunn’s post-test for further multiple comparisons. A strong positive expression of all cytokeratins was detected in both normal and xenografted vaginal tissues. Human markers that exhibited weak to moderate positive expression were collagen IV, laminin, fibronectin and VEGFR-3. Human elastin and human Langerhans cells exhibited strong and varying expression patterns respectively. Weak expression patterns for all murine markers were reported, with an exception of VEGFR-2 which was negatively expressed in all xenografted vaginal tissues. Significant differences (P<0.05) in the mean staining intensities between normal and xenografted vaginal tissues were reported for cytokeratin 1, fibronectin and Langerhans cells. There were no statistical differences (P>0.05) in the mean staining intensities for other markers.In conclusion, immunohistochemical studies proved that human vaginal tissue could not only survive in nude mice, but could also become active and develop structures necessary for survival, in this case, a newly formed stromal layer. The epithelium and stromal layer exhibited a human ecosystem.

Introduction: Knee flexion contractures have a negative impact on function for patients with osteoarthritis (OA). Those with contracture treated with total knee arthroplasty (TKA) have more post-operative pain and worse outcome. Little knowledge is available about patient demographic factors or gene expression in the knee joint capsule in the setting of contracture and severe OA. // Methods: Subjects with primary severe knee OA awaiting a TKA were recruited. We collected subject demographic factors that may be associated with preoperative knee contracture. Subjects’ posterior knee capsule was harvested intraoperatively. Capsule histological analysis was performed using light microscopy. Gene expression analysis was performed using whole genome microarray and immunohistochemistry was used for protein production analysis comparing those with contracture to those without. // Results: Twenty subjects were recruited for the demographics portion of the study (13 contractures and 7 controls), and capsules from 12 subjects (6 contractures, 6 controls) were used for histology, microarray, and IHC analyses. Contracture subjects had longer duration of OA, reduced extension in the contralateral knee, and showed a trend toward elevated body mass index. Tissue cross-sectional areas of adipose, non-adipose and synovial tissues were not statistically different histologically between the two groups. There was increased expression in the contracture group for the genes CHAD, Cyr61, and Sox9. There was a corresponding increase in protein production for CHAD and Sox9. // Conclusions: Screening for OA duration and bilateral knee range of motion (ROM) could be functionally beneficial. When a knee joint contracture is present, correcting for the resulting leg length discrepancy pre- and post-operatively could improve patient outcome. Gene protein products linking capsular cells to the ECM can influence capsular fibrosis and potentially impact ROM.

The sigma-1 receptor (σ-1R) is an endoplasmic reticulum (ER) protein that modulates a number of ion channels. It is hypothesized that σ-1Rs activated with agonist translocate to the plasma membrane. The σ-1R potentiates N-methyl-D-aspartate Receptors (NMDARs), important constituents of synaptic plasticity. NMDARs are anchored in the plasma membrane by Postsynaptic Density Protein-95 (PSD-95). The mechanism behind σ-1R modulation of NMDARs is not known. The results of my investigation confirm that σ-1Rs localize extrasomatically. Following σ-1R activation, σ-1R localization to dendrites and postsynaptic densities (PSDs) is upregulated. Unpublished work from our lab has shown that σ-1Rs associate with PSD-95 and NMDARs. Furthermore, immunocytochemistry (ICC) showed σ-1R colocalization with PSD-95 and NMDAR subunits. After σ-1R activation there was significantly increased colocalization between σ-1R, PSD-95, and GluN2B. Overall, this study may have provided insight into the molecular mechanism behind σ-1R modulation of NMDARs, which could have implications in the understanding of synaptic plasticity.

Orientadores: Pablo Agustin Vargas, Luiz Paulo Kowalski / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-22T08:56:49Z (GMT). No. of bitstreams: 1 Gondak_RogeriodeOliveira_D.pdf: 3115921 bytes, checksum: 6a0ba84f4ad5ecaa9e8dbd843e421cf0 (MD5) Previous issue date: 2013 / Resumo: Na infecção pelo HIV, as células dendríticas (CDs) podem desempenhar vários papéis, incluindo a provável captação inicial do HIV, transporte para os linfonodos, e posterior transferência para células T, desempenhando um importante papel no sistema imune. As manifestações orais observadas em pacientes infectados pelo HIV, incluindo aquelas associadas ao HSV-1 podem estar diretamente relacionadas à injúria das CDs. A proposta deste estudo foi identificar e quantificar as CDs intersticiais na língua de pacientes autopsiados com AIDS e portadores de infecção herpética lingual (n=10), pacientes com AIDS e sem lesões linguais (n=10) e pacientes sem AIDS e sem lesões linguais (n=10) por meio de reações imunoistoquímicas. Além disso, investigamos a população de CDs nos linfonodos e tonsilas palatinas de pacientes com AIDS (n=32) e sem AIDS (n=21). Nos tecidos linguais, foram utilizados os anticorpos contra CD1a e CD83 para identificação das CDs e o anticorpo contra HSV-1 para detecção do vírus da herpes simples tipo 1. Nos linfonodos e tonsilas palatinas foi utilizados além dos anticorpos contra CD1a e CD83, o anticorpo contra fator XIIIa. Para a quantificação das CDs nos tecidos linguais foi utilizado análise histomorfométrica convencional e nos tecidos linfóides foi aplicado o método analítico Positive Pixel Count (software Image Scope). Os resultados mostraram uma intensa depleção na população de CDs em tecidos linguais e linfóides de pacientes com AIDS e a infecção lingual pelo HSV-1 não potencializou a redução de CDs / Abstract: During HIV infection, dendritic cells (DCs) may play several roles, including the probable initial uptake of HIV, transport to the lymph nodes, and subsequent transfer to T cells. Oral opportunistic infections observed in HIV-infected patients, including those associated with HSV-1 may be directly related to injury of DCs. The purpose of this study was to identify and quantify the interstitial DCs in the tongue of autopsied patients with AIDS and lingual herpes (n = 10), AIDS patients with normal tongues (n = 10) and non-AIDS patients with normal tongues (n = 10) by immunohistochemistry. Furthermore, we investigated the DCs population in lymph nodes and palatine tonsils of AIDS patients (n = 32) and non-AIDS patients (n = 21). CD1a and CD83 antibodies were carried out to identify DCs in lingual tissues and HSV-1 antibody for detection of herpes simplex virus type 1. In lymphoid tissues, CD1a, CD83 and factor XIIIa antibodies were carried out to identify DCs. Interstitial DCs were measured by conventional histomorphometry whereas the lymphoid DCs were measured by Positive Pixel Count Algorithm method using ImageScope software. The results showed a decreased population of DCs in lingual and lymphoid tissues of AIDS patients independently of the presence of concomitant infection by HSV-1 / Doutorado / Patologia / Doutor em Estomatopatologia

No description available.

Zebrafish is a frequently used model organism with an array of transgenic lines that have been used indevelopmental and physiological studies. We aim to generate novel transgenic zebrafish reporter lines to study subpopulations of spinal neurons in vivo. The gene editing system called CRISPR/Cas9 system was used to knock in reporter genes such as green fluorescent protein (GFP) or Gal4 transcription factor, to generate transgenic fish lines. Zebrafish embryos were injected with gRNAs targeting gabrb1 or nr4a2a and GFP or Gal4 plasmid, respectively. F0 larvae were screened, positive fish were raised until sexual maturity, and founders characterized to verify germline insertion. Three founders were found for gabrb1 and the location and the direction of the insert verified. The GFP expression was studied during development and differential expression patterns were identified whereas all founders had expression in brain and spinal cord. In parallel, positive fish from the Gal4 injections were raised and will be screened. Immunohistochemistry was performed to check if nr4a2a is expressed in the same cells as known neuronal markers. However, no co-localization was detected. The three gabrb1 founders identified in this study highlight the challenges into creating stable transgenic lines recapitulating true expression of the gene of interest. Sequencing, in-situ hybridization and immunohistochemistry should be performed to verify the line. A possible reason for the varying expression may be that through the knock-in we may interfere with regions regulating gene. The nr4a2a-Gal4 line will be used to perform functional studies. Those experiments will be performed using reporter genes, such as opsins or GCaMP, controlled by Upstream Activation Sequence (UAS). These transgenic lines will provide important insights regarding neuronal subpopulations that express gabrb1 and nr4a2a to unravelhow the locomotor network is formed.

English: Common warthogs (Phacochoerus africanus) and bushpigs (Potamochoerus larvatus), were experimentally infected with classical swine fever virus (CSFv) following the diagnosis of classical swine fever (CSF) subtype 2.1 in 2005 in domestic pigs in South Africa. At that time, no data regarding their susceptibility or the potential lesions in these wild suids were available. Seven sub-adult warthogs and six bushpigs were captured, taken to the high containment facilities of the Transboundary Animal Diseases Programme of the Agriculture Research Council (ARC) - Onderstepoort Veterinary Research Institute, and infected intranasally with the South African isolate. In each experiment, two in-contact control animals of the same species verified intraspecies transmission, while two domestic pigs were used to demonstrate virus virulence and viability. Surviving animals were euthanized 44 days post infection. Formalin-fixed tissue samples collected from all experimental animals were evaluated for histological lesions. The warthogs, which remained clinically normal throughout the study, developed histological lesions that were inconsistently present and sometimes subtle. Three warthogs, including one in-contact control, developed distinct perivascular lymphoplasmacytic cuffing in their brains. Subtle lesions included scant lymphoplasmacytic infiltration of various organs, occasionally accompanied by perivascular cuffing. In contrast, the bushpigs developed overt clinical signs similar to CSF in domestic pigs. Four animals out of six, including two in-contact controls, died or were euthanized during the trial. On post mortem examination, intestinal necrosis and ulceration, purulent rhinitis and pneumonia were present. Acutely affected animals developed lymphoid necrosis and depletion whilst surviving individuals showed perivascular lymphoplasmacytic cuffing in multiple organs. Immunohistochemical demonstration of CSFv antigen using a commercially available mouse monoclonal antibody, WH303, revealed intense, widespread labelling in most tissues of all the warthogs and bushpigs as well as the four domestic pigs used as controls during the trial. A wide range of cell types and tissues reacted with the antibody. These included: mononuclear cells (monocyte-macrophages, lymphocytes and plasma cells), follicular reticular cells, epithelial cells, vascular endothelial cells, mesothelial cells, smooth muscle cells and fibroblasts. Tissues that were labelled included tonsil, lymph nodes, spleen, third eyelid, adrenal gland, urinary bladder, skin, liver, kidney, lung, certain cells within central nervous tissue like the choroid plexus, various parts of the gastro-intestinal tract as well as glandular tissue like the pancreas and salivary gland. The tonsils were the most consistently labelled tissue, while no labelling was noted in myocytes of skeletal or cardiac muscle. From the present work, it was concluded that these wild Suidae are susceptible to CSFv and intra-species transmission under experimental conditions can occur. / Afrikaans: Wilde Afrika varke, nl. vlakvarke (Phacocoerus africanus) en bosvarke (Potamochoerus larvatus) was eksperimenteel infekteer met europese varkpes virus nadat die siekte in kommersiële mak varke diagnoseer is in 2005 (dit was tipeer as subtipe 2.1). Geen inligiting oor die vatbaarheid of potensiële letsels weens europese varkpes infeksie in hierdie wilde varke was beskikbaar nie. Sewe wilde onvolwasse vlakvarke en ses bosvarke is gevang, na die isolasie eenheid van die Onderstepoort Veterinêre Instituut se oor-grens siekte afdeling geneem en intranasal geïnfekteer met die Suid-Afrikaanse isolaat van 2005. Twee in-kontak kontrole diere van dieselfde spesie is gebruik in elke eksperiment om intra-spesie oordraging vas te stel en twee mak varke om virus lewensvatbaarheid en virulensie te demonstreer. Oorlewende diere is uitgesit na 44 dae. Formalien gefikseerde weefsel monsters is versamel van hulle, sowel as van diere wat uitgesit is tydens die eksperiment. Die vlakvarke was klinies normal regdeur die eksperiment, maar het wel histologiese letsels ontwikkel wat subtiel was en ook nie altyd teenwoordig in alle gevalle nie. Drie vlakvarke, waarvan een ‘n in-kontak dier was, het prominente limfo-plasmasitiese perivaskulêre flensing in hul breine ontwikkel. Subtiele letsels het klein hoeveelhede limfoplasmasitiese infiltrasies in verskeie organe en somtyds perivaskulêre flensing ingesluit. In teenstelling, het die bosvarke uitgesproke kliniese tekens soortgelyk aan Europese varkpes in mak varke, ontwikkel. Vier uit die ses diere, insluitend twee in-kontak diere is dood of uitgesit tydens die eksperiment. Met nadoodse ondersoek is daar intestinale nekrose en ulserasie, purulente rinitis en pneumonie gevind. Diere wat dood is, het limfoïede nekrose en limfoïede uitputting getoon, terwyl die oorlewende bosvarke perivaskulêre flensing met limfo-plasma selle in verskeie organe ontwikkel het. Immunohistochemiese demonstrasie van Europese varkpes virus antigen deur gebruik van ‘n kommersieël beskikbare muis monoklonale teenligaam, WH303, het duidelike wydverspreide kleuring in meeste weefsel van die die vlakvarke, bosvarke en mak varke getoon. ‘n Wye reeks van weefsel en sel tipes het met die teenliggam reageer naamlik: mononukliêre selle (monosietmakrofage en limfo-plasma selle), follikulêre retikulêre selle, epiteel, vaskulêre endoteel, mesoteel, gladde spier selle en fibroblaste. Weefsel wat gemerk is met die teenliggaam het ingesluit: mangels, limfknope, milt, derde ooglid, adrenaal klier, urienblaas, vel, lewer, nier, long, sekere selle in die sentrale senuwee stelsel, soos die koroïed pleksus, verskeie dele van die gastro-intestinale stelsel sowel as klier weefsel soos die pankreas en speekselklier. Die mangels was die mees konsekwent gemerkte weefsel, terwyl geen kleuring gevind is in miosiete van skelet of hartspier nie. Uit hierdie werk kon daar afgelei word dat vlakvarke en bosvarke vatbaar is vir Europese varkpes en dat intra-spesie oordraging plaasvind onder eksperimentele omstandighede. / Dissertation (MMedVet)--University of Pretoria, 2011. / Paraclinical Sciences / Unrestricted