Made available in DSpace on 2014-10-09T12:24:01Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:07:55Z (GMT). No. of bitstreams: 1 00481.pdf: 828758 bytes, checksum: 1c5f5457d167dcdae526c01f18297ab6 (MD5) / Dissertacao (Mestrado) / IEA/D / Instituto de Biociencias, Universidade de Sao Paulo - IB/USP

Cyst enlargement is an important part of the initial stage of organ formation. in vitro experiments have shown that the speed of expansion of this nearly spherical object is affected by many factors. Moreover, in many cases, bursting of the cyst cell layer takes place from time to time, leading to nearly periodic deflation of the cyst sphere. Biophysical models have been proposed that take into account the build-up of osmotic pressure in the lumen and cell proliferation in the cyst layer. Here we integrate two previous models for Hydra cyst swelling and collapse cycle and MDCK cyst growth saturation respectively to describe the Caco-2 cyst swelling and rupture cycle in a series of experiments carried out in Prof. Jian-Dong Huang’s lab at HKU. Gene expression analysis is also carried out to identify pathways that are over-expressed and active during cyst enlargement.

Orientador: Paulo Traiman / Banca: Fred Ellinger / Banca: Ana Glória Pontes / Resumo: Estudo dos fatores de risco das pacientes com citologia oncológica cervical e anal alteradas. 64 pacientes encaminhadas ao Ambulatório de Colposcopia da Faculdade de Medicina de Marília-SP foram estudadas prospectivamente de fevereiro de 2006 até fevereiro de 2007, com diagnóstico citológico de HSIL. Suas histórias clínicas foram escolhidas, as citologias cervical e anal coletadas, e a colposcopia e anuscopia realizadas com Tido acético a 2%, lugol e azul de toluidina a 1%, respectivamente. A espátula de Ayres e a cytobrush foram usadas para a coleta das citologias no colo e a cytobrush, no ânus. As lâminas foram fixadas em álcool 70% e as biópsias foram realizadas com a pinça Gaylor-Medina e fixadas em formol a 10%. O Teste do Qui-Quadrado de Pearson e o Teste Exato de Fisher foram empregados para a análise estatística. Os perfis sócio-demográfico e epidemiológico das pacientes foram traçados. Nas 64 pacientes da amostra estudada, no perfilsócio-demográfico, a idade média encontrada foi de 36,6 anos. 60,9% das pacientes eram casadas; 84,4% tiveram até cinco filhos. 100% tinham baixo nível sócio-econômico. Segundo perfil epidemiológico, em média o número de parceiros sexuais foi 2,9 por paciente. A menarca ocorreu aos 12,8 anos; a coitarca, aos 16,5 anos. 76,6% das pacientes usaram método Dntraceptivo hormonal. 75% não praticavam coito anal. 70,3% não fumavam e 100% não bebiam nem usavam drogas. 68,8% colhiam citologia de rotina e 89,1% não tinham história de câncer familiar. Nas cinco pacientes com citologia anal alterada, no perfil sócioográfico, a idade média foi de 30,6 anos. 60% das pacientes eram casadas; 60% tiveram . dois filhos. 100% tinham baixo nível sócio-econômico. Segundo o perfil epidemiológico... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Study of the risk factories in the patients with changed cervical and anal oncological cytology. Methods: 64 guided patients to the Colposcopy Ambulatory from Marília Medical School (SP) were prospectively studied between February 2006 and February 2007, with HSIL cytological diagnoses. Their clinical histories were picked up, the anal and cervical cytologies were gathered and the colposcopy and the anuscopy were done with acetic acid 2%, Lugol's iodine and blue of toluidine 1%. Ayres spatula and the cytobrush were used to the cytology collects at the cervix and the cytobrush at the anus. The microscopic slides were fixed in 70% alcohol and the biopsies were done with Gaylor-Medine's pincers and fixed in formaldehyde 10%. Pearson's Chi-Squared test and Fisher Exact test were used to the statistic analyses. Results: the patients' social demography and epidemiological profile were braided. In the 64 patients from the studied sample, at the social demography profile, the medium age found was 36,6 years old. 60,9% patients were married; 84,4% had got until five children. 100% were low socio-economic leveI. At the epidemiological profile, in medium the number of sexual partners was 2,9 per patient. The menarche happened at 12,8 years old; the first coitus at 16,5 years old. 76,6% patients used hormonal contraceptive method. 75% didn't practice anal coitus. 70,3% didn't smoke and 100% patients didn't used alcohol or any drugs. 68,8% patients picked routine cytology up and 89,1% didn't have familiar cancer history. In the five patients with changed anal cytology at the social demography profile, the medium age found was 30,6 years old. 60% patients were married; 60% had got until two children. 100% were low socio-economic leveI. At the epidemiological profile, in medium... (Complete abstract click electronic access below) / Mestre

Dissertação(mestrado) - Universidade Federal do Rio Grande, Programa de Pós- Graduação em Educação em Ciências: Química da Vida e Saúde, Instituto de Educação, 2011. / Submitted by EDUARDO PENA (edupenaa@hotmail.com) on 2012-10-26T01:02:06Z No. of bitstreams: 1 O LIVRO DIDÁTICO DE BIOLOGIA E A TEMÁTICA CITOLOGIA.pdf: 1531992 bytes, checksum: 20a745db700fb35357237ba01995979a (MD5) / Approved for entry into archive by Bruna Vieira(bruninha_vieira@ibest.com.br) on 2013-06-19T14:57:59Z (GMT) No. of bitstreams: 1 O LIVRO DIDÁTICO DE BIOLOGIA E A TEMÁTICA CITOLOGIA.pdf: 1531992 bytes, checksum: 20a745db700fb35357237ba01995979a (MD5) / Made available in DSpace on 2013-06-19T14:57:59Z (GMT). No. of bitstreams: 1 O LIVRO DIDÁTICO DE BIOLOGIA E A TEMÁTICA CITOLOGIA.pdf: 1531992 bytes, checksum: 20a745db700fb35357237ba01995979a (MD5) Previous issue date: 2011 / O presente trabalho objetivou analisar como o tema Citologia é abordado em livros didáticos de Biologia, fornecidos pelo Programa Nacional do Livro para o Ensino Médio 2009 (PNLEM 2009). Para isso, foi elaborado um roteiro que conteve aspectos gerais e específicos, sendo os aspectos gerais relacionados à estrutura básica do livro, como a presença de figuras, tabelas e quadros comparativos, bem como a presença de leituras complementares. Já os específicos estão relacionados ao conteúdo, quais temas foram sugeridos como leituras complementares, a abordagem do tema e também a análise dos conceitos discutidos. A escolha por este objeto de estudo é devido ao mesmo ser utilizado amplamente por profissionais da área de educação, seja ela Pública ou Privada, e também pelos estudantes como referência para trabalhos escolares. Além disso, este recurso é fornecido gratuitamente aos professores e estudantes da rede Pública de Ensino, desde que as suas escolas estejam devidamente cadastradas para o recebimento dos mesmos. Com esse estudo, percebeu-se que em alguns livros as figuras utilizadas não são mencionadas ao longo do corpo textual, há a presença de leituras complementares em todos os livros analisados, bem como sugestões de experimentação. Porém, também foram encontrados alguns erros conceituais e constatados a utilização de muitos termos técnicos que, embora próprio desta área do saber, algumas vezes estes termos não foram explicados nos livros. / This work has set as a goal to analize how the issue of cytology is approached on textbooks of biology, provided by the Book's National Program for the High School 2009 (PNLEM 2009). For this, it was prepared a guide with general and specific aspects. The general aspects were related to the basic structure of the book, such as the presence of images, tables, comparative tables and supplementary readings. On the other hand, the specific aspects are related to the content, which issues were suggested as supplementary readings, the approach of the issue and also the analysis of the concepts discussed. The reason to choose this theme is due to the same is also widely used by education professionals, from the public or private schools, and also by students, as a reference. Besides, this feature is provided free to teachers and students of public schools, since their schools are properly registered to receive them. With this work, it was noticed that in some books the images used are not mentioned in the text's body, there are supplementary readings in all books analyzed and trial suggestions. However, it was also noticed some misconceptions and found the use of many technical terms that, even though from this area of knowledge, sometimes these terms were not explained on the books.

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Philosophiae Doctor - PhD / The role of male germ cell death in spermatogenesis is an important one as it removes dysfunctional or genetically damaged germ cells and is necessary to maintain an optimal germ cell to Sertoli cell ratio. The formation of the bloodtestis barrier requires the elimination of excessive germ cells and a surge of germ cell apoptosis occurs prior to puberty regulating the ratio of germ cells to Sertoli cells. The aim of this study was to evaluate the presence of four apoptotic markers on sperm from patients with various grades of fertility using flow cytometry. Furthermore, any correlations between the apoptotic marker assays and the standard semen analysis results were identified. This study compares early and late parameters of apoptosis with morphological features in spermatozoa in the same samples. The three sample groups were identified as: teratozoospermic [G-pattern] (n=26), teratozoospermic [P-pattern] (n=98) and oligoteratozoospermic [Ppattern] (n=36). Standard semen analysis was conducted on the semen samples according to the WHO guidelines. Four apoptotic marker assays using flow cytometry was applied in this study to examine the apoptotic alterations in ejaculate sperm. These assays included the Annexin-V staining for the determination of phosphatidylserine exposure, APO-Direct to identify DNA fragmentation, caspase-3 to detect expression of this active protease during early apoptosis and Fas expression. For the Annexin-V and caspase-3 assays, statistically significant differences (P<0.05) were evident between the three groups. No significant differences (P>0.05) were found between the groups with respect to the APO-Direct assay. A significant difference (P<0.05) was found when comparing the teratozoospermic [G-pattern] group and the oligoteratozoospermic [P-pattern] group for the Fas assay. A strong positive correlation was evident between the Fas and the caspase-3 assays in the teratozoospermic [G-pattern] group. For the teratozoospermic [P-pattern group] the following positive correlations existed between the APO-Direct and the Fas assays, APO-Direct and caspase-3 assays and between caspase-3 and Fas assays. The only strong positive correlation was between the caspase-3 and APO-Direct assays in the oligoteratozoospermic [P-pattern] group. The presence of spermatozoa showing microscopic features resembling apoptosis has been identified in ten human ejaculate samples per sample group. Electron microscopy was used to identify morphological features of apoptosis in these human sperm samples. Classical apoptosis as observed in diploid cells could be identified in sperm and these included: loose fibrillarmicrogranular chromatin network, presence of vacuoles in the nuclear chromatin, membranous bodies within the vacuoles of the chromatin, partially disrupted nuclear membranes, plasma membrane protuberances and apoptotic bodies containing cytoplasmic vacuoles and dense masses. This study has confirmed that semen samples with abnormal semen parameters exhibit the presence of apoptotic markers in sperm. The identification of apoptotic markers on the sperm suggests that abnormalities occur during their developmental process, however, the exact mechanism thereof remains unclear. These findings may suggest that certain apoptotic markers may be an indicator of abnormal sperm function and possibly indicative of male infertility. / South Africa

Histology and electron microscopy were used to describe and compare the structure of the dorso-lateral pedal defensive glands of three species of marine Basommatophora, Siphonaria capensis, S. serrata and S. gigas. All three species possessed multi-cellular glands that were larger and most abundant in S. capensis. In S. capensis and S. serrata, defensive glands were composed of two types (type I and II) of large secretory cells filled with product and some irregularly shaped support cells that surrounded a central lumen. The product of both cell types was produced by organelles confined to the bases of the cells. The entire gland was surrounded by a well developed layer of smooth muscle and collagen. Type I cells stained positively for neutral and sulphated mucins, and at the transmission electron microscope level the product had a reticulated appearance. By contrast type II gland cells stained very positively for acidic mucins and the secretory product was formed as large granular vesicles. The product from both types of cell, which appeared to be secreted by holocrine secretion, mixed in the lumen of the duct. Individuals of Siphonaria gigas had two types of lateral pedal glands, a large multi-cellular type and a tubular unicellular gland. The multi-cellular glands, which were surrounded by poorly developed muscle, contained one type of gland cell that stained for neutral and sulphated mucins only, as well as some support cells. The tubular glands contained a heterogeneous product that stained very positively for neutral and sulphated mucins. In addition two species of shell-less marine Systellommatophorans, Onchidella capensis and O. hildae, were examined. Onchidellids also posses large marginal, multi-cellular, epidermal glands that produce a repugnatorial secretion. Like the multi-cellular epidermal glands of siphonariids, those of onchidellids are surrounded by layers of smooth muscle. The muscular capsule was particularly well developed in both species of onchidellid, but more so in O. hildae. In addition, this study has shown that unlike siphonariids, muscle fibres run between the gland cells of O. capensis and O. hildae. Unlike siphonariids, onchidellids have a layer of epithelial cells lining the lumen of the gland. The well developed muscle layer and the strands of muscle running between the different gland cells indicates that the glands can be constricted to forcibly propel their secretions along the length of the duct and away from the body of the animal. Based on their product, glands of O. capensis were comprised of five different types of secretory cell and O. hildae only four. Histological and histochemical staining of the glands of showed that the secretory product is largely made up of acidic mucopolysaccharides and neutral and sulphated mucins. A single species from the order Eupulmonata, Trimusculus costatus, was examined and the glands were very different to the species from the siphonariids and onchidellids. Trimusculus costatus does not have large multi-cellular glands encapsulated in a well developed muscle layer, but based on their cell contents, three different types of large unicellular gland cell can be recognised. The glands of T. costatus gave positive results for acid, neutral and sulphated mucins, but negative results for carboxylated mucin. It is possible that the mucous secreted by T. costatus is also an anti-bacterial agent and whilst not totally eliminating bacteria may prevent the accumulation of epibionts on these sedentary limpets. The acidic or sulphated nature of the secretions may help in this role. The defensive mucous secretions of Siphonaria and Onchidella contain polypropionate derivatives, whilst the active ingredients of Trimusculus mucus have been identified as labdane diterpenes, similar to those produced by opisthobranchs. The structure of the glands thought to produce these repungnatorial secretions is very different, with the glands of T. costatus resembling those of the opisthobranchs.

Basic Helix-Loop-Helix proteins are transcriptional regulators crucial for many development processes. Using gain- and loss-of-function analysis in zebrafish, the functional role of two members of this protein family, lyl1 (Lymphoblastic leukaemia 1) and Id4 (Inhibitor of differentiation 4) in stem cell fate was determined. Ectopic overexpression of lyl1 resulted in the expansion of haematopoietic stem cell pool and its progeny promoting erythrocyte differentiation and suppressing myeloid differentiation. TALEN-mediated lyl1-/¬- embryos developed normally but displayed distinct marker gene expression during primitive and definitive haematopoiesis establishing a role for lyl1 in both waves of haematopoiesis. During primitive haematopoiesis expression of scl/tal1 and gata1 was unaltered but expression of pu.1 was increased suggesting that lyl1 antagonises myeloid differentiation. Lyl1-deficiency resulted in reduction of Gfi1aa expression during primitive and definitive haematopoiesis. In addition, a reduction in the expression of c-myb in the caudal hematopoietic tissue and rag1 in the thymus was observed indicating that lyl1 is required to maintain the definitive haematopoietic stem cell pool and to drive T lymphopoiesis. In adult zebrafish lyl1 regulates lineage choice driving lymphopoiesis and suppressing myelopoiesis. Morpholino-mediated knockdown of Id4 alone or in combination with p53 resulted in reduced cell proliferation, increased cell death and premature neuronal differentiation. Phenotypic analysis of TALEN-mediated Id4 mutants confirmed that Id4 plays a crucial role in the expansion of neural stem cells and timing of neuronal differentiation. Inhibition of p38MAPK in Id4 morphants as well as Id4-/- mutants resulted in a phenotypic rescue establishing that Id4 negatively regulates p38MAPK activity to ensure normal neurogenesis.

Stable telomeres play a key role to the survival of cancer cells; therefore, different cancer chemotherapeutic approaches have been developed in order to disrupt or destabilise telomeres or telomerase. One of the newest methods is the disruption of vital protein–protein interactions in the telomere, such as that between shelterin components TRF1 and TIN2. The principal aim of this project was to obtain a novel peptide-like molecule, an analogue of a key interacting region of TIN2 that could compete effectively for the binding sites on TRF1 and so lead to the destabilisation of telomere structure. Molecular modelling and simulations were undertaken as the starting point of the project. Structure-based drug design was applied, starting from the available crystal structure data. A library of peptide analogues of the TRF1-binding motif in TIN2 was designed using the MM-GBSA simulation method to predict binding affinities. Then, a number of the peptide analogues were selected from the library for further investigations. The secondary goal was to investigate the accuracy of the predicted ΔGbinding values and try to optimise them; the latter aim was set out after finding a significant difference in the predicted binding free energy values after repeating the identical protocol for the same complex system. Therefore, different approaches were applied to optimise the predicted ΔGbinding values. Subsequently, selected TIN2 peptide analogues were synthesised in the laboratory using Fmoc solid-phase peptide synthesis. Then, the hTRF1 protein was expressed and purified in preparation for the development of the in vitro assay. Finally, biophysical evaluations and screening of the peptide analogues were performed using fluorescence polarisation assay. One of the peptide analogues developed in this study was identified as an early lead compound. In addition, the findings of this research showed that the ΔGbinding values of the peptide analogues have significantly improved accuracy after optimisation. As a result of these investigations, suggestions were identified for future research.

An in vitro phagocytosis assay system was developed and characterized for studying the specific receptor-mediated phagocytosis of bovine ROS by bovine RPE cells. The phagocytosis of ROS was detected qualitatively by electron microscopy and quantitatively by treating RPE cells with radioiodinated ROS or by probing ROS-treated RPE cells with a radiolabeled antirhodopsin monoclonal antibody. The binding sites for various antirhodopsin monoclonal antibodies were localized as an essential step in their application as immunochemical probes for analysis of the structure and function of rhodopsin. Five monoclonal antibodies raised against rhodopsin have been shown to be directed against the N-terminal regions on the basis of their reactivity to an immunoaffinity purified 2-39 glycopeptide, a 2-16 tryptic glycopeptide and a 1-16 synthetic peptide as measured by radioimmune competition assays. Limited proteolysis, immunogold-dextran labeling and competitive inhibition studies identified two antirhodopsin monoclonal antibodies which bound to internal cytoplasmic loop regions of rhodopsin. Finally, the binding sites for these and other C-terminal specific antirhodopsin monoclonal antibodies were used to elucidate the proposed transmembrane helical model of rhodopsin. An antirhodopsin monoclonal antibody (rho 4D2), which bound to rhodopsin in glutaraldehyde-fixed ROS plasma membranes, was employed as an immunocytochemical probe in studying the possible role of rhodopsin in the binding and phagocytosis of rod outer segments. An immunoaffinity purified 2-39 N-terminal rhodopsin glycopeptide, a synthetic 1-16 peptide analogue of rhodopsin and phospholipid vesicles reconstituted with rhodopsin were all found to be ineffective in inhibiting the phagocytosis of ¹²⁵I-labeled ROS by RPE cells. In essence, these results provided compelling evidence that rhodopsin in the ROS plasma membrane does not function as the ligand for recognition by RPE cells. The molecular properties of the ROS cell surface ligand(s), which are involved in recognition by bovine RPE cells, were studied by limited-proteolytic digestion in conjunction with quantitative phagocytosis assays. Mildly trypsin-treated ROS were found to be less effectively phagocytized than untreated ROS by bovine RPE cells. Moreover, the glycopolypeptides (34kD and 24kD) released from the ROS cell surface by trypsin were capable of inhibiting ROS phagocytosis. The ROS plasma membrane specific, ricin-binding, 230kD glycoprotein was observed by SDS-gel electrophoresis and western blotting to be highly trypsin sensitive under these conditions. Hence, ricin affinity chromatography and immunoaffinity chromatography were employed in an attempt to purify this 230kD glycoprotein from ROS membranes. Enriched preparations of the 230kD glycoprotein were reconstituted into phospholipid vesicles and effectively used to inhibit the phagocytosis of ROS by RPE cells. In summary, a ROS plasma membrane specific, 230kD glycoprotein has been identified and isolated; this protein may act as a ligand in specific ligand-receptor interactions between ROS and RPE cells. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate