<P align="justify">Dans les conditions physiologiques normales, la glycémie est maintenue dans d'étroites limites. La sécrétion d'insuline par les cellules B pancréatiques joue un rôle majeur dans ce contrôle. En effet, l'insuline est la seule hormone capable d'empêcher une élévation excessive de la glycémie. La cellule B pancréatique constitue un exemple d'oscillateur cellulaire dont la dynamique non linéaire permet l'apparition d'une activité électrique "en salves" ("bursting"). Lors de la sécrétion, les cellules B présentent des oscillations du potentiel membranaire, alternances de phase actives, où la membrane est dépolarisée et durant laquelle des potentiels d'action sont produits, et de phases silencieuses, où le potentiel membranaire est stable et hyperpolarisé. La durée relative de la phase active est directement carrelée au taux de sécrétion d'insuline. Cette activité électrique est produite par une modification des flux ioniques transmembranaires. Il est maintenant établi que l'élévation de la concentration intracellulaire de Ca2+ ([Ca2+]i) qui se produit lors de la phase active est impliquée dans le déclenchement de l'exocytose. Néanmoins, les mécanismes impliqués dans la régulation de l'activité électrique restent mal compris.</P><p><p><P align="justify">Dans ce travail, nous examinons le rôle d'une protéine présente dans la membrane de la cellule B, l'échangeur Na+/Ca2+ ,dans la modulation des oscillations du potentiel membranaire et de la [Ca2+]i .Depuis sa découverte au niveau des cellules cardiaques et de l'axone de calmar, l'échange Na+/Ca2+ a été identifié dans de nombreux autres types cellulaires dont la cellule B pancréatique. Ce transporteur utilise le gradient électrochimique du Na+ comme source d'énergie pour l'expulsion des ions Ca2+ du cytosol. Dans les cellules cardiaques, il représente un mécanisme important d'expulsion du Ca 2+ intracellulaire. De plus, étant donné la stoechiométrie de la réaction de transport, son activité induit un courant (I Na/Ca) qui est impliqué dans la prolongation des potentiels d'action cardiaques. Au niveau de la cellule B pancréatique, le rôle de l'échange Na+/Ca2+ reste mal compris. L'absence d'inhibiteur spécifique de la protéine handicape sérieusement l'approche expérimentale.</P><p><p><P align="justify">Dès lors, nous avons utilisé une approche basée sur la modélisation mathématique afin de clarifier l'impact de l'activité de l'échange Na+/Ca2+ sur l'activité électrique et la régulation de [Ca2+]i de la cellule B pancréatique. La modélisation de l'activité électrique de la cellule B pancréatique repose sur le formalisme développé par Hodgkin et Huxley pour la cellule nerveuse. Dans ce contexte, l'application de la conservation de la charge au circuit équivalent de la membrane cellulaire fournit un système d'équations différentielles ordinaires, non linéaires.</P><p><p><P align="justify">Lors de la première partie de notre travail, nous avons mis au point un dispositif expérimental permettant de mesurer le courant électrique lié aux phénomènes de transport ioniques dans les membranes cellulaires. Nous avons pu détecter I Na/Ca et étudier son activation par la [Ca2+]i et le potentiel membranaire. Ces données expérimentales nous ont permis de proposer un modèle minimal pour I Na/Ca. D'autre part, afin d'évaluer expérimentalement l'effet de l'échange Na+/Ca2+ sur l'activité électrique de la cellule B pancréatique, nous avons étudié l'effet d'une réduction de la concentration extracellulaire de Na+ sur les oscillations du potentiel membranaire.</P><p><p><P align="justify">D'un point de vue théorique, à partir de notre modèle minimal pour I Na/Ca ,nous avons élaboré différents modèles mathématiques de l'activité électrique des cellules B. Ces modèles fournissent une prédiction correcte, qualitativement et quantitativement, de l'effet d'une réduction de la concentration extracellulaire de Na+ sur l'activité électrique périodique de la cellule B pancréatique. D'autre part, nos simulations numériques nous ont permis de démontrer la capacité de l'échange Na+/Ca2+ à moduler la durée relative de la phase active des oscillations du potentiel membranaire. De plus, nous avons pu mettre en évidence un mécanisme physiologique original liant la concentration extracellulaire de glucose et l'activité du transporteur. Enfin, nous nous sommes intéressés aux effets induits par la présence de l'échange Na+/Ca2+ sur l'activité électrique périodique et la régulation de [Ca 2+]i de cellules B couplées électriquement et hétérogènes en leurs paramètres. En effet, dans les conditions physiologiques, les cellules B constituent une population de cellules hétérogènes, électriquement couplées au sein des îlots pancréatiques. Il est établi que ce couplage joue un rôle essentiel dans l'apparition et la régulation de l'activité électrique périodique. Nous avons étudié différents modèles mathématiques correspondant à un réseau de cellules électriquement couplées. Nos simulations numériques nous ont permis de démontrer que l'échange Na+/Ca 2+ joue un rôle clef dans la régulation de la [Ca2+]i au sein d'un réseau de cellules B couplées électriquement. Il prévient, localement, l'apparition de niveaux de [ca2+]i trop élevés, potentiellement dangereux pour le métabolisme cellulaire, causés par l'hétérogénéité des paramètres cellulaires.</P><p><p> / Doctorat en sciences, Spécialisation physique / info:eu-repo/semantics/nonPublished

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus which causes acute infectious mononucleosis and is etiologically associated with malignant lymphoproliferative disorders including Burkitt's lymphoma, nasopharyngeal carcinoma, B-cell lymphomas in immunocompromised hosts, Hodgkin's disease, T cell lymphomas, and smooth muscle tumors in allograft recipients. The medical significance of EBV is underscored by its potent growth transforming effects on human B-lymphocytes in-vitro and the potentially oncogenic consequences of infection in-vivo. The majority of EBV-associated malignancies occur in the setting of chronic infection and strong virus-specific humoral immunity, suggesting that cellular immunity is primarily responsible for preventing the outgrowth of EBV-transformed B cells in-vivo. Similarly, primary EBV infection in adolescents and adults stimulates an intense cytotoxic-T-lymphocyte (CTL) response which coincides with a marked reduction in the number of infected B cells in the peripheral blood. Evidence of previous EBV infection can be confirmed by the presence of EBV-specific, HLA-restricted memory T cells in the peripheral blood which inhibit the outgrowth of newly EBV-transformed B cells and efficiently lyse established autologous B-lymphoblastoid cell lines. Worldwide, EBV is responsible for substantial morbidity, comparable to measles, mumps and hepatitis virus, for which vaccines exists. Accordingly, the potential public health impact of an EBV vaccine has reinforced our efforts to identify the immunodominant virus-encoded T-cell epitopes which stimulate naive CTL effectors during acute infection and maintain memory CTL surveillance during convalescence. The EBV-encoded antigens against which the memory CTL response is directed have been partially defined, and include most of the EBV latent proteins (EBNA-2, 3a, 3b, 3c, LP, and LMP-l, 2a, 2b) consistently expressed by in-vitro EBV-transformed B lymphocytes (type-III latency). Importantly, all EBV-associated malignancies express EBNA-1, and as yet no EBNA-1-specific memory CTL have been convincingly demonstrated. Additionally, many EBV-specific CTL lines and clones have been described which do not recognize any of the known latent proteins or other EBV protein antigens tested thus far. Thus while much is known about CTL-mediated immunity against EBV, our knowledge of EBV-derived CTL epitopes remains incomplete. In contrast to the EBV-specific memory CTL response, very little is known about the source of viral epitopes recognized during the primary CTL response to EBV. In this regard, acute infectious mononucleosis represents an ideal model system to study virus-specific, cell-mediated immunity. Acute IM is a self-limited illness characterized by the appearance of "atypical" lymphocytes (CD3+/CD8+/HLADR+), including both virus-specific and alloreactive CTL, which undoubtedly contribute to virus elimination and provide CTL precursors for life-long immunity to EBV. Like other herpesvirus, EBV can undergo either lytic or latent cycle replication. During primary EBV infection many lytic cycle genes are expressed which are likely responsible for stimulating the intense cellular immune response associated with acute infectious mononucleosis. During convalescence a minor population of circulating B cells remain latently infected, harbor multiple EBV episomes, and express only EBNA-1 and possibly LMP-2a (type-I latency). Thus, latency type-I infected B cells in-vivo express a much more restricted spectrum of latent proteins and are therefore not subject to elimination by the same virus-specific CTL as are type-III EBV latently infected cells. Accordingly, many mechanisms have been proposed to explain EBV persistence including; restricted expression of EBV latent genes, reduced levels of cellular adhesion molecules, downregulation of MHC class-I molecules, absence of EBNA-1 T-cell-epitopes, and most recently, EBNA-1-mediated inhibition of antigen processing. While these mechanisms may contribute to ineffective T cell surveillance against latency type-I EBV- infected cells, B cells expressing the full spectrum of latent proteins (type-III) also exist transiently in vivoand maintain detectable humoral and CTL responses to most latent proteins. Our first goal was to identify the virus-encoded immunodominant antigens recognized by in-vivoactivated MHC class-I restricted CTL isolated from college students experiencing primary EBV infection, manifested as acute IM. Following a prodromal period of several weeks, newly EBV infected patients present with signs and symptoms of acute IM, including elevated numbers of activated CD8+ T cells in their peripheral blood, many of which, like memory CTL, are EBV-specific and HLA-restricted. In order to address the issue of EBV persistence and the immune control of EBV-induced lymphoproliferation, we also studied the long-term EBV-specific memory CTL response in these same individuals. Blood from acute IM patients and healthy EBV seropositive donors served as a source of peripheral blood lymphocytes to generate bulk CTL cultures and autologous target cells. The infecting strain of EBV was determined for each patient by DNA-PCR amplification of virus from saliva. Lymphocytes were isolated from whole blood by Ficoll-Paque density centrifugation and T- and B-cell enriched populations were obtained by AET-sheep red cell rosette selection. Autologous B cell blasts served as a source of target cells and recombinant vaccinia virus constructs were used to introduce individual EBV latent genes into target cells. Expression of individual EBV genes in target cells was confirmed by both western blot and immunofluorescence. Primary CTL responses to EBV were evaluated in standard 5lCr release assays using freshly isolated, T-cell enriched PBL from acute IM patients as effector cells. EBV-specific memory CTL responses were evaluated with bulk CTL culture generated by in-vitro restimulation with autologous B-LCLs. FACS analyses were routinely performed on bulk cultures of effector CTL populations in order to more clearly characterize their phenotype. Lastly, monoclonal antibody blocking studies and cold target competition assays were performed in order to accurately identify the viral antigen and MHC components responsible for target cell recognition. Our results based upon evaluation of 35 acute IM patients and 32 convalescent patients demonstrate that the virus-specific primary CTL response is broadly directed against the full spectrum of latent proteins, including EBNA1 and the viral coat glycoprotein gp350, while the memoryCTL response, which essentially lacks EBNA1 reactivity, is directed primarily against the EBNA 3 family of proteins (3A, 3B, 3C). Importantly, the immunodominant response by both primary and memory CTL was directed against the EBNA3 proteins. CTL from 7 of the 35 acute IM patients evaluated recognized EBNA1 expressing targets, and in 4 of these 7 patients, EBNA1 was an immunodominant antigen. Similarly, CTL from 7 of 35 acute IM patients recognized gp350 transfected targets, while no gp350-specific memory CTL responses were observed. While the phenotype of in-vivo primed CTL effectors were CD8+/HLA-DR+/CD11b+, the major subpopulation of memory CTL were CD8+/HLA-DR+/CD11b-. The CD11b "memory marker" reached peaked levels on the first sample day for all patients and gradually declined to baseline levels over a period of several months. In contrast, the CD11b marker was quickly shed from in vitropropogated CTL, over a period of 5-10 days. Target cell lysis by in-vivoactivated CTL was almost completely blocked by antibody directed againt [against] class-I molecules (BBM.1), whereas the effect of blocking target cell lysis by anti-CD8 mAb varied between 40-75%. These findings are consistent with an absolute need for class-I restricted antigen presentation, and imply that CD8 was variably required, likely for the lower affinity TCR/ Ag combinations. Cell lysis mediated by in-vitro-restimulated memory CTL was also largely inhibited by anti-class-I mAb, while anti-CD8 mAb was only mild/moderately effective in blocking target cell lysis, in keeping with the concept that memory CTL bear higher avidity TCR which can recognize antigen independent of CD8. Our detection of only one EBNA1-specific memory CTL response among the 32 patients tested supports the theory that latently infected B cells in-vivo, expressing only EBNA1, escape CTL recogition and thus might serve as a reservoir for viral persistence and/or reactivation. The rare ability to detect an EBNA1-specific memory CTL responses remains a relatively unexplained phenomenon and may involve a number of tolerizing mechanisms including the induction of anergy by presentation of EBNA-1 in the absence of costimulation, clonal deletion of low affinity T cells, the absence of dominant T cell epitopes within EBNA1 or a result of the recently described inhibiting properties of EBNA-1 on antigen processing and presentation. Alternatively, the absence of detectable EBNA1-specific memory CTL may be the result of insufficient or inappropriate restimulation of memory CTL in vitro. We addressed this possibility by attempting to selectively restimulate and expand EBNA1-specific CTL from acute IM patients by using EBNA1 expressing B cells blasts as a stimulus. Effector cells generated in this manner killed target cells in an MHC class-I restricted manner but were specific for an unspecified vaccinia antigen. Interestingly, the phenotype of the effector cells was predominantly CD3+/CD4-/CD8-/γδ T cells. In summary, our findings suggest that a multitude of previously unrecognized, EBV-specific CTL are present in the peripheral blood during acute IM, and include EBNA-1-specific CTL. The importance of accurately defining the in-vivo immune response to EBV is underscored by the ever-growing list of EBV associated malignancies. In addition to providing insights into the oncogenesis and potential treatment of NPC, a newly described link between precursor lesions and EBV infection raises the possibility that heightened immunity to EBV or EBV-infected cells may prevent the development of NPC. An obvious expectation would include extension of such knowledge to other EBV associated malignancies such as B and T cell lymphomas, Hodgkin's lymphomas, and smooth muscle tumors. First however, existing gaps in knowledge regarding the immune response to EBV and EBV-associated malignancies must be closed. Details about the viral gene products which are involved in stimulating a broadly protective, virus-specific immune response in a large number of individuals is fundamental to the design of an effective EBV vaccine. Since the presence of activated CD8+ T cells correlates with the rapid decline of EBV infected B cells in the peripheral blood, a concise description of the EBV-specific CTL response in the setting of acute infection will be necessary for the rational design of an effective acute IM vaccine. Increased understanding of viral escape mechanisms is also likely to contribute to therapeutic modalities to treat autoimmune disorders.

Nas secreções glandulares de anfíbios, muitos dos compostos isolados apresentam ação antifúngica e/ou antibacteriana, cuja função é protegê-los contra infecções, uma vez que estes animais habitam ambientes inóspitos. Os peptídeos têm-se destacado dentre os compostos com atividade antimicrobiana isolados de venenos de diversos animais. Além disso, compostos de natureza não proteica presentes nestas secreções têm demonstrado ação antimicrobiana. O objetivo deste estudo foi, primeiramente, isolar e caracterizar, estruturalmente, compostos com atividade antifúngica relevante. Em um segundo momento, foi realizar um estudo comparativo, através de técnicas ômicas, dos compostos presentes nas secreções das glândulas mucosas e parotoides. Os ensaios antimicrobianos, in vitro, foram realizados com fungos, leveduras e bactérias. O material de baixa massa molecular da água de diálise das secreções das glândulas parotoides apresentou atividade antimicrobiana sobre, principalmente, os fungos filamentosos. Este mesmo material foi submetido a uma filtração em gel, resultando em 11 frações as quais foram testadas contra Microsporum canis no ensaio de diluição seriada. A fração 9 mostrou atividade antifúngica e foi submetida a uma cromatografia de fase reversa, resultando em 9 frações, as quais foram testados contra M. canis. Destas frações, as frações 1 e 9 demonstraram, respectivamente, atividade fungicida e fungistática contra M. canis. No segundo momento, as secreções de ambas as glândulas foram analisadas por nanocromatografia líquida de ultra eficiência acoplada a um espectrômetro de massas de alta resolução. Desta análise, foram obtidos 11.034 compostos, distribuídos entre as secreções de ambas as glândulas. Todos os íons obtidos foram fragmentados (MS/MS) e submetidos à análise no programa PEAKS 6. Foram obtidas 511 sequências de peptídeos e/ou sequências parciais de proteínas das secreções das glândulas mucosas, enquanto que para as secreções das glândulas parotoides foram obtidas 110 sequências. Nas secreções das glândulas mucosas, a maior parte das sequências de aminoácidos são de compostos com massas moleculares entre 1 e 3 kDa, o que corresponde a 82% das sequências obtidas. Já para as secreções das glândulas parotoides, a maioria das sequências de aminoácidos são correspondentes aos peptídeos/proteínas com massas moleculares entre 1 e 2 kDa, o que representa 55% do total de sequências obtidas para esta secreção. As sequências foram analisadas pelo BLAST e revelou que um peptídeo presente nas secreções das glândulas parotoides apresenta 66,7% de identidade com o peptídeo antimicrobiano conhecido como ponericin-G2. Entre as sequências encontradas para as secreções das glândulas mucosas, o destaque foi para o precursor do peptídeo brevinin-1, com identidade de 31,2%. Todas as frações obtidas da filtração em gel das secreções das glândulas mucosas e parotoides foram direcionadas à análise por espectrometria de massas MALDI-TOF, o que resultou em maiores informações sobre a natureza dos compostos presentes nestas frações. A fração 8 das secreções das glândulas mucosas foi fracionada por cromatografia de fase reversa, resultando em 7 frações denominadas Pep1-Pep7. O composto denominado Pep7 teve sua massa molecular (6,012 kDa) determinada através da espectrometria de ressonância ciclotrônica de íons por transformada de Fourier com ionização por electrospray. Os peptídeos Pep4, Pep6 e Pep7 foram sequenciados pelo método de degradação de Edman. Estes peptídeos mostraram identidades com peptídeos antimicrobianos das famílias das tigerinin, shepherin e microcin e brevinins, respectivamente. Este estudo demonstrou que há compostos com atividade antimicrobiana significativa nestas secreções, principalmente para fungos filamentosos. Além disso, foi o primeiro a fazer uma análise comparativa dos compostos presentes nas secreções das glândulas mucosas e parotoides de R. schneideri, destacando a presença de peptídeos antimicrobianos / In amphibian glandular secretions, many of the isolated compounds have antifungal and/or antibacterial action, whose function is to protect the animal against infections, since they usually inhabit inhospitable environments. Peptides have been highlighted among antimicrobial compounds isolated from several venomous animals. In addition, non-proteic compounds present in these secretions have demonstrated antimicrobial action. The aim of this study was to isolate and structurally characterize antifungal compounds from Rhinella schneideri parotoid and mucous secretions. Moreover, in this study it was also performed a comparative study of the composition of mucous and parotid gland secretion by omics techniques. The low molecular weight material from parotoid gland secretion showed antimicrobial activity against filamentous fungi and bacteria. The low molecular weight material that showed antimicrobial activity was subjected to gel filtration, resulting in 11 fractions that were tested against Microsporum canis in serial dilution assay. The fraction 9 showed antifungal activity and it was subjected to a reversed phase chromatography, resulting in 9 fractions, which were tested against M. canis. Among them, fraction 1 and 9 showed fungicidal and fungistatic activity against M. canis, respectively. Gland secretion and the respective low molecular weight material were subjected to gel filtration and, each obtained fraction was analyzed by SDSTricine- PAGE. Secretion of the both glands were also quantitatively analyzed by nano ultraperformance liquid chromatography hyphenated to a high-resolution mass spectrometer. It was obtained 6,460 compounds distributed among secretions of both glands. All obtained ions were fragmented (MS/MS) and analyzed by PEAKS 6 program. 511 sequences were obtained from mucous gland secretion, against 110 sequences from parotoid gland secretion. In mucous gland secretion, most of the amino acid sequences (82%) are compounds with molecular masses between 1 and 3 kDa, while for parotoid gland secretion, most of sequences (55%) correspond to peptides/proteins with molecular masses between 1 and 2 kDa. Sequences were analyzed by BLAST, reveling a peptide in parotoid gland secretion that presents 66.7% of identify with an antimicrobial peptide known as ponericin-G2. Among sequences found in mucous gland secretion, the highlight was to one presenting 31.2% of identify with brevinin-1 precursor. All fractions obtained from gel filtration of the mucous and parotoid gland secretions were analyzed by MALDI-TOF mass spectrometry, which resulted in more informations about the nature of the compounds present in these secretions. Fraction 8 from mucous gland secretion was fractionated by reversed phase chromatography on C18 column, resulting in seven fractions denominated Pep1-Pep7. Pep7 presented a molecular mass of 6.012 kDa when analyzed by Fourier transform ion cyclotron resonance mass spectrometry. In this analysis, six precursor ions were identified, but no sequence was obtained. Pep4, Pep6 and Pep7 were sequenced by Edman degradation method. These peptides showed identity with tigerinin, shepherin and microcin and brevinins, respectively. This study revealed compounds with significant antimicrobial activity in the analyzed secretions, especially to filamentous fungi. In addition, it was the first to perform a comparative analysis of compounds present in mucous and parotoid gland secretion from R. schneideri toad, highlighting the presence of antimicrobial peptides

Indiana University-Purdue University Indianapolis (IUPUI) / Polycystic kidney diseases (PKD) are genetic disorders characterized by fluid filled cysts in the kidney tubules and liver bile ducts. There are two forms of PKD, autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD). The focus of the studies in this thesis has been on ADPKD. The disease progresses slowly and the fluid-filled cysts grow in size due to increased rates of cell proliferation and fluid secretion into the cyst lumen. The expanding cysts compromise the normal kidney function and result in a decrease of renal function to the point of end-stage renal failure in midlife. Cyst enlargement is due, at least in part, to chloride secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Currently therapy is limited to renal cyst aspiration, dialysis, and eventually renal transplantation after organ failure, thus it has critical to determine possible drug therapies for the treatment of PKD. Previous studies showed that cyst fluid caused a secretory response in cells lining the cysts. We hypothesized that once the cyst have expanded and become so large that they burst or leak, which could also occur due to renal injury or aging, the cyst fluid may stimulate additional cyst growth. Lysophosphatidic Acid (LPA) was determined to be the active component of human cyst fluid, and we investigated the LPA stimulated signaling pathway. Our data suggest that the LPA stimulates chloride and fluid secretion by a combination of CFTR and Calcium-Activated chloride channels (CaCC) and that the two channels may functionally be linked to each other. The secretion is not occurring through a cAMP stimulated pathway, and it is possible that TMEM16A, a CaCC, plays a larger role than previously expected. Previous studies demonstrated that PPARγ agonists, insulin sensitizing drugs used to treat diabetes, inhibit chloride secretion by the collecting duct principal cells by decreasing CFTR synthesis. It was logical therefore to considered PPARγ agonists as long-term treatment for PKD. The first preclinical studied showed that high (20 mg/kg BW) dose pioglitazone, a PPARγ agonist, inhibited cyst growth in the PCK rat model, a slow progressing model, of PKD. To continue to look at the effects of the PPARγ agonists another preclinical study was completed, which tested if there was a class action of PPARγ agonists and if a lower dose was effective in treating the cystic burden. Using the PCK rat model, and another PPARγ agonist, rosiglitazone, a 24 week study was completed using 3 doses (4, 0.4, and 0.04 mg/kg BW). 4 mg/kg BW rosiglitazone is analogous to 20 mg/kg BW pioglitazone. The data indicated that the rosiglitazone is effective in lowering the cystic burden, and importantly the low dose proved to be effective. An additional rat model, the W-WPK rapidly progressing model was used to determine efficacy across multiple models, and to determine if there was a way to track the progress of the disease in a manner analogous to that used in human patients. The animals were treated with pioglitazone using 2 doses (2 and 20 mg/kg BW), and were imaged using CT scans to track the progress of the disease. The data suggest that pioglitazone was not as effective in the W-WPK rat model as it was the PCK rat model. There was a trend however, that low dose PPARγ agonist was as effective ad high dose. Even more important, the CT scans proved to be an effective way to track the progress of the disease in animal models.

Yue Gar-Lee Grace. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 113-120). / Abstracts in English and Chinese. / Abstract (English version) --- p.i / Abstract (Chinese version) --- p.iii / Acknowledgements --- p.v / Table of contents --- p.vi / List of figures --- p.x / List of tables --- p.xiii / List of abbreviations --- p.xiv / Chapter Chapter I: --- Introduction --- p.1 / Chapter 1.1. --- Transepithelial electrolyte transport in colon --- p.1 / Chapter 1.1.1. --- Intestinal fluid secretion --- p.1 / Chapter 1.1.2. --- Cellular mechanism of chloride secretion --- p.3 / Chapter 1.2. --- Biological activities of flavonoids --- p.6 / Chapter 1.2.1. --- Classification and general activities of flavonoids --- p.6 / Chapter 1.2.2. --- Bioavailability and pharmacokinetic properties of flavonoids --- p.8 / Chapter 1.3. --- "What is Scutellariae radix""?" --- p.9 / Chapter 1.3.1. --- Usage in Traditional Chinese Medicine --- p.9 / Chapter 1.3.2. --- Relationship with Coptidis rhizoma --- p.9 / Chapter 1.4. --- Effect of flavonoids on gastrointestinal activities --- p.12 / Chapter 1.4.1. --- Genistein and quercetin --- p.12 / Chapter 1.4.2. --- Baicalein --- p.12 / Chapter 1.5. --- Possible intracellular signaling pathway involved in the secretory response by Scutellariae radix (SR) in T84 cells --- p.14 / Chapter 1.5.1. --- Human colonic T84 cell --- p.14 / Chapter 1.5.2. --- Intracellular signaling pathway --- p.14 / Chapter 1.6. --- Aim of study --- p.17 / Chapter Chapter II : --- Methods and Materials --- p.18 / Chapter II.1. --- Culture technique of the T84 cells --- p.18 / Chapter II.2. --- Simultaneous measurement of short-circuit current (Isc) and intracellular calcium ([Ca2+]i) --- p.21 / Chapter II.2.1. --- Experimental setup --- p.21 / Chapter II.2.2. --- Preparation of the permeable supports --- p.23 / Chapter II.2.3. --- Cell seeding --- p.27 / Chapter II.2.4. --- Dye loading --- p.27 / Chapter II.2.5. --- Simultaneous measurement of Isc and [Ca2+]i- --- p.30 / Chapter II.3. --- Conventional short-circuit current (Isc) measurement --- p.34 / Chapter II.3.1. --- Experimental setup --- p.34 / Chapter II.3.2. --- Preparation of the permeable supports --- p.36 / Chapter II.3.3. --- Cell seeding --- p.36 / Chapter II.3.4. --- Measurement --- p.38 / Chapter II.4. --- Measurement of cAMP --- p.39 / Chapter II.5. --- Solutions and chemicals --- p.40 / Chapter II.6. --- Statistical analysis --- p.42 / Chapter Chapter III : --- Results --- p.43 / Chapter III. 1. --- Effects of baicalein and its interaction with calcium and cAMP-dependent secretagogues --- p.43 / Chapter III. 1.1. --- Effects of baicalein on baseline Isc and [Ca2+]i --- p.43 / Chapter III. 1.2. --- Ionic basis of baicalein-evoked Isc --- p.43 / Chapter III. 1.3. --- Effect of baicalein on carbachol-evoked Isc --- p.47 / Chapter III. 1.4. --- "Effect of baicalein on Isc stimulated by another calcium mobilizing agonist, histamine" --- p.58 / Chapter III. 1.5. --- Effect of carbachol on Isc response stimulated by baicalein --- p.61 / Chapter III. 1.6. --- Chronic effect of baicalein on carbachol-evoked increase in Isc --- p.63 / Chapter III.1.7. --- Interaction of baicalein with forskolin --- p.65 / Chapter III.2. --- Effects of baicalein on cAMP generation in T84 cells --- p.69 / Chapter III.2.1. --- Effects of baicalein on cAMP production --- p.69 / Chapter III.2.2 --- Effects of baicalein on forskolin-induced cAMP production --- p.70 / Chapter III.3. --- Effects of Scutellariae radix extract on ion transport activities in T84 cells --- p.73 / Chapter III.3.1. --- Effects of Scutellariae radix extract (SRE) on baseline Isc --- p.73 / Chapter III.3.2. --- Ionic basis of SRE-evoked Isc --- p.77 / Chapter III.3.3. --- Effects of adenylate cyclase inhibitor and PKA inhibitor --- p.77 / Chapter III.3.4. --- PKC modulation --- p.86 / Chapter III.3.5. --- Involvement of intracellular calcium --- p.86 / Chapter III.3.6. --- Involvement of cAMP --- p.94 / Chapter Chapter IV : --- Discussion --- p.98 / Chapter IV. 1. --- Effects of baicalein on ion transport in human colonic T84 cells --- p.98 / Chapter IV. 1.1. --- Roles of baicalein in chloride secretion in intestinal epithelial cells --- p.98 / Chapter IV. 1.2. --- Potentiation effect of baicalein on calcium-mediated chloride secretion --- p.100 / Chapter IV. 1.3. --- Potentiation effect of carbachol on baicalein-stimulated chloride secretion --- p.102 / Chapter IV. 1.4. --- Interaction between baicalein and forskolin --- p.104 / Chapter IV.2. --- Effects of Scutellariae radix extract on ion transport in human colonic T84 cells --- p.107 / Chapter IV.2.1 --- Characteristcs of Isc induced by Scutellariae radix extract --- p.107 / Chapter IV.2.2. --- Possible signaling mechanism involved in Isc induced by Scutellariae radix extract --- p.108 / Chapter IV.3. --- Comparison of the effects on ion transport in human colonic T84 cells produced by baicalein and Scutellariae radix extract --- p.110 / Chapter IV.3.1. --- Properties of baicalein- and Scutellariae radix extract- induced Isc response --- p.110 / Chapter IV.3.2. --- Summary --- p.111 / Chapter Chapter V : --- References --- p.113 / Publications --- p.120

Primatas da subespécie A. g. clamitans, popular bugio, possuem um dimorfismo sexual evidenciado na fase adulta com machos de cor ruiva e fêmeas de cor castanho com nuanças avermelhadas. Em estudos de cativeiro com esta subespécie descobriu-se uma secreção epidérmica avermelhada, semelhante à cor da pelagem dos MA. A partir desta constatação diferentes hipóteses têm sido levantadas sobre a função e a origem desta secreção. Assim, o presente estudo objetivou: 1- Analisar se esta secreção é responsável pela coloração do pêlo dos animais e se a água é capaz de descolorir os pêlos 2- Verificar se a liberação de secreção sofre influencia das temperaturas corpórea e ambiente; 3- Identificar através de microscopia óptica e eletrônica as características morfológicas das glândulas produtoras de secreção, 4- Mapear as regiões do corpo dos animais que possuem tal glândula, 5- Definir qual sexo e faixa etária possui a glândula; 6- Determinar se a secreção é capaz de corar o pêlo mesmo após estocada a -4C; 7- Avaliar a cor dos campos cromatogênicos, durante um ano em animais adultos, sub adultos e juvenis; 8- Analisar a composição da secreção; 9- Dosar o hormônio testosterona em animais de diferentes sexo e faixas etárias e 10- Verificar uma possível relação entre comportamento social em bugios silvestres e a cor observada. Constatou-se que MA, MSA, MJ , FA e FSA liberam secreção. A liberação em quase todas as regiões do corpo sofre influência da temperatura corpórea, diferindo apenas na região do hióide em MA e da mandíbula em MSA, regiões que a temperatura corpórea possuem maior influência. Verificou-se que pêlos de MSA quando mantidos em estufa, com a secreção fresca liberada por este animal, muda de cor, de escuro para ruivo, igualando a cor do pêlo de MA; uma vez pigmentado a água não muda a cor do pêlo,. A secreção estocada muda apenas à cor da região central do pêlo. Identificou-se a GPP (glândula produtora de secreção colorida), na região do osso hióide e mandíbula, em MA e MSA. Em FA e FSA identificou-se estrutura glandular menos desenvolvida, denominada de GPPi (glândula produtora de pigmento em fase intermediária) nas regiões do hióide, mandíbula, esterno e região inguinal. As GPPi estão também presentes na região inguinal e nuca de MA e MSA. Os animais juvenis e infantes apresentam somente glândulas sudoríparas normais em todas as regiões do corpo. Na secreção colorida não existem carotenóides. As secreções de MA, FA, MAS e MJ possuem diferentes concentrações de ferro. A secreção de MA apresentou maior concentração de ferro quando comparada às secreções dos animais de outro sexo e/ou idade. A microscopia eletrônica confirmou a característica secretora da GPP e evidenciou estruturas cristalinas dentro das células do ácino semelhantes as inclusões de ferro observadas no citoplasma de células de outros organismos. Os animais que sofreram maior variação de cor nos campos cromatogênicos foram os MSA, e foram os animais que tiveram maiores índices de esfregação, sugerindo um mecanismo de espalhamento da secreção Os níveis de testosterona, foram proporcionais ao sexo e idade dos animais, sendo os maiores valores encontrados em MA. O estudo do comportamento social mostrou que os MA ruivos possuem um elevado status hierárquico e desempenham um papel de guardião do grupo. Os resultados indicam que a secreção colorida, liberada na epiderme de A. g. clamitans, são as responsáveis pela coloração do pêlo de MA devido à concentração de ferro. Um cromóforo que contém ferro seria um dos agentes responsáveis pela coloração. A diferença de cor nesta subespécie é uma característica sexual secundária, e parece ocorrer por um mecanismo ativacional do hormônio testosterona, ativando o desenvolvimento das glândulas GPP. O nível de testosterona, a presença de GPP e a coloração ruiva intensa possuem uma forte correlação com o status hierárquico de MA. / Adult brown howler monkeys, Alouatta guariba clamitans, have a clear sexual dimorphism, with red-haired males and chestnut females with red nuances. Captivity studies with this subspecies revealed an epidermic secretion of red coloration, similar to the coat color of adult males. Different hypotheses have been proposed on the function and origin of this secretion. The present work aimed to: 1 analyze if fresh secretion is responsible for hair coloration in this animals and if water is capable to decolorize hair; 2 verify if secretion release is affected by body and atmospheric temperatures; 3 identify by optic and electronic microscopy the morphologic characteristics of the secretion-producing glands; 4 map the areas of the animal body that have such glands; 5 define which sex-age categories have this gland; 6 determine if secretion is able to color hair after stored at -4º C; 7 evaluate during one year the coloration of cromatogenic fields in adult, subadults and juvenile animals; 8 analyze the secretion composition; 9 measure testosterone levels in animals of different sex-age categories; 10 verify the possible relationship between the social behavior of wild animals and the observed coloration. It was found that adult, subadult and juvenile males and adult and subadult females release secretion. The release in almost all body areas is under influence of the corporal temperature that is greater in adult male hyoid and subadult male jaw areas. The hair of subadult males of A. g. clamitans maintained in a stove treated with fresh secretion changes from dark to reddish color, similar to the adult males hair color. The water does not change the color of the hair, once pigmented. The stored secretion changes only the color of the central area of hair. The gland producing colored secretion (GPP) was identified in the area of the hyoid bone and jaw in adult and subadult males. In adult and subadult females a less developed glandular structure was found, denominated GPPi in the hyoid, jaw, breastbone and inguinal areas. GPPi are also present in the inguinal area and nape of adult and subadult males. The juvenile and infant animals presented only normal sweat glands in all body areas. There are no carotenoids in the colored secretion. The secretions of adult, subadult and juvenile males and adult females have different concentrations of iron and adult males presented larger concentration of iron in their secretions when compared to other sex and age categories. The electronic microscopy confirmed the secretory features of GPP and evidenced crystalline structures inside the acinus cells that resemble iron-containing structures evidenced in other cellular structures. Subadult males suffered the greater degree in color variation in cromatogenic fields, and they were also the animals that presented larger rubbing index, suggesting that this would be the mechanism of secretion dispersal. The testosterone levels were proportional to the sex and age categories of the animals, with the largest values found in adult males. The study of the social behavior showed that the reddish adult males have a superior hierarchic status and play the role of group guardians. Our results indicate that the colored secretions, released in the epidermis of A. g. clamitans, are responsible for the hair coloration in adult males due to iron concentration. An iron-containing chromophore would be one of the agents promoting the coloration. The color difference in this subspecies is a secondary sexual trait that seems to occur through a testosterone-activated mechanism, promoting the development of GPP glands. Testosterone levels, the presences of GPP and the intense red-haired coloration have a strong correlation with the hierarchical status of adult males.

Primatas da subespécie A. g. clamitans, popular bugio, possuem um dimorfismo sexual evidenciado na fase adulta com machos de cor ruiva e fêmeas de cor castanho com nuanças avermelhadas. Em estudos de cativeiro com esta subespécie descobriu-se uma secreção epidérmica avermelhada, semelhante à cor da pelagem dos MA. A partir desta constatação diferentes hipóteses têm sido levantadas sobre a função e a origem desta secreção. Assim, o presente estudo objetivou: 1- Analisar se esta secreção é responsável pela coloração do pêlo dos animais e se a água é capaz de descolorir os pêlos 2- Verificar se a liberação de secreção sofre influencia das temperaturas corpórea e ambiente; 3- Identificar através de microscopia óptica e eletrônica as características morfológicas das glândulas produtoras de secreção, 4- Mapear as regiões do corpo dos animais que possuem tal glândula, 5- Definir qual sexo e faixa etária possui a glândula; 6- Determinar se a secreção é capaz de corar o pêlo mesmo após estocada a -4C; 7- Avaliar a cor dos campos cromatogênicos, durante um ano em animais adultos, sub adultos e juvenis; 8- Analisar a composição da secreção; 9- Dosar o hormônio testosterona em animais de diferentes sexo e faixas etárias e 10- Verificar uma possível relação entre comportamento social em bugios silvestres e a cor observada. Constatou-se que MA, MSA, MJ , FA e FSA liberam secreção. A liberação em quase todas as regiões do corpo sofre influência da temperatura corpórea, diferindo apenas na região do hióide em MA e da mandíbula em MSA, regiões que a temperatura corpórea possuem maior influência. Verificou-se que pêlos de MSA quando mantidos em estufa, com a secreção fresca liberada por este animal, muda de cor, de escuro para ruivo, igualando a cor do pêlo de MA; uma vez pigmentado a água não muda a cor do pêlo,. A secreção estocada muda apenas à cor da região central do pêlo. Identificou-se a GPP (glândula produtora de secreção colorida), na região do osso hióide e mandíbula, em MA e MSA. Em FA e FSA identificou-se estrutura glandular menos desenvolvida, denominada de GPPi (glândula produtora de pigmento em fase intermediária) nas regiões do hióide, mandíbula, esterno e região inguinal. As GPPi estão também presentes na região inguinal e nuca de MA e MSA. Os animais juvenis e infantes apresentam somente glândulas sudoríparas normais em todas as regiões do corpo. Na secreção colorida não existem carotenóides. As secreções de MA, FA, MAS e MJ possuem diferentes concentrações de ferro. A secreção de MA apresentou maior concentração de ferro quando comparada às secreções dos animais de outro sexo e/ou idade. A microscopia eletrônica confirmou a característica secretora da GPP e evidenciou estruturas cristalinas dentro das células do ácino semelhantes as inclusões de ferro observadas no citoplasma de células de outros organismos. Os animais que sofreram maior variação de cor nos campos cromatogênicos foram os MSA, e foram os animais que tiveram maiores índices de esfregação, sugerindo um mecanismo de espalhamento da secreção Os níveis de testosterona, foram proporcionais ao sexo e idade dos animais, sendo os maiores valores encontrados em MA. O estudo do comportamento social mostrou que os MA ruivos possuem um elevado status hierárquico e desempenham um papel de guardião do grupo. Os resultados indicam que a secreção colorida, liberada na epiderme de A. g. clamitans, são as responsáveis pela coloração do pêlo de MA devido à concentração de ferro. Um cromóforo que contém ferro seria um dos agentes responsáveis pela coloração. A diferença de cor nesta subespécie é uma característica sexual secundária, e parece ocorrer por um mecanismo ativacional do hormônio testosterona, ativando o desenvolvimento das glândulas GPP. O nível de testosterona, a presença de GPP e a coloração ruiva intensa possuem uma forte correlação com o status hierárquico de MA. / Adult brown howler monkeys, Alouatta guariba clamitans, have a clear sexual dimorphism, with red-haired males and chestnut females with red nuances. Captivity studies with this subspecies revealed an epidermic secretion of red coloration, similar to the coat color of adult males. Different hypotheses have been proposed on the function and origin of this secretion. The present work aimed to: 1 analyze if fresh secretion is responsible for hair coloration in this animals and if water is capable to decolorize hair; 2 verify if secretion release is affected by body and atmospheric temperatures; 3 identify by optic and electronic microscopy the morphologic characteristics of the secretion-producing glands; 4 map the areas of the animal body that have such glands; 5 define which sex-age categories have this gland; 6 determine if secretion is able to color hair after stored at -4º C; 7 evaluate during one year the coloration of cromatogenic fields in adult, subadults and juvenile animals; 8 analyze the secretion composition; 9 measure testosterone levels in animals of different sex-age categories; 10 verify the possible relationship between the social behavior of wild animals and the observed coloration. It was found that adult, subadult and juvenile males and adult and subadult females release secretion. The release in almost all body areas is under influence of the corporal temperature that is greater in adult male hyoid and subadult male jaw areas. The hair of subadult males of A. g. clamitans maintained in a stove treated with fresh secretion changes from dark to reddish color, similar to the adult males hair color. The water does not change the color of the hair, once pigmented. The stored secretion changes only the color of the central area of hair. The gland producing colored secretion (GPP) was identified in the area of the hyoid bone and jaw in adult and subadult males. In adult and subadult females a less developed glandular structure was found, denominated GPPi in the hyoid, jaw, breastbone and inguinal areas. GPPi are also present in the inguinal area and nape of adult and subadult males. The juvenile and infant animals presented only normal sweat glands in all body areas. There are no carotenoids in the colored secretion. The secretions of adult, subadult and juvenile males and adult females have different concentrations of iron and adult males presented larger concentration of iron in their secretions when compared to other sex and age categories. The electronic microscopy confirmed the secretory features of GPP and evidenced crystalline structures inside the acinus cells that resemble iron-containing structures evidenced in other cellular structures. Subadult males suffered the greater degree in color variation in cromatogenic fields, and they were also the animals that presented larger rubbing index, suggesting that this would be the mechanism of secretion dispersal. The testosterone levels were proportional to the sex and age categories of the animals, with the largest values found in adult males. The study of the social behavior showed that the reddish adult males have a superior hierarchic status and play the role of group guardians. Our results indicate that the colored secretions, released in the epidermis of A. g. clamitans, are responsible for the hair coloration in adult males due to iron concentration. An iron-containing chromophore would be one of the agents promoting the coloration. The color difference in this subspecies is a secondary sexual trait that seems to occur through a testosterone-activated mechanism, promoting the development of GPP glands. Testosterone levels, the presences of GPP and the intense red-haired coloration have a strong correlation with the hierarchical status of adult males.

Insects have developed many strategies of defence against predators in the course of evolution. The evolutionarily oldest and most widely used type of defence is chemical defence, followed by acoustic or optical defence. However, many species of insects use simultaneously multiple types of warning signals, which affect different sensory receptors of the given predator. Such a complex method of warning signals is called multimodal method. It may consist of a combination of simultaneous chemical and optical signals, or a combination of acoustic and optical signalling. The combination of chemical and optical signalling used against a predator is probably the most common form of multimodal signalling. The presented work deals with the analysis of biologically active substances, which participate in the defence mechanisms of a widespread species of insects - true bugs (Heteroptera). Pterin derivatives represent a large group of natural compounds derived from pteridin, bicyclic heterocycle, and they are found in virtually all living organisms from bacteria to vertebrates. In insects, they primarily serve as pigments, resulting for example in striking coloration of cuticles of Heteroptera. The first part of the dissertation was focused on identification and quantification of pterin derivatives in cuticles...

Vascular Endothelial Growth Factor Receptor-3 (VEGFR3/Flt4) and its ligand Vegfc are necessary for development of both blood and lymphatic vasculature in vertebrates. In zebrafish, Vegfc/Flt4 signaling is essential for formation of arteries, veins, and lymphatic vessels. Interestingly, Flt4 appears to utilize distinct signaling pathways during the development of each of these vessels. To identify components of this pathway, we performed a transgenic haploid genetic screen in zebrafish that express EGFP under the control of a blood vessel specific promoter. As a result, we indentified a mutant allele of vascular endothelial growth factor c (vegfc), vegfcum18. vegfcum18 mutants display defects in vein and lymphatic vessel development but normal segmental artery (SeA) formation. Characterization of this allele led to the finding that the primary defect in vegfcum18 mutants was a general failure in vein and lymphatic vessel sprouting. Further genetic and biochemical analysis of this mutant revealed profound paracrine defects, which likely result in the observed loss of lymphatic and venous structures. Furthermore, double mutant analysis demonstrated that defects during SeA formation in vegfcum18 mutants were masked by inputs from the Vegfa signaling pathway. Endothelial cell autonomous expression of vegfcum18 induced angiogenic effects on blood vessels while endothelial cells lacking vegfc displayed defects in tip cell occupancy, suggesting a cell autonomous-autocrine role for Vegfc during developmental angiogenesis. Finally, we present genetic evidence that links processing of Vegfc by Furin during the formation of lymphatics in zebrafish. Together the data presented here suggest two discrete modes of signaling during blood and lymphatic vessel development, thus implying that regulation of Vegfc secretion and processing may play a pivotal role in the formation of these distinct vessel types in zebrafish.