Tesis para optar al Grado de Magíster en: Enología y Vitivinicultura / La transformación del mosto de uva en vino, es un complejo proceso microbiológico realizado principalmente por levaduras del género Saccharomyces, responsables de la fermentación alcohólica. Actualmente, las co-inoculaciones con levaduras comerciales no Saccharomyces (Torulaspora delbrueckii) y Saccharomyces cerevisiae, son una alternativa para la incorporación de enzimas β-glucosidasas, pectinasas y proteasas al mosto. El mosto alberga una diversa microbiota inicial; esta heterogeneidad microbiológica dificulta el control del proceso fermentativo. Por ello, se desarrolló un método de monitoreo de levaduras vínicas en fermentaciones de co-inoculación, aplicando métodos de taxonomía molecular. Se analizaron las regiones ribosomales, sobre las cuales se diseñó una estrategia “in silico”, mediante perfiles de restricción (RFLP), empleando endonucleasas específicas; lográndose una fácil y fidedigna diferenciación de los microorganismos involucrados. De manera experimental se trabajó con un mosto del cultivar Syrah; que se separó en dos cubas, una fue inoculada únicamente con Saccharomyces cerevisiae y otra fue co-inoculada con Torulaspora delbrueckii y Saccharomyces cerevisiae. Se examinaron distintas etapas del proceso de fermentación, logrando en todos los casos extraer ADN de forma directa desde las muestras de vino en una aproximación independiente de cultivo. El ADN extraído fue sujeto a un proceso de PCR para amplificar la región ITS 5.8S. Estos amplicones se sometieron a digestión con las enzimas AluI, ApaI, HaeIII, SphI, lo que permitió definir e identificar la presencia de las levaduras involucradas en cada fase de la fermentación alcohólica. Todo esto permitió demostrar que la variación genética de las regiones ribosomales, permite la diferenciación a través de métodos moleculares, de las levaduras vínicas: Saccharomyces cerevisiae y Torulaspora delbrueckii, comúnmente usadas en co-inoculación. / Transformation of grape must into wine is a complex microbiological process done mainly by Saccharomyces yeasts, responsible for the alcoholic fermentation. Currently, co-inoculated with Saccharomyces commercial yeasts (Torulaspora delbrueckii) and Saccharomyces cerevisiae, are an alternative to the incorporation of β-glucosidase enzymes, pectinase and protease to the must. The must has a diverse microbiota initial, this heterogeneity hinders this fermentation process control. Therefore, we developed a method of monitoring fermentations wine yeasts in co-inoculation, using methods of molecular taxonomy. Ribosomal regions were analyzed, over which strategy was designed “in silico” by restriction profiles (RFLP) using specific endonucleases; achieving an easy and accurate differentiation of the microorganisms involved. Experimentally we worked with Syrah grape cultivar, which split into two tanks, one was inoculated with Saccharomyces cerevisiae and other was co-inoculated with Torulaspora delbrueckii and Saccharomyces cerevisiae. We examined various stages of the fermentation process, obtaining in all cases extract DNA directly from the wine samples in a culture-independent approach. The extracted DNA was subjected to a process of PCR to amplify the region ITS 5.8 S. These amplicons were digested with the enzyme AluI, ApaI, HaeIII, SphI, allowing define and identify the presence of yeast involved in each stage of the fermentation. All this helped to show that the genetic variation of ribosomal regions, allows differentiation through molecular methods of wine yeasts: Saccharomyces cerevisiae and Torulaspora delbrueckii, commonly used in co-inoculation.

Made available in DSpace on 2014-06-11T19:26:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-23Bitstream added on 2014-06-13T20:55:08Z : No. of bitstreams: 1 ferreira_ls_me_arafcf.pdf: 537259 bytes, checksum: 4756eac4f37ae0739a8b59e969ac32a1 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Universidade Estadual Paulista (UNESP) / A esporotricose é uma micose de distribuição universal cujo agente é o fungo termodimórfico Sporothrix schenckii. A forma mais comum da doença é a linfocutânea, que compromete pele, tecido subcutâneo e gânglios linfáticos regionais. Os principais constituintes da parede celular de S. schenckii são compostos peptídeo-polissacarídicos contendo ramnose, manose e galactose. Estes compostos se arranjam na parede celular de modo a formar duas subcamadas distintas na forma leveduriforme do fungo, uma das quais, a mais interna e fixa delas, é denominada como peptídeo-polissacarídeo da parede celular (PPC) e foi utilizada como antígeno neste estudo, juntamente com a levedura termo-inativada (SSTI). Os antígenos liberados pelo fungo participam diretamente do processo de escape do sistema imune e também servem como alvos para a eliminação do mesmo por anticorpos ou ligação a receptores presentes em células da imunidade inata, como os macrófagos. Nossos resultados mostraram que o PPC induz citocinas de padrão inflamatório de forma mais pronunciada que o SSTI, que por sua vez induziu maior liberação de IL-10. Sugerimos também que no modelo experimental utilizado a IL-10 não atua provocando supressão da resposta proliferativa dos esplenócitos, e que a IL-4 atua apenas na fase de resolução da infecção, não sendo induzida como forma de escape imunológico pelo fungo. Também mostramos que os antígenos testados são capazes de induzir a liberação de IL-17 em culturas de esplenócitos, num perfil semelhante àquela das citocinas Th1 / Sporotrichosis is a micotic infection of universal distribution, which is caused by dimorphic fungus Sporothrix schenckii. It usually happens as a lymphocutaneous disease, compromising skin, subcutaneous tissues and regional lymphatic nodules. Major constituents of S. schenckii cell wall are peptido-polysaccharide complexes containing rhamnose, mannose and galactose. These complexes are organized in two distinct layers in the fungus yeast cell wall, of which the inner one is called cell wall peptido-polysaccharide (PPC), being used as antigen in this study, along with the heat-killed yeast (SSTI). Antigens released by the fungus directly participate in evasion of the immune system, also serving as targets for fungus elimination by binding of antibodies or innate immune cells like macrophages. Our results showed that PPC induces inflammatory cytokines in a more pronounced way when compared to SSTI, which induced higher levels of IL-10. We also suggest that, in the animal model used, IL-10 doesn’t act as a suppressor of splenocyte proliferative response, and that IL-4 play a role at the resolution phase of the infection, being not induced as a means of immunologic escape by the fungus. Also, we showed that the assayed antigens are capable of inducing IL-17 secretion in splenocyte cultures, in a fashion close to that of Th1 cytokines

Living amidst a milieu of pathogenic organisms, vertebrates are in constant threat of contracting one or the other disease. As a mechanism of protection against such ‘invasions’, the vertebrate immune system has evolved to serve two main functions. One, to generate a specific immune response against the invading pathogen (in the from of specific antibodies and cell mediated immune responses). And two, to ‘remember’ the pathogen after the first exposure and mount a heightened and quicker immune response upon subsequent encounters. This phenomenon is called immunological memory, or anamnestic response and is achieved by the generation of memory B and T cells. The generation of specific Immunological memory is indeed the most important requirement/purpose of prophylactic vaccination Though different mechanisms are known to operate to maintain memory B and T cells, some aspects are still debatable. The ‘relay hypothesis’ (Nayak etal., Immunology.102(4)(2001); Nayak R etal., Microbes. Infect.(2005)), addresses some of those key issues. It describes that antigen specific memory B cells can be maintained by the interaction of membrane bound idiotypic (Id, Ab1) and anti-idiotypic (α-Id, Ab2) antibodies on B cells. Anti-Ids binding to idiotopes on Abs (Ab1) are known to be potential regulators of immunity in a variety of diseases, such as autoimmunity, cancer as well as viral, bacterial or parasitic infections. The relay hypothesis outlines the mechanism of persistence of memory cells in the absence of persisting antigen. This is achieved through the ‘internal image’ of the antigen on the Ab2 variable region, which serves as surrogate antigen thus helping in maintenance of immunological memory even in the absence of persisting antigen. It also explains that all antigens, protein or nonprotein can be converted to the common “coinage” of internal image peptides, otherwise called peptido-mimics. Peptido-mimics that have similar binding properties to MHC as the antigenic epitope, will ensure that the antigen specific memory T cells are also maintained. Hence T cell activation could also occur in the absence of nominal antigen, a potentially important process in T-B cooperation and immune regulation. Scope and objectives of this work: To demonstrate the presence of idiotypic and cognate anti-idiotypic antibody for the given antigen  To examine the likelihood of the three dimensional structural similarity between antigen and Ab2 variable region  To demonstrate the presence of peptido-mimics of the antigenic epitope in the Ab2 variable region; and if those peptido-mimics have structural and functional similarity with antigenic peptides when bound to MHC-I To examine the (immunological) memory associated phenotype of thediotypic anti-idiotypic B cells. The antigen of choice for the current study is Heamagglutinin-Neuraminidase (HN) protein of peste des petits ruminants virus (PPRV). Idiotypic (Id, Ab1), antiidiotypic (α-Id, Ab2) hybridoma against a deletion mutant of PPRV HN were generated and characterized. These hybridoma served as surrogate B cells for the study of Id α-Id B cell interactions. Anti-anti-idiotypic (Ab3) lymphocytes were also generated by immunizing syngenic BALB/c mice with Ab2 hybridoma. Results not only indicated the interplay of idiotypic and anti-idiotypic B and T cells in this cascade but also the mimicry of the antigen by Ab2. Ab2 Mab could recognize idiotopes of anti-PPRV HN Ab1 raised in diff species of animals, thus demonstrating that Ab2 was indeed an antigen mimic that interacts with Ab1 paratope irrespective of which species the Ab1 originates from. Ab2 Mab also mimicked the antigen (Hemagglutinin-neuraminidase) in functional assays by bringing about hemagglutination. Similarly, Polyclonal Ab3 which reacts with Ab2 Mab and with antigen, inhibits hemagglutination, just as Ab1 does, albeit to a lesser extent. This suggests Ab3 has functional similarity with Ab1. It is imperative that T cells be involved in this network of B cells for the maintenance of antigen specific immunological memory. This is because B cells require T cell help in the form of cytokines for proliferation and Cytotoxic T Lymphocytes (CTLs) are needed to control the specific population of Id and anti-Id B cells to maintain homeostasis. The Ab2 hybridoma as well as soluble Ab2 stimulated the proliferation of antigen specific T cells. Similarly, Ab3 splenocytes were stimulated to proliferate by the Ab2 as well as the antigen. Peptides generated from monoclonal Ab2 heavy and light chain variable regions (VH and VL) showed structural and functional similarity to the antigenic peptides in terms of p-MHC binding. These peptides stimulated the proliferation of antigen and Ab2 specific T cells, and also triggered 4-5 times higher CTL targeted cell lysis of peptide pulsed RMAS-Kd cells, as compared to a control peptide. VH, VL and antigenic peptides stabilized MHC-I on the cell surface of the TAP deficient, RMAS-Kd cell line for upto six hrs as compared to the ‘empty’ MHC-I, which remained on the surface only for one hr. The presence of peptido-mimics in the Ab2 variable region, which have structural similarity with antigenic peptide (when bound to MHC I), was also established using insilico software tools. Antigenic peptides and VH and VL peptides were modeled onto MHC-I crystal structures using the molecular modeling software InsightII and the minimization program, CNS. Putative MHC-I binding peptides from these sequences were generated using the p-MHC-I binding prediction algorithm, BIMAS. By replacing these peptides in the respective crystal structure of MHC I and superimposing the two structures, we have tried to establish that through structural similarity in binding to MHC-I, peptidomimics have a role in the maintenance of antigen-specific CTL memory. Consequently CTL memory specific to antigenic epitope can be preserved even in the absence of antigen by its peptidomimic. Following long-term immunizations, as expected of a secondary immune response, the serum Ab1 titre was found to be higher than the titer during primary response. It was also noted that though the number of Ab1 and Ab2 cell number was comparable in the total splenocyte population, Ab1 titre in the serum was higher than Ab2, immaterial of Ag/Ab2 booster. The same trend was noticed in prolifertion assay and CTL assays when the splenocytes were stimulated by Ag/Ab2 pulsed bone-marrow derived dendritic cells (BMDCs) as APCs. That is, irrespective of immunization and boost with Ag/Ab2, Ag pulsed BMDCs stimulated the proliferation and CTL lysis of long term immunized splenocytes to a greater extent than Ab2 pulsed BMDCs. Memory markers present on B and T cell surface might help maintain their close interactions in the idiotypic network. CD27/CD70 (CD27L) might play a role in maintaining these cells in a memory state. The Id α-Id B cells in addition to being triggered through the membrane bound Id, α-Id antibodies, can also be activated through CD27/CD70 to differentiate into plasma cells upon activation by antigen. Id and α-Id B cells were demonstrated to possess the CD27 memory marker on their surface in addition to the membrane bound IgM. Antigen specific IgM and CD27 double positive cells were detected in the range of 1-3% in the total splenocyte population. In conclusion: PPRV HN immunization triggered the generation of Ab1, Ab2, Ab3 (Id, α-Id, α-α-Id) cascade through the interaction of membrane bound immunoglobulin of the corresponding B cells. Ab2 was demonstrated to be a significant structural and functional mimic of the antigen. Peptidomimics of the antigenic epitope, present in the Ab2 variable region, can serve the purpose of maintaining antigen specific T cell memory response. These findings re-confirm the importance of anti-id antibodies in the regulation of immune responses. Ever since the concept of antigen mimicry by anti-Id antibody has been confirmed by several laboratories, the utility of anti-Ids as surrogate antigens for the purpose of prophylactic vaccination has received great attention. The results of the current work are especially significant for the purpose of development of vaccines for diseases related to antigens that are very cumbersome to purify (for ex., in case of several cancers) or when it is too dangerous to immunize with the antigen itself (for ex., in case of some pathogenic organisms). The results also signify that immaterial of the nature of the antigen, their respective petidomimics can establish and maintain immunological memory.

Orientador: Iracilda Zeppone Carlos / Banca: Angela Maria Victoriano de Campos Soares / Banca: Eduardo Bagagli / Resumo: A esporotricose é uma micose de distribuição universal cujo agente é o fungo termodimórfico Sporothrix schenckii. A forma mais comum da doença é a linfocutânea, que compromete pele, tecido subcutâneo e gânglios linfáticos regionais. Os principais constituintes da parede celular de S. schenckii são compostos peptídeo-polissacarídicos contendo ramnose, manose e galactose. Estes compostos se arranjam na parede celular de modo a formar duas subcamadas distintas na forma leveduriforme do fungo, uma das quais, a mais interna e fixa delas, é denominada como peptídeo-polissacarídeo da parede celular (PPC) e foi utilizada como antígeno neste estudo, juntamente com a levedura termo-inativada (SSTI). Os antígenos liberados pelo fungo participam diretamente do processo de escape do sistema imune e também servem como alvos para a eliminação do mesmo por anticorpos ou ligação a receptores presentes em células da imunidade inata, como os macrófagos. Nossos resultados mostraram que o PPC induz citocinas de padrão inflamatório de forma mais pronunciada que o SSTI, que por sua vez induziu maior liberação de IL-10. Sugerimos também que no modelo experimental utilizado a IL-10 não atua provocando supressão da resposta proliferativa dos esplenócitos, e que a IL-4 atua apenas na fase de resolução da infecção, não sendo induzida como forma de escape imunológico pelo fungo. Também mostramos que os antígenos testados são capazes de induzir a liberação de IL-17 em culturas de esplenócitos, num perfil semelhante àquela das citocinas Th1 / Abstract: Sporotrichosis is a micotic infection of universal distribution, which is caused by dimorphic fungus Sporothrix schenckii. It usually happens as a lymphocutaneous disease, compromising skin, subcutaneous tissues and regional lymphatic nodules. Major constituents of S. schenckii cell wall are peptido-polysaccharide complexes containing rhamnose, mannose and galactose. These complexes are organized in two distinct layers in the fungus yeast cell wall, of which the inner one is called cell wall peptido-polysaccharide (PPC), being used as antigen in this study, along with the heat-killed yeast (SSTI). Antigens released by the fungus directly participate in evasion of the immune system, also serving as targets for fungus elimination by binding of antibodies or innate immune cells like macrophages. Our results showed that PPC induces inflammatory cytokines in a more pronounced way when compared to SSTI, which induced higher levels of IL-10. We also suggest that, in the animal model used, IL-10 doesn't act as a suppressor of splenocyte proliferative response, and that IL-4 play a role at the resolution phase of the infection, being not induced as a means of immunologic escape by the fungus. Also, we showed that the assayed antigens are capable of inducing IL-17 secretion in splenocyte cultures, in a fashion close to that of Th1 cytokines / Mestre

Hemos inducido en ratas wistar dos modelos de hipertensión arterial (HTA): hipertensión vasculorrenal 2 riñones-1 clip (2R-1C) e hipertensión por déficits crónico de óxido nítrico (NO). La angiotensina II (AII) participa en el desarrollo y mantenimiento de la hipertensión vasculorrenal y por déficit crónico de NO. La administración crónica de losartán, aún previniendo el desarrollo de la hipertensión por déficit crónico de NO, no confiere protección completa frente a las alteraciones hemodinámicas que dicha hipertensión conlleva. Los bloqueantes de los receptores de la AII, saralasina y losartán, poseen efectos hemodinámicos similares, aunque de distinta potencia, sobre los grupos de HTA. En condiciones normales, los efectos hemodinámicos del péptido natriurético auricular (PNA) no son modulados por los receptores de AII. La HTA potencia los efectos hemodinámicos del PNA por un efecto en el que intervienen los receptores de AII. / We have induced in Wistar rats two models of experimental hypertension: two kidney-one clip hypertension (2K-1C) and hypertension induced by chronic inhibition of nitric oxide production (L-NAME-induced hypertension). In both, angiotensin II (AII) participates in the development and maintenance of the hypertension. Chronic administration of losartan prevents the development of the L-NAME-induced hypertension, although it does not confer complete protection to the hemodynamic alterations that this hypertension causes. The AII receptor antagonists, saralasin and losartan, have similar hemodynamic effects on the hypertensive groups, although in different degrees. Moreover, the hemodynamic effects of the atrial natriuretic peptide (ANP) under normal conditions are not modulated by the AII receptor. However, hypertension increases the hemodynamic effects of the ANP by an effect partially due to the AII receptors