Orientadores: Sophie Françoise Mauricette Derchain, Luiz Carlos Zeferino / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-11-08T16:11:29Z (GMT). No. of bitstreams: 1 Gontijo_RenataClementino_M.pdf: 325627 bytes, checksum: 39c39b0e15ab2522b5d3e7cb150d69ed (MD5) Previous issue date: 2003 / Resumo: Objetivo: avaliar o desempenho da citologia oncológica (CO) convencional, captura de híbridos II (CH II) e inspeção visual com ácido acético (IVA) na detecção de lesões cervicais neoplásicas e pré-neoplásicas numa população de uma Unidade Básica de Saúde da região de Campinas. Sujeitos e métodos: para este estudo descritivo de corte transversal foram incluídas 684 mulheres. Após responderem a um questionário pré-estruturado, foram submetidas a coleta de material para CO, CH II e IVA. As mulheres com pelo menos um exame positivo eram convocadas para colposcopia com biópsia dirigida das áreas anormais. Foram comparadas as mulheres com pelo menos um exame positivo com aquelas com todos os exames normais segundo as características sociodemográficas e reprodutivas, utilizando-se o cálculo do odds ratio com intervalo de confiança a 95%, o cálculo do p valor e a análise de regressão logística em stepwise. O desempenho da CO, CH II e IVA foi calculado através da sensibilidade, especificidade, e valores preditivos positivo e negativo e seus respectivos intervalos de confiança a 95%. Para esta avaliação, tomou-se como padrão ouro a colposcopia. As mulheres cujo diagnóstico histológico final foi compatível com NIC 1 ou mais foram consideradas como diagnóstico final positivo. Quando a colposcopia foi normal ou quando a biópsia apresentou cervicite ou metaplasia foram consideradas como diagnóstico final negativo. Resultados: setenta e uma (10,4%) mulheres apresentaram atipias celulares na CO, a CH II foi positiva em 131 (19,2%), e foram encontradas à IVA alterações em 55 (8%) mulheres. Embora 198 mulheres apresentassem pelo menos um teste de rastreamento alterado, apenas sete apresentaram os três exames positivos. Entre as 150 mulheres que fizeram colposcopia, 91 não apresentaram imagem suspeita. Das mulheres com resultado histológico, 38 apresentaram cervicite/metaplasia escamosa, 10 NIC 1, oito NIC 2, dois NIC 3 e um carcinoma invasor. Comparando as mulheres com exames de rastreamento positivos e negativos na análise multivariada observamos que iniciar a atividade sexual com menos de 18 anos e ter tido mais que um parceiro no último ano foram fatores com associação positiva e significativa com a presença de pelo menos um exame alterado. Entretanto, apenas o fato de nunca ter sido submetida à citologia anteriormente esteve significativamente associado com a presença de doença histológica. A sensibilidade dos três testes foi semelhante, a especificidade da IVA e da CO foi maior que a da CH II. Nos casos que apresentaram resultado de CO negativo, a IVA apresentou melhor desempenho quando comparado com a CH II. Conclusão: apesar da alta prevalência de resultados alterados dos exames de rastreamento, a detecção de lesão histológica foi baixa. Embora algumas características sociodemográficas e reprodutivas se mostrassem associadas com a positividade dos exames, apenas a realização de CO prévia associou-se com ausência de doença. O desempenho da CO em associação com a IVA foi melhor que o da CO com a CH II e que a CO isolada neste estudo / Abstract: Objective: to evaluate the performance of Pap smear, hybrid capture II (HC II) and visual inspection with acetic acid (VIA) in detecting neoplastic and pre-neoplastic cervical lesions in one Basic Public Health Unit from Campinas. Subjects and methods: in this descriptive cross-sectional study, 684 women were evaluated. They answered a questionnaire and were submitted to a clinical exam, including Pap smear, VIA and HC II for HPV DNA detection. When at least one of the screening tests was positive, colposcopy was performed and targeted biopsies were taken from any suspicious lesions. Women with at least one positive exam and with all exams negative were compared regarding sociodemographic and reproductive factors, using odds ratio, calculated with 95% confidence interval, p value and stepwise logistic regression analysis. Sensitivity, specificity, positive and negative predictive value with respective confidence intervals of each test were evaluated. Colposcopy was considered as the gold standard for the performance evaluation. Women with histologic diagnosis of CIN 1 or higher were considered as positive final diagnostic. When colposcopy result was normal or biopsy result was cervicitis/metaplasia it was considered as negative final diagnostic. Results: Pap smear, VIA and HC II were positive in 10.4%, 8% and 19.2% of all women screened respectively. Although at least one positive test was found in 198 women, only seven presented the three tests with positive results. Among the 150 women who were submitted to colposcopy, the result was normal in 91. The biopsies results were 38 cervicitis/metaplasia, 10 CIN 1, eight CIN 2, two CIN 3 and one invasive carcinoma. Comparing women with at least one positive or all negative screening tests results, we observed that age less than 18 years at the first intercourse and have had more than one sexual partner in last year were significantly associated with the positive results. However, only have never having a Pap smear taken was associated with the presence of histological disease. The sensitivity of the three tests was similar. VIA and Pap smear presented higher specificity than HC II. In women with negative Pap smear result, VIA have showed better performance than HC II. Conclusions: besides the high prevalence of abnormal screening tests results, histological disease detection rate was low. Although some sociodemographic and reproductive factors were associated with positive tests results, only have never had a Pap smear taken before was associated with disease. Pap smear combined with VIA performed better than Pap smear with HC II and then Pap smear alone this study / Mestrado / Tocoginecologia / Mestre em Tocoginecologia

Orientador: Wilter Ricardo Russiano Vicente / Coorientador: Frederico Ozanan Barros Monteiro / Banca: Rodrigo del Rio do Valle / Banca: Marcus Antonio Rossi Feliciano / Resumo: Realizou-se o exame ultrassonográfico de macacos-da-noite para avaliar os volumes uterino e ovariano e analisar a interação entre diferentes faixas etárias e número de partos. Foram realizados exames ultrassonográficos da região pélvica, a fim de comparar as dimensões uterinas com o peso, idade (infantil, juvenil, subadulta, adulta jovem e adulta) e número de partos (nulípara, primípara e multípara) e comparar as dimensões ovarianas com o peso e a idade. O volume uterino (VU) foi diretamente proporcional ao número de partos, fator mais importante no crescimento uterino das fêmeas adultas (p < 0,05). O peso e a idade demonstraram uma correlação positiva com o VU (r = 0.5354, r = 0.6489, p < 0.01), respectivamente. O volume dos ovários cresceu proporcionalmente a idade das fêmeas (p < 0,05). A puberdade foi o período de maior crescimento tanto do útero como do ovário / Abstract: We aimed to evaluate the uterine and ovarian volumes of owl monkeys in different age groups with different numbers of live births and to analyze the interaction between both. We performed pelvic ultrasound exams to compare the uterine measurements with weight, age (infant, juvenile, subadult, young adults, and adults) and the number of live births (nulliparous, primiparous, and multiparous) and to compare the ovarian measurements with weight and age. The uterine volume (UV) was directly proportional to the number of parturitions, which was the most important factor in the uterine growth of adult females (P < 0.05). The body weight and age of the animals showed a high positive correlation with UV (r = 0.5354, r = 0.6489, P < 0.01), respectively. The volume of the ovaries grew in proportion to the age of the females (P < 0.05). Puberty was the period of greatest uterine and ovarian growth / Mestre

No description available.

S. cerevisiae (budding yeast) is particularly powerful to study the relation between the molecular mechanisms of actin regulation and its biological significance due to its powerful molecular and genetic methods, well-established biochemical techniques, and development of live cell imaging and immuno-electron microscopy. Further, budding yeast has a simple cytoskeleton but the molecular mechanisms controlling its remodeling are conserved from yeast to mammals. Interestingly, a dynamic actin cytoskeleton is mandatory for endocytosis in S. cerevisiae. In this organism the formation of the primary endocytic profiles in yeast occurs concomitant with the assembly of a complex actin structure (the endocytic actin module), which includes the actin nucleator Arp2/3 complex. Consistent with observations indicating that the myosin-I Myo5 in complex with the WIP homolog Vrp1 is a major Arp2/3-dependent actin nucleating promoting factor (NPF) driving productive membrane invagination, our laboratory has demonstrated that the C-terminal domain of Myo5 immobilized on the surface of Sepharose beads induces assembly of the endocytic actin module in the presence of yeast extracts. Analysis of the in vitro actin assembly assay demonstrated that Myo5 is phosphorylated by the casein kinase CK2 at the serine 1205 during the assay. In this study, we have characterized the CK2 activity that phosphorylates Myo5 at the C-terminus and investigated the functional significance of this phosphorylation in vivo and in vitro. Our results indicate that a non-tetrameric and particulate-associated CK2 activity that includes the catalytic subunit Cka2 but not Cka1 or the regulatory subunits Ckb1 and Ckb2 phosphorylates Myo5 S1205. Our results also indicate that this phosphorylation event down-regulates the assembly of complex actin structures in vitro and slows down the internalization process and the dissociation of the myosin from the plasma membrane in vivo. The Cka2-mediated phosphorylation at Myo5 S1205 does not seem to regulate the affinity of the NPF for the Arp2/3 complex but rather down-regulates the interaction of Myo5 with its co-activator Vrp1. This phosphorylation also increases directly or indirectly the binding of the myosin to the endocytic coat components Sla1 and Pan1 and to the syndapin Bzz1, suggesting that the phosphorylation event occurs late during the maturation of the endocytic invagination. Finally, our data suggest that Cka2 have endocytic targets other than Myo5 and that its activity might also regulate early steps during the assembly of the endocytic coat. / La levadura S. cerevisiae es un organismo modelo muy utilizado para estudiar los mecanismos moleculares que regulan el citoesqueleto actina y su función biológica, ya que los mecanismos moleculares que controlan la organización del citoesqueleto de actina están conservados, por lo que muchos de los descubrimientos realizados en S. cerevisiae son aplicables también en eucariotas superiores. Una de las principales funciones biológicas de la actina en S. cerevisiae es la formación de una vesícula endocítica. En este organismo, la formación de los perfiles endocíticos primarios ocurre de forma simultánea con el ensamblaje de una estructura de actina compleja (módulo de actina) que incluye al complejo nucleador de actina Arp2/3. Diferentes estudios indican que la miosina-I Myo5 junto su co-activador Vrp1 activan al complejo Arp2/3 produciendo la elongación del perfil endocítico. Nuestro laboratorio ha demostrado que el dominio C-terminal de Myo5 inmovilizado en la superficie de bolas de sefarosa induce el ensamblaje del módulo de actina en presencia de extracto de levadura. El análisis del ensayo demostró además que Myo5 está fosforilada por la quinasa CK2 en la S1205. En este estudio hemos caracterizado la actividad CK2 que fosforila a Myo5 en la S1205 y hemos investigado el significado funcional de dicha fosforilación in vivo e in vitro. Nuestros resultados indican que una actividad CK2 asociada a partículas, que incluye la subunidad catalítica Cka2 pero no Cka1 ni las subunidades reguladoras Ckb1 y Ckb2 fosforila la S1205 de Myo5. Esta fosforilación perturba la interacción entre Myo5 y su co-activador Vrp1, regulando negativamente la formación de las estructuras de actina complejas in vitro, y consistentemente, disminuyendo la velocidad de la internalización endocítica in vivo. Además, nuestros resultados también sugieren que Cka2 seguramente tiene otras dianas además de Myo5, y que su actividad podría regular otras fases iniciales durante la formación de la vesícula endocítica.

The reproductive biology of poriferans is still poorly understood. We have investigated the sexual reproductive biology of seven demosponge species, six of them from the Mediterranean (Corticium candelabrum, Crambe crambe, Raspaciona aculeata, Axinella damicornis, Chondrosia reniformis, and Petrosia ficiformis), and one from the Pacific coast of Canada (Asbestopluma occidentalis). The thesis consists of a general introduction, 7 different chapters and a general discussion.Chapter 1. It is well established that in temperate regions invertebrates restrict their reproductive cycles to the warm periods. The sexual cycle of A. damicornis, C. candelabrum, C. reniformis, and R. aculeata is very different in timing and duration, despite all of them shared habitat and thermal regime. While the gametogenesis of R. aculeata and C. reniformis underwent during summer and autumn (warm periods in the Mediterranean), the gametogenesis of C. candelabrum and A. damicornis occurred during winter. Therefore, the relationship between gametogenesis and temperature in temperate waters was not straightforward, and many different relationships appeared.Chapter 2. The oogenesis of C. candelabrum resulted in a surprising long process, with continuous production of oocytes. However, the oocyte maturation extended for 7/8 months during autumn and winter. Spermatogenesis occurred during 4 or 5 at the end of the oocyte maturation (spring and summer). Spermatozoans were primitive but possessed a true C-shaped acrosome. Chapter 3. The gametogenesis of the oviparous demosponge P. ficiformis occurred during autumn and early winter. Large oocytes and round primitive spermatozoans with proacrosomal vesicles were released to the water on December. Recently fertilised eggs were placed in Petri dishes. Since no free-swimming larva was observed, the development in this demosponge was suggested to be direct.Chapter 4. The reproduction of the carnivorous sponge A. occidentalis was studied under light and electron microscopy. It was a contemporaneous hermaphroditic sponge with clusters of oocytes and very complex spermatic cysts. The fertilisation mechanism was unusual for the phylum Porifera, and shared many similarities with the feeding mechanism of this carnivorous sponge.Chapter 5. The spermatogenesis of the common Mediterranean demosponge C. crambe was investigated by light and electron microscopy. The mature spermatozoan was a extremely modified cell, with the body bent at the level of the flagellum insertion, a true acrosome, and a striated rootlet that connected the basal body to the mitochondrion. In addition, such modifications resembled to the sperm morphology of a phoronid, in a well example of adaptive convergence. Chapter 6. The oogenesis in A. damicornis and R. aculeata was very similar, except for the duration. While it extended for 5 months in R. aculeata, A. damicornis required 7 months to complete it. The differences resided in the vitellogenesis. Yolk was almost exclusively auto-synthesized in A. damicornis from digestion of bacteria. However, in R. aculeata the process shortened because of the help of nurse cells in creation of yolk. Chapter 7. Unspawned sperm and precursors of sperm cells were phagocytosed by motile phagocytic cells in the spermatic cysts of P. ficiformis and R. aculeata. All these features observed for the first time in demosponges revealed that sponges contain many complex features and are capable of complex processes that are usually regarded to higher invertebrates and vertebrates.

El colesterol regula la localització subcel.lular de l'Anx6, i intervé en el transport de la caveolina de Golgi fins a la membrana plasmàtica. En aquest treball hem demostrat com l'Anx6 regula el transport de caveolina produint una alteració en el transport de colesterol intracel.lular des del compartiment endocític tardà fins a l'aparell de Golgi. Es va investigar la localització de la caveolina i el tràfic d'aquesta proteïna en el nostre model cel.lular, una línia transfectada de forma estable per a la sobreexpressió d'Anx6 (CHOAnx6). Hem demostrat que nivells elevats d'Anx6 provoquen una acumulació de caveolina al complex de Golgi. Aquest increment de caveolina s'associa a una acumulació de colesterol lliure als endosomes tardans, que alhora provoca una disminució dels nivells de colesterol a la membrana plasmàtica i a les membranes de Golgi. La disminució de colesterol al Golgi impedeix que la caveolina monomèrica pugui formar oligomers que és el requisit necessari perquè pugui sortir del Golgi i arribar a la membrana plasmàtica. Per això, en les cèl.lules CHOAnx6 el nombre de caveoles a la membrana està reduït fins a un 50%. Afegint consistència a aquests fets, tant l'expressió ectòpica de la proteïna NPC1 salvatge (que transporta el colesterol cap a fora dels endosomes tardans) com l'adició de colesterol soluble exogen, reverteixen l'acumulació de caveolina , que arriba amb normalitat a la membrana, i es recupera el nombre de caveoles fins a nivells control. En ambdós casos, també desapareix l'acumulació de colesterol lliure als endosomes. Aquesta reversió del fenotip també es produeix inhibint totalment la funcionalitat de l'Anx6 amb l'RNA d'interferència específic per a aquesta proteïna. Tots aquests resultats en conjunt demostren que la sobreexpressió d'Anx6 produeix una acumulació de colesterol als endosomes tardans, que fa que no arribi prou colesterol al Golgi, retenint la caveolina en aquest compartiment, i inhibint la formació de caveoles a la membrana plasmàtica. / "ANNEXIN A6-INDUCED ALTERATIONS IN CHOLESTEROL TRANSPORT AND CAVEOLIN EXPORT FROM THE GOLGI COMPLEX"TEXT:Annexin A6 (AnxA6) belongs to a family of Ca 2+ dependent membrane-binding proteins and is involved in the regulation of endocytic and exocytic pathways. We previously demonstrated that AnxA6 regulates receptor mediated endocytosis and lysosomal targeting of low-density lipoproteins and translocates to cholesterol-enriched late endosomes (LE). As cholesterol modulates he membrane binding and the cellular location of nxA6, but also affects the intracellular distribution of caveolin, we investigated the localization and trafficking of caveolin in AnxA6-expressing cells. Here, we show that cells expressing high levels of AnxA6 are characterized by an accumulation of caveolin-1 (cav-1) in the Golgi complex. This is associated with a sequestration of cholesterol in the LE and lower levels of cholesterol in the Golgi and the plasma membrane, both likely contributing to retention of caveolin in the Golgi apparatus and a reduced number of caveolae at the cell surface. Further strengthening these findings, knock down of AnxA6 and the ectopic expression of the Niemann-Pick C1 protein in AnxA6-overexpressing cells restore the cellular distribution of cav-1 and cholesterol, respectively. In summary, this study demonstrates that elevated expression levels of AnxA6 perturb the intracellular distribution of cholesterol, which indirectly inhibits the exit of caveolin from the Golgi complex.

Tesi realitzada a l'Institut de Recerca Biomèdica de Barcelona (IRBB) / El tráfico de membrana en el cono crecimiento axonal es clave durante el desarrollo del sistema nervioso. La familia de proteínas SNARE son una familia de moléculas cuya función principal es mediar la fusión entre membranas. Este hecho hace que las proteínas SNARE tengan potencialmente tengan un papel en el desarrollo axonal y otros procesos que impliquen una gran actividad en los procesos de tráfico de membrana: un ejemplo de casos como así es la progresión de tumores con un alto grado de recambio de membrana como el glioblastoma, que es el caso más común y mortal de astrocitoma de grado IV. Las proteínas SNAREs se clasifican en dos grupos t-SNARE (localizadas en las membranas objetivo) y v-SNARE (localizadas en las membranas vesiculares) y juntas median la fusión entre membranas a través de la formación del complejo SNARE. El complejo SNARE mejor caracterizado es el tetrámero formado por Sintaxina1 (Stx1), dos SNAP-25 y VAMP2. En esta tesis doctoral estudiamos en primer lugar el papel de las proteínas SNARE en los procesos de guía axonal. Para ello utilizamos como modelo el desarrollo del sistema nervioso embrionario en Drosophila melanogaster y obtuvimos como resultado que problemas en las proteínas SNARE, especialmente en Sintaxina1A (Syx1A), generan problemas de guía axonal en el sistema nervioso. Si nos centramos en los estudios del desarrollo del sistema nervioso en mutantes nulos para Syx1A, todas las estructuras nerviosas desde el sistema nervioso central, los nervios motores y el sistema periférico desarrollaron problemas de guía axonal. Además, estudios de interacción génica y una detalla caracterización del fenotipo sugieren que la alteración de las proteínas SNARE, especialmente Syx1A, genera problemas en la vía de guía axonal Slit/Robo. En segundo lugar, estudiamos el rol de las proteínas SNARE en la progresión tumoral. Pare ello analizamos los efectos in vitro e in vivo que tiene el bloqueo de la función de Stx1 en líneas celulares y modelos murínicos del glioblastoma Como resultado, detectamos una pérdida de la proliferación in vitro e in vivo al bloquear Stx1. Además, el bloqueo de Stx1 disminuye la capacidad invasiva in vitro de las células de glioblastoma. / The membrane delivery/traffic in the growth cone is a key element during axons development. SNARE family proteins may be important players in this process since they mediate the fusion of membrane vesicles with their targets. Moreover, since membrane trafficking is a key element during the progression of several tumors, SNAREs may be important also during tumoral progression. Two groups form the SNARE family: t-SNARE (Syntaxins, SNAPs…) located in the target membranes, and, v-SNARE (VAMPs), located in the vesicles. Complexes between specific t- and v- SNAREs are formed which ultimately leads to the fusion of vesicles with their target membranes. The best-characterized SNARE complex is the tetramer formed by syntaxin1 (Stx1), two SNAP-25 and VAMP2, which mediates the fusion of synaptic vesicles during the exocytosis of neurotransmitters. In order to study the role of SNARE in axon guidance we used as a model Drosophila melanogaster embryonic nerve system development. Embryos mutant for SNARE proteins developed axon guidance defects. From all the stocks, Syx1A had a severe phenotype in comparison with the other mutants. Syx1A null embryos developed axon guidance problems in all the nerve system structures: ventral nerve cord, motor axons or peripheral nerves. In addition, a deep analysis of the phenotype and genetic interaction studies suggest that Syx1A mutants have problems in Slit/Robo axon guidance pathway. In order to study the role of SNARE protein in tumoral progression we studied in vitro and in vivo the effect of Stx1 inactivation in glioblastoma models (glioblastomas are the most common and deadliest astrocitomas). Our results show that the loss of function in Stx1 decreases in vitro and in vivo glioblastoma progression and it blocks in vitro cellular invasion.

Tesi realitzada a l'Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) / The quality of life of a significant proportion of IBD patients is poor as a result of persistent disease activity and repeated surgery, among others. Current treatments for Crohn’s disease are not able to neither prevent this serious impact nor improve the long term prognosis of a significant proportion of patients. Therefore, new therapeutic approaches are needed in order to modify the immune response of these patients. We hypothesize that administration of ex-vivo generated autologous tol-DCs to Crohn’s disease patients may arrest Th1 lymphocyte proliferation and therefore may restore specific tolerance against non-pathogenic antigens in the gut. The overall objective of this thesis was to generate and characterize tol-DCs for the purpose of implementing an autologous immunotherapy treatment for Crohn’s disease patients. In the first study, we described the generation of tol-DCs from healthy donors and Crohn’s disease patients by use of clinical-grade reagents in combination with dexamethasone as immunosuppressive agent and characterized their functional properties. Our main findings demonstrated that the combination of dexamethasone with a specific cytokine cocktail yields clinical-grade DCs with the following characteristics: a semi-mature phenotype, a pronounced shift towards anti-inflammatory versus inflammatory cytokine production and low T-cell stimulatory properties. This characteristic tolerogenic profile is maintained when tol-DCs are activated using heat-inactivated Gram-negative bacteria as maturative stimulus. Whole microorganisms contain multiple PAMPs capable of stimulating DCs by different pathways. Our results clearly showed a strong inhibitory effect on DC phenotype, a robust inhibition of pro-inflammatory cytokines, increased IL-10 secretion, and inhibition of T-cell proliferation and Th1 induction. Interestingly, we showed that tol-DCs have reduced immunogenic capacity in autologous, allogeneic and antigen-specific T-cell responses. We further evaluated the ability of tol-DCs to induce CD4+ T-cell hypo-responsiveness. Our results demonstrated that T-cells or antigen-specific T-cells previously cultured with tol-DCs are anergic exhibiting a reduced capacity to proliferate as well as reduced IFN-gamma secretion when rechallenged with fully competent mDCs. With regard to tol-DCs clinical application, we importantly found that their tolerogenic properties remain stable after washing out dexamethasone and subsequent restimulation with LPS, CD40L or different Gram-negative enterobacteria strains. All these properties led us to conclude that this cell product is suitable to be tested in clinical trials of immune-based diseases such as Crohn’s disease. We further identified a positive biomarker for tol-DCs, MERTK receptor is highly expressed on clinical grade dexamethasone-induced human tol-DCs and contributes in their tolerogenic properties. Our results demonstrated that MERTK expression in human DCs is regulated by glucocorticoids and described a new function of this receptor in directly regulating T-cell response. Interestingly, our findings showed that neutralization of MERTK with monoclonal antibodies in allogeneic MLR cultures leads to increased T-cell proliferation and IFN-gamma production. The direct regulation of T-cell response was confirmed by the use of recombinant MERTK-Fc protein, used to mimic MERTK on DCs. Our results remarkably showed that MERTK-Fc suppresses naïve and antigen-specific memory Tcell proliferation and activation. These findings identified a new non-cell autonomous regulatory function of MERTK expressed on DCs. Additionally, we described that this regulation is mediated by the neutralization of MERTK soluble ligand PROS1. We also found that MERTK is expressed on T-cell surface and that PROS1 drives an autocrine pro-proliferative effect on these cells. In summary, the results of this work demonstrated that MERTK on DCs regulates T-cell activation and expansion through the competition for PROS1 interaction with MERTK in the T-cells. We showed that MERTK expression in human DCs has a key role in instructing adaptive immunity and identified MERTK as a potent suppressor of T-cell response. Therefore targeting MERTK may provide an interesting approach to effectively increase or suppress tolerance for the purpose of immunotherapy. / Esta tesis doctoral estudia el proceso de generación de células dendríticas tolerogénicas en grado clínico, con el objetivo de establecer un protocolo destinado al tratamiento de la enfermedad de Crohn. El estudio realizado ha permitido la caracterización de dichas células y sus propiedades tolerogénicas, incluyendo la descripción novedosa de un marcador de células tolerogénicas y el estudio de sus propiedades funcionales relacionadas con la inducción de tolerancia.

Al llarg dels darrers anys, s’ha demostrat que la fragmentació del DNA espermàtic té un paper important en la infertilitat masculina. A més, sembla lògic que trencaments en el DNA espermàtic poden comprometre la viabilitat del zigot, afectant al desenvolupament embrionari. Tenint en compte la hipòtesi que els trencaments de cadena senzilla i doble del DNA poden tenir efectes diferencials en la clínica del pacient, s’han desenvolupat diferents objectius: i) quan a metodologies, ii) efectes clínics en diferents grups de pacients, iii) aspectes bàsics dels trencaments de doble cadena, i iv) la relació amb la compactació del DNA. Com a aspectes rellevants, s’han establert valors llindar per la detecció d’infertilitat mitjançant les diferents metodologies d’anàlisi de la fragmentació del DNA, i s’ha descrit l’efecte clínic de la fragmentació de doble cadena del DNA espermàtic en humans i s’ha proposat un mecanisme bàsic, utilitzant un model de ratolí. Els diferents resultats han conclòs que aquest tipus de trencaments és responsable d’un increment en el risc d’avortament associat al factor masculí. Aquests descobriments han permès desenvolupar noves eines diagnòstiques que permeten un millor consell reproductiu, així com la possibilitat de recerca de possibles tractaments per pal·liar aquest efecte. / In the recent years, it has been demonstrated that sperm DNA fragmentation plays an important role in male infertility. Moreover, it seems logical that sperm DNA breaks can compromise the zygote viability and affect to embryonic development. In this sense, taking into account that single and double stranded DNA breaks may have different clinical effects, different objectives have been developed: i) regarding methodologies, ii) clinical effects on patients, iii) basic aspects of double stranded DNA breaks, and iv) in relation to sperm DNA compaction. The most relevant results have been the establishment of cut-off values for male infertility diagnosis for the most common DNA fragmentation methodologies, and the description of the clinical effect of double stranded DNA breaks in humans, and its basic mechanism using a murine model. The different results concluded that these double stranded DNA breaks are responsible from an increase on male factor miscarriage risk. These findings allowed developing new diagnostic tools for a better reproductive counseling, as well as to open new research pathways on male infertility treatments

Tesis por compendio de publicaciones / Para poder entender el efecto de los parásitos sobre los hospedadores y la evolución de las interacciones parásito-hospedador es necesario comprender primero el efecto de los factores ambientales sobre estos organismos y su interacción, lo que a su vez requiere considerar tales factores a la escala adecuada según los organismos implicados. El objetivo principal de esta tesis es estudiar el efecto de factores abióticos, particularmente la temperatura, en la regulación de la diapausa del ectoparásito Carnus hemapterus. Este insecto es un díptero hematófago generalista con amplia distribución geográfica que parasita a los pollos de una gran variedad de especies aviares (en su mayoría trogloditas). El trabajo se desarrolló en una localidad árida del sureste de España (desierto de Tabernas), donde coexisten entremezclados tres tipos de hábitats (oquedades naturales en taludes de ramblas, cavidades en construcciones humanas, - principalmente puentes - y cajas nido) ocupados por Carnus hemapterus y sus hospedadores aviares. Como primer paso se determinó mediante sensores el microclima de las tres cavidades donde Carnus desarrolla su diapausa. Las características microclimáticas mostraron notables diferencias, siendo las cajas nido más inestables (frías y con marcada oscilación térmica), mientras que las otras oquedades fueron mucho más estables, presentando las cavidades en taludes condiciones más cálidas y secas que las de puentes. Esta heterogeneidad ambiental tiene importantes implicaciones tanto para los organismos endotermos que las habitan como para los ectotermos. Mediante experimentos de campo realizados con diversos hospedadores se comprobó en condiciones naturales la coexistencia de diapausas largas y cortas. Dado que la duración del ciclo de vida y número de generaciones al año (voltinismo) está determinado por la duración de la diapausa, registramos consecuentemente la coexistencia de moscas uni y bivoltinas tanto a nivel de población como de nido. Estos resultados muestran la naturaleza facultativa de la diapausa de Carnus así como el papel de las variables abióticas en la modulación de aquella. El estudio con cámaras climáticas de la influencia de la temperatura en la finalización de la diapausa y el patrón de emergencia mostró que aunque los choques de frío no son necesarios para que se termine la diapausa de Carnus, sí tienen importantes efectos, acelerando el desarrollo de la diapausa, adelantando la emergencia y acelerando su progreso respecto a condiciones constantes. Además, mediante experimentos en campo, comprobamos que las características térmicas particulares de los diversos microhábitats modularon el progreso de la diapausa, el comienzo de la emergencia y su patrón. En microclimas inestables (cajas nido) la fenología de emergencia se adelantó en la estación, pero su progreso fue más lento, alargando el periodo de emergencia en comparación con los microhábitats estables (puentes y taludes) entre los que no hubo diferencias en la emergencia. Comprobamos experimentalmente la consistencia de estos resultados cuando las diferencias microclimáticas fueron anuladas (homogeneizadas) en dos momentos de la fase de la diapausa; durante la diapausa invernal y al final de la fase de desarrollo de la post-diapausa. Estos resultados sugieren posibles procesos de adaptación o aclimatación a los microclimas. También se reveló la mayor sensibilidad de Carnus a cambios térmicos justo antes de la emergencia, lo que favorecería la percepción del cambio ambiental por la presencia del hospedador en el nido y consecuentemente la sincronización de la emergencia del parásito. El estudio de los requerimientos térmicos del parásito desveló que el desarrollo de la post-diapausa necesitó acumular menor cantidad de unidades de calor (grados día) en cajas nido, pero debido a las condiciones térmicas (fuerte oscilación con temperaturas extremas), el desarrollo fue ralentizado, necesitando más tiempo para desarrollarse que en cavidades térmicamente estables. En estas últimas cavidades los requerimientos térmicos fueron mayores (sobre todo en taludes), mostrando una tasa de emergencia similar entre ellas, pero acelerada con respecto a cajas nido. En definitiva, la presente tesis revela la importancia de considerar los factores abióticos a pequeña escala, así como los mecanismos que están actuando en la regulación del ciclo vital de parásitos para poder abordar futuros estudios fisiológicos, ecológicos o evolutivos entre otros. / Understanding the impact of parasites in their hosts and the evolution of host-parasite interactions requires previous knowledge on the effect of environmental factors on such organisms and its interaction. For this, it is critical to consider such factors at the appropriate scale, defined by the organisms under study. The main objective of this thesis is to study the effect of abiotic factors, particularly temperature, on the regulation of diapause of the ectoparasite Carnus hemapterus. This insect is a generalist haematophagous dipteran with a large geographic distribution that parasitizes nestling of a wide variety of bird species (mainly troglodyte ones). This work was developed in an arid locality in the southeast of Spain (Tabernas´ desert), where three types of habitats (natural burrows at sandy cliffs in the ramblas, cavities in human constructions, - mainly bridges - and nest boxes) coexist interspersed and are occupied by Carnus hemapterus and its avian hosts. Firstly, the microclimate of the three cavity types, where the diapause of Carnus takes place, was studied by data-loggers and considerable differences were found: nest boxes were colder and with marked thermal oscillation, while the temperature in the other cavities was much more stable. Moreover, burrows showed warmer and drier conditions than cavities in bridges. This environmental heterogeneity has important implications for the organisms (both endotherms and ectotherms) that inhabit these cavities. Field experiments performed with different host species in natural conditions revealed the co-occurrence of long and short diapauses. Since the duration of the life cycle and the number of generations per year (voltinism) is determined by the length of the diapause, we registered consequently, the co-occurrence of uni and bivoltine flies at both population and nest level. These findings illustrate the facultative nature of diapause of Carnus and the modulating role of abiotic variables. Experimental tests with climatic chambers of the influence of temperature in the termination of diapause and the emergence pattern showed that, although cold shocks are not necessary for the end of the diapause, they stimulate the development of diapause, advance the emergence and accelerate its progress when compared to constant conditions. Furthermore, by means of field experiments, we checked that the particular thermal features of the microhabitats modulated the progress of the diapause, the emergence start and its pattern. In habitats with fluctuating microclimate (nest boxes) the phenology of emergence occurred earlier in the season, but its emergence progress was slower, making the emergence longer compared with stable microhabitats (cavities in bridges and burrows) where no differences in emergence were found. We confirmed experimentally the consistency of such results when the microclimatic differences were removed in two different moments of the diapause: during the winter diapause and at the end of the development phase of the post-diapause. These findings suggest adaptation or acclimatization of individuals to specific microclimates. Also, a higher sensitivity of Carnus to thermal changes just before emergence was revealed, what would favor the perception of environmental changes due to the presence of the host in the nest and, consequently, the synchronization of the parasite emergence. The study of the thermal requirements of the parasite revealed that its post-diapause development needed to accumulate less heat units (degree day) in nest boxes than in the other cavity types. Yet, due to sharp thermal oscillation with extreme temperatures, the development was likely decelerated by development arrests. Thus, flies from nest boxes needed more time to develop than flies from thermally stable cavities. Flies from cavities in bridges and burrows showed higher thermal requirements (mainly in burrows), being the emergence rate similar between them, but faster when compared with flies from nest boxes. Finally, this thesis reveals the importance of considering the abiotic factors at a small scale and the mechanisms acting in the regulation of the life cycle of parasites before addressing future physiological, ecological or evolutionary studies.