FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / As pneumonias sÃo problemas de saÃde publica mundial, especialmente em crianÃas menores que cinco anos de idade. Os vÃrus parainfluenza (VPI-1, 2 e 3) sÃo agentes frequentes de pneumonia, pouco se conhecendo sobre a participaÃÃo do VPI-4 devido a dificuldades do seu isolamento em cultura de cÃlulas, a ausÃncia de antÃgenos especÃficos para este vÃrus nos painÃis de rotina de detecÃÃo dos vÃrus respiratÃrios, alÃm de serem relacionados apenas a casos de infecÃÃes respiratÃrias leves. O objetivo do presente estudo à descrever o perfil epidemiolÃgico e clÃnico das pneumonias causadas pelos quatro tipos de VPI na populaÃÃo de estudo, no perÃodo de janeiro de 2013 a dezembro de 2014. Para tanto, aspirados nasofarÃngeos de 542 crianÃas de atà cinco anos atendidas no Hospital Infantil Albert Sabin (HIAS), que receberam o diagnÃstico de pneumonia, foram submetidos à RT-PCR para a detecÃÃo dos VPI-1, 2 e 3 e 4. Estas amostras tinham sido analisadas anteriormente por imunofluorescÃncia indireta para vÃrus sincicial respiratÃrio (VSR), influenza (A e B), adenovÃrus e VPI (1, 2 e 3). Os VPI foram detectados em 165 casos, seguido de VSR (136), adenovÃrus (34) e influenza (30). As caracterÃsticas clÃnicas e epidemiolÃgicas de pneumonias pelos VPI foram analisadas em 104 amostras que apresentaram infecÃÃo isolada por um dos quatro tipos de VPI. Os VPI mais frequentemente detectados, em ordem decrescente, foram os tipos VPI-3 (64,42%), VPI-4 (19,23%), VPI-1(14,42%) e VPI-2 (1,92%). O VPI-4 foi o mais associado a co-infecÃÃes. O VPI-4 foi o Ãnico VPI cuja circulaÃÃo esteve associada à estaÃÃo chuvosa dos dois anos de estudo (p<0,0001). O VPI-3 e o VPI-1 apresentaram pico de circulaÃÃo associado à estaÃÃo seca. Os VPI sÃo agentes frequentes de pneumonias em crianÃas menores que cinco anos na cidade de Fortaleza. / Pneumonia are public health problems worldwide, especially in children younger than five years old. Human parainfluenza virus (HPIV-1, 2 and 3) are common agents of pneumonia, little was know about the involvement of HPIV-4 due to difficulties of isolation in cell culture, the absence of antigens specific for this virus in panels routine detection of respiratory viruses, and are associated only with cases of mild respiratory infections. The aim of this study is to describe the clinical and epidemiological profile of pneumonia caused by four types of HPIV in the study population, from January 2013 to December 2014. To this end, nasopharyngeal aspirates of 542 children under five treated at Hospital Infantil Albert Sabin (HIAS), who were diagnosed with pneumonia, were subjected to RT-PCR for the detection of HPIV-1, 2, 3 and 4. These samples had been previously analyzed by indirect immunofluorescence for respiratory syncytial virus (RSV), influenza (A and B), adenovirus, and HPIV (1, 2 and 3). The HPIV were detected in 165 cases, followed by RSV (136), adenovirus (34) and influenza (30). Clinical and epidemiological characteristics of pneumonia by HPIV were analyzed in 104 samples with isolated infection with one of four types of HPIV. The HPIV most frequently detected, in descending order, were the HPIV-3 types (64.42%), HPIV-4 (19.23%), HPIV-1 (14.42%) and HPIV-2 (1.92 %). The HPIV-4 was the most associated with co-infection. The HPIV-4 was the only HPIV whose circulation was associated with the rainy season of two years of study (p <0.0001). The HPIV-3 and HPIV-1 had a circulation peak associated with the dry season. The HPIV are frequent agents of pneumonia in children younger than five years in the city of Fortaleza.

Made available in DSpace on 2014-06-11T19:24:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2006Bitstream added on 2014-06-13T20:51:50Z : No. of bitstreams: 1 watanabe_asa_me_botfm.pdf: 435949 bytes, checksum: e2d8389a510d8ed3643dbe728b2b723a (MD5) / From about 200 virus that cause respiratory infections, only 8 are responsible for severe illness in children, immunodeficient adults and elderly, inc1uding Adenovirus, Influenza A and B, Parainfluenza 1, 2 and 3, Human RespÍratory Syncytial Vírus (HRSV) and Human Metapneumovirus (hMPV). Three of these virus are responsible for a significant morbidity in elderly: Influenza, HRSV and hMPV. The nosocomial infection caused by these vÍruses can be fatal in hospitalized children and patients with other pathologies. With the advance of the of molecular biology techniques, the diagnosis and the characterization of these vírus became more effective. Beside vírus detection by PCR, isolation of vírus in specific cellular cultures to increase the amount of pathogens have been used in severallaboratories. Studies on prevalence of respÍratory vírus in elderly are rare. The objective of this research was to evaluate the occurrence of Influenza, HRSV and of the hMPV and the risk factors involved in the diseases caused by these vÍruses. Nasopharyngeal swabs were collected and used for inoculation in cells culture and dÍrect analysis by RT-PCR. The results from RT-PCR of Influenza Vírus and RSV were negative. We also tested the samples with GeneScan, that is a technique based on fluorescent primers specific to the studied vírus. Results for Flu virus and HRSV were the same ofRT-PCR, but 1 MPV sample was positive. 55,32% ofthe 47 studied elderly were vaccinated against Influenza; 14,9% had tabagism habits and 70,2% were women. These risk factors might had influenced in the absence of positive samples for the Influenza vírus andHRSV.

Las Candidas forman uno de los grupos con mayor n´umero de hongos pat´ogenos en humanos. La relaci ´on que tienen desde un punto de vista filogen´etico demuestra que la capacidad de infectar humanos ha surgido varias veces de forma independiente en este clado. El complejo de Candida parapsilosis es ideal para investigar la aparici´on de virulencia puesto que contiene tres especies cercanas que muestran diferentes grados de virulencia y de importancia: C. parapsilosis, C. orthopsilosis y C. metapsilosis. En esta tesis presento la secuenciaci´on y posterior an´alisis de quince cepas de origen cl´ınico y medioambiental que forman parte de este clado. Cabe remarcar que los genomas de C. orthopsilosis tipo 1 y de C. metapsilosis no se hab´ıan secuenciado previamente. Nuestros resultados muestran evidencia gen´omica de la existencia de recombinaci´on, apareamiento e hibridaci´on en este clado, que previamente se habia considerado asexual. Proponemos que las cepas cl´ınicas emergieron de forma independiente en linajes de cepas encontradas en el medioambiente y una posible relaci ´on entre la hibridaci´on y la adquisici´on de caracter´ısticas relevantes para la virulencia. Finalmente, para estudiar la aparici´on de virulencia en este clado, hemos comparado, por primera vez, los genomas de las tres especies que se encuentran dentro del complejo de Candida parapsilosis. Hemos encontrado expansiones de familias g´enicas previamente implicadas en virulencia, como es el caso de las adhesinas, transportadores de membrana y enzimas extracelulares. Tambi´en hemos observado expansiones de familias de genes que hasta el momento no han estado relacionadas con virulencia. En resumen, nuestros resultados aportan una base para poder elaborar numerosas hip´ otesis sobre la aparici´on de virulencia en las especies de Candida y para que estas hip´ otesis puedan ser comprobadas posteriormente en el laboratorio. / Candida species constitute one of the most prevalent groups of fungal pathogens. From their phylogenetic relationships it is clear that virulence to humans has emerged in this clade several, independent times. The Candida parapsilosis complex is particularly suitable to investigate the emergence of virulence, with three closely-related species of varying degree of pathogenicity and of growing relevance: C. parapsilosis, C. orthopsilosis and C. metapsilosis. In this thesis I present the genome sequencing and analysis of fifteen strains from this clade, sampled from clinical and environmental sources. Notably, genomes of C. orthopsilosis Type 1 and C. metapsilosis were sequenced for the first time. Our results show for the first time the genomic evidence for the existence of recombination, mating and hybridization in this clade, previously considered asexual. We propose the independent emergence of clinical isolates from environmental lineages and a possible role of hybridization in the acquisition of relevant traits for pathogenesis. Finally, in order to gain insight into the emergence of virulence in this clade, we have compared the genomes of all three species of C. parapsilosis complex. We have found expansions in gene families known to be involved in virulence, like adhesins, membrane transporters and extracellular enzymes, as well as expansions in gene families not implicated in virulence so far. Altogether, our findings provide the grounds for numerous hypotheses about the emergence of virulence in Candida spp. and for their future experimental testing. 1

Antecedentes: La infección por el virus del papiloma humano de alto riesgo HPV (HR) es un factor necesario para las lesiones cervicales (SIL) y el cáncer cervical invasivo (ICC). En las mujeres infectadas por el HIV, la infección por HPV es más frecuente y hay un mayor riesgo de ICC. Los objetivos de este estudio son: estimar la prevalencia de HPV (HR) y de lesiones cervicales, describir la distribución de los diferentes genotipos, conocer las características clínico-epidemiológicas y de la historia de cribado de las mujeres coinfectadas por el HIV y el HPV (HR) e identificar los factores asociados a la infección por HPV (HR) y a las alteraciones citológicas en mujeres HIV positivas de Cataluña. Métodos: 479 mujeres HIV positivas provenientes de la cohorte PISCIS participaron en el estudio. Las participantes se sometieron a una exploración ginecológica, citología cervical, captura híbrida (HC2, Digene), genotipado de HPV (Roche lineal-Array PCR) y colposcopia y biopsia, si era necesario. Los datos fueron obtenidos a través de cuestionarios con variables sociodemográficas, conductuales, clínicas y de cribado. Para conocer los factores asociados se utilizó un modelo de regresión logística multivariante. Resultados: La prevalencia de infección por HPV (HR) fue de 33.2%. La prevalencia de ASCUS, LSIL y HSIL fueron 7.9%, 13.8% y 3.8%, respectivamente. Los genotipos más frecuentes fueron HPV16 (23%), HPV53 (20.3%) y HPV52 (16.2%). La frecuencia de cribado anual y la cobertura de cribado dentro de los últimos 2 años en las mujeres HIV positivas coinfectadas por el HPV (HR) fueron de 45.1% y 55.1% respectivamente. Los factores asociados a la infección por HPV (HR) fueron: la edad (OR: 0.9 IC: 0.94- 0 .99), último PAP anormal (OR: 8.2 IC: 4.0-16.9) y 1 versus > 11 PAP a lo largo de la vida (OR: 2.0 IC: 1.1-3.8). Los factores asociados a las alteraciones citológicas fueron: la primera relación sexual ≤18 años (OR: 2.3 IC: 1.1-5.0), último PAP anormal (OR: 10.3 IC: 3.5-30.1), recuento de CD4 <200 cel/mm3 versus >500 cel/mm3 (OR: 5.7 IC: 2.2-14.8) y recuento de CV >10000 copias/mL versus <400 copias/mL (OR: 3.1 IC: 1.4-6.9). Conclusiones: Se confirma en nuestro medio la elevada prevalencia de infección por HPV (HR), de lesiones cervicales y de genotipos con alto potencial oncogénico. Existe un cribado inadecuado consistente con la alta incidencia de ICC entre las mujeres HIV positivas en Cataluña. Es urgente aumentar la sensibilización entre los profesionales de la salud y las mujeres HIV positivas para optimizar la detección y prevención del ICC. Es necesario valorar en esta cohorte de manera longitudinal la asociación entre el HIV y el HPV (HR) para conocer los efectos de la terapia antirretroviral en el desarrollo y progresión de las lesiones cervicales. / Background: High-risk type Human papillomavirus HPV (HR) infection is a necessary factor for cervical squamous intraepithelial lesions (SIL) and invasive cervical cancer (ICC). In HIV infected women, HPV infection is more prevalent and a higher risk of ICC has been identified. The objectives of this study are: to estimate the prevalence of HPV (HR) and cervical lesions, describe the distribution of genotypes, to determine the clinico-epidemiological and screening history of women coinfected by HIV and HPV (HR) and identify the factors associated with HPV (HR) infection and cytological changes in HIV positive women in Catalonia. Methods: 479 HIV infected women were identified from ongoing prospective HIV infected patients cohort. Participants underwent a gynecologic examination, cervical PAP smear, hybrid-capture (HC2, Digene), HPV genotyping (Roche Linear-Array PCR) and colposcopy and biopsy, if necessary. Questionnaires on sociodemographic, behavioral, clinical and cervical cancer screening information were obtained. Multivariable logistic regression modeling was used. Results: HPV (HR) infection prevalence was 33.2%. The prevalence of ASCUS, LSIL and HSIL were 7.9%, 13.8% and 3.8% respectively. Most frequent genotypes were HPV16 (23%), HPV53 (20.3%) and HPV52 (16.2%). The frequency of annual screening and screening coverage within the last two years in HIV positive women coinfected with HPV (HR) were 45.1% and 55.1% respectively. The factors associated to HPV (HR) infection were: age (OR:0.9 CI:0.94-0.99), last PAP smear abnormal (OR:8.2 CI:4.0-16.9) and 1 versus >11 PAP smear lifetime (OR:2.0 CI:1.1-3.8). The factors associated to abnormal cytology were: first sexual intercourse ≤ 18 years (OR: 2.3 CI: 1.1-5.0), the last PAP abnormal (OR: 10.3 CI: 3.5-30.1), CD4 count <200 cells/mm3 versus > 500 cells/mm3 (OR: 5.7 CI: 2.2-14.8) and CV count > 10000 copies/ mL versus <400 copies / mL (OR: 3.1 CI: 1.4-6.9). Conclusion: It is confirmed in our area the high prevalence of HPV (HR) infection, cervical lesions and genotypes with high oncogenic potential. The history of poor cervical cancer screening found is consistent with the high incidence of ICC among HIV infected women in Spain. It is urgent to increase awareness among both health professionals and HIV infected women to optimize the screening and prevention of ICC. It is necessary a longitudinal assessment in this cohort to know the association between HIV and HPV (HR) to determine the effects of antiretroviral therapy in the development and progression of cervical lesions.

La infecció pel virus de la immunodeficiència humana (VIH) provoca una deficiència progressiva del sistema immunològic, caracteritzada per una destrucció massiva de les cèl•lules T CD4 i, una activació i inflamació immune mantinguda. La Teràpia Antiretroviral de Gran Activitat (o TARGA) indueix una supressió sostinguda de la replicació viral en individus infectats pel VIH, i una reducció de l’activació immune, encara que no es normalitza comparat amb individus no-infectats. Les causes d’aquesta activació immune persistent malgrat la supressió viral són encara desconegudes. Els nostres resultats demostren una dicotomia en les forces que indueixen l’activació immune en els limfòcits T CD4 i CD8 en individus suprimits per la TARGA. La replicació residual del VIH indueix l’activació immune en les cèl•lules T CD8. Per aquest motiu, la intensificació de la TARGA amb raltegravir produeix una reducció específica però reversible de l’activació de les cèl•lules CD8; i per tant, aquestes cèl•lules semblen ser sensors de la replicació viral (en particular l’expressió de CD38). Curiosament, l’expressió de CD38 està sota el control dels interferons de tipus I, el que suggereix que el virus també controla altres respostes inflamatòries, i que aquestes respostes estan íntimament lligades als marcadors d’activació de les cèl•lules T CD8. Contràriament, en individus tractats, la persistència viral té pocs efectes en el compartiment de cèl•lules T CD4, que encara pateix les conseqüències de la depleció pre-TARGA i que determina la recuperació immune. De fet, l’activació de cèl•lules CD4 no es redueix després d’un any de intensificació amb raltegravir. En canvi, la resposta homeostàtica a la depleció de cèl•lules CD4 sembla induir l’activació en aquest compartiment, especialment en pacients tractats que presenten una resposta immunològica deficient malgrat una supressió viral completa (pacients immunodiscordants). Els nostres resultats demostren que els pacients immnodiscordant tenen un menor producció de novo de cèl•lules T CD4, i una major translocació microbiana, activació i mort cel•lular comparat amb pacients amb una bona recuperació immune; malgrat aquestes diferències la immunodiscordància sembla està associada a l’increment de la destrucció cel•lular. L’activació i inflamació persistent en aquests individus procura un ambient que accelera la l’esgotament de les cèl•lules T CD4 i la immunosenescència de la resta del sistema immunològic, contribuint a llarg termini amb les co-morbiditats i l’envelliment prematur. Per aquest motiu, és important determinar les causes de l’activació immune incrementada (i mort cel•lular), per definir les estratègies terapèutiques que poden ser útils per millorar la recuperació immune. / Human immunodeficiency virus (HIV-­‐1) infection causes a progressive impairment of the immune system, characterized by a massive CD4 T-­‐cell depletion and sustained immune activation and inflammation. Highly active antiretroviral therapy (HAART) induces a sustained effective suppression of viral replication in HIV-­‐infected subjects and reduces immune activation, but does not normalize it. The causes of this persistent immunehyperactivation despite viral suppression remain unknown. Our results show a dichotomy between CD4 and CD8 T-­‐cell driving forces of immune activation in HIV-­‐infected HAART-­‐suppressed individuals. The low (but detectable) levels of residual replication drive immune activation in CD8 T-­‐cell compartment. Therefore, raltegravir intensification of HAART results in specific and reversible reduction of CD8 T-­‐cell activation, which seems to be a sensor of replication events (in particular CD38 expression). Interestingly, CD38 expression is under the control of Type I IFN, suggesting that the virus also controls inflammatory responses and that these responses are intimately linked to CD8 T-­‐cell activation markers. Conversely, in treated individuals, viral persitence has low effects on CD4 T-­‐cell compartment, which still show the consequences of pre-­‐HAART depletion and determine immune recovery. In fact, CD4 T-­‐cell activation is not reduced after one year of raltegravir intensification. Instead, the homeostatic response to CD4 T-­‐cell depletion might drive activation in this compartment, particularly in HAART-­‐treated individuals with satisfactory virological response but poor immune recovery (immunodiscordant subjects). Our results suggest that, even though immunodiscordant individuals showed lower CD4 T-­‐cell production and higher microbial translocation, activation and cell death, immunodiscordance seems to be related to increased cell-­‐destruction of CD4 T-­‐cells. The persistent immune activation and inflammation in these subjects provide a milieu of accelerated immunoexhaustion of CD4 T-­‐cells and immunosenescence of the whole immune system, contributing to long-­‐term co-­‐morbidities and accelerated ageing observed in these subjects. Therefore, determining the causes of this increased hyperactivation and cell-­‐death may be important for the development of therapeutic strategies aiming to improve immune recovery.

Translation control is a vital aspect of gene expression for both viruses and their cellular hosts. We have previously shown that the cellular mRNA decay activators Dhh1 and Lsm1-7 promote translation of positive-strand RNA [(+)RNA] viral genomes and their subsequent transport from the cellular translation machinery to replication complexes, a process that requires translation repression. These key steps in the replication of all (+)RNA viruses require profound rearrangements of the viral ribonucleoprotein (RNP) composition. How cellular decapping activators promote translation and replication of viral genomes remains unknown. Using the replication of the Brome mosaic virus in yeast, a fruitful model system for (+)RNA viral replication, we show that Dhh1 and Lsm1-7 function differentially in viral RNA translation and replication by assembling alternative mRNP complexes. The dependence on Dhh1 for viral RNA translation initiation is mediated by specific cis-acting sequences in the viral UTRs and a stem-loop in the ORF. Excitingly, by ribosome profiling analyses we identify a specific subset of cellular mRNAs that also depends on Dhh1 for translation. These mRNAs have as (+)RNA genomes long 5´UTR and highly structured 5´UTRs and ORFs. Moreover, they are enriched in mRNAs related to ribosome biogenesis. Interestingly, ribosome biogenesis is often altered in cancer cells and we and others determine that DDX6, the human ortholog of Dhh1, is indeed overexpressed in pancreatic and colon cancer. In conclusion, our results demonstrate that components of the cellular decapping machinery have a broad function in translational regulation. This enables fast fine-tuning of gene expression in response to perturbations. / El control de la traducción es un aspecto vital de la expresión génica tanto para los virus como para sus huéspedes celulares. Nuestro laboratorio ha demostrado que los activadores de la degradación del ARN mensajero celular, Dhh1 y Lsm1-7, promueven la traducción de los genomas de virus de ARN de cadena sencilla y polaridad positiva [(+)ARN] así como su transporte desde la maquinaria de traducción celular a los complejos de replicación, un proceso que necesita represión de la traducción. Estos pasos clave en la replicación de todos los virus (+)ARN requieren de una profunda reorganización en la composición de la ribonucleoproteina (RNP) viral. Cómo los activadores del decapping celular promueven la traducción y replicación de los genomas virales aún es desconocido. Utilizando la replicación del virus del mosaico del Bromus en levadura, un modelo muy usado para el estudio de la replicación de virus (+)ARN, hemos demostrado que Dhh1 y Lsm1-7 funcionan de manera distinta en la traducción y replicación del ARN viral mediante el ensamblaje de complejos alternativos de RNP. La dependencia de Dhh1 para la iniciación de la traducción del ARN viral está mediada por secuencias específicas cis-acting localizadas en las regiones no traducidas (RNTs) del virus y un stem-loop en el marco de lectura. Sorprendentemente, mediante el uso del ribosome profiling hemos identificado un grupo específico de ARN mensajeros celulares que también dependen de Dhh1 para su traducción. Estos ARN mensajeros tienen, como los genomas de los virus (+)ARN, 5’RNT largos y altamente estructurados, así como marcos de lectura altamente estructurados. Además, entre ellos abundan los ARN mensajeros relacionados con biogénesis ribosomal. Es interesante mencionar que la biogénesis ribosomal está normalmente alterada en células cancerosas y nosotros, y otros grupos, hemos determinado que DDX6, el ortólogo en humanos de Dhh1, está sobreexpresado en cáncer pancreático y de colon. En conclusión, nuestros resultados demuestran que componentes de la maquinaria de decapping celular tienen una amplia función en la regulación de la traducción. Este hecho permite un rápido y preciso ajuste de la expresión génica en respuesta a perturbaciones.

Made available in DSpace on 2014-06-12T15:53:19Z (GMT). No. of bitstreams: 2 arquivo5109_1.pdf: 4981987 bytes, checksum: 45983940c169b7f60379907b2e4ab868 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2004 / Os lentivírus de pequenos ruminantes (SRLV) causam doença progressiva multissistêmica crônica, com longo período de incubação, que acomete caprinos e ovinos de todas as raças e idades. Estes vírus podem se manifestar de forma artrítica, respiratória, mamária e nervosa ou mesmo atingir vários órgãos, causando perda de peso e debilidade até a morte do animal. A infecção é persistente apesar da maioria dos animais infectados não desenvolver sintomas clínicos, com risco de disseminar o vírus para outros animais. A principal forma de transmissão do vírus do vírus é através da ingestão de colostro e/ou leite proveniente de animais infectados. Pode ocorrer transmissão horizontal. Os SRLV infectam células do sistema fagocítico-mononuclear e células dendríticas in vivo e infectam e replicam em macrófagos e em células derivadas de vários tecidos in vitro. O cultivo primário de células de membrana sinovial (MS) é amplamente utilizado para isolamento e replicação de SRLV. A capacidade de divisão destas células é limitada até atingir seu estado de senescência celular. Objetivando a otimização da produção, foi celular feito o reaproveitamento de explantes das articulações metacarpianas de feto caprino soronegativos para SRLV. Os explantes foram reaproveitados por seis vezes. As células de MS obtidas mantiveram-se em níveis satisfatórios de produtividade e permissividade à replicação de amostras de SRLV. Estas células de MS foram utilizadas para estudo da replicação de amostras brasileiras de SRLV BrPe1-01 e BrMg2-03, de forma comparativa com as amostras padrão CAEV Cork e Maedi-visna K1514. Não se obtiveram títulos viral e antígênico significativos das amostras brasileiras nas várias tentativas de replicação das amostras BrPe1-01 e BrMg2-03. Visando um melhor entendimento da epidemiologia dos SRLV em caprinos do Estado de Pernambuco, foi feito um levantamento sorológico nos anos de 2002 e 2003. Foram testados, por técnica de imunodifusão em gel de agar (IDGA), todos os animais de 31 rebanhos do Estado de Pernambuco. Dos 2206 animais testados, 15,3% (n= 338) estavam infectados por SRLV. Os animais soropositivos estavam distribuídos em 74,2% dos rebanhos (n=23).A avaliação do perfil lipídico de animais infectados por SRLV sem sintomatologia clínica, quando comparados aos de animais controle, sob as mesmas condições de manejo e alimentação, apresentaram os seguintes resultados: a) Os níveis de colesterol total foram significativamente mais elevados (p<0.005); b) O colesterol das lipotroteínas de elevada densidade (HDL) estava levemente aumentado, não sendo significativo; c) O colesterol das lipoproteínas de muito baixa densidade (VLDL) estava levemente baixo; d) O nível de triglicerídeo foi similar nos dois grupos; e) O colesterol das lipoproteínas de baixa densidade (LDL) estava significativamente elevado (p<0.005)

Orientador: João Manuel Grisi Candeias / Resumo: Não disponível. / Abstract: From about 200 virus that cause respiratory infections, only 8 are responsible for severe illness in children, immunodeficient adults and elderly, inc1uding Adenovirus, Influenza A and B, Parainfluenza 1, 2 and 3, Human RespÍratory Syncytial Vírus (HRSV) and Human Metapneumovirus (hMPV). Three of these virus are responsible for a significant morbidity in elderly: Influenza, HRSV and hMPV. The nosocomial infection caused by these vÍruses can be fatal in hospitalized children and patients with other pathologies. With the advance of the of molecular biology techniques, the diagnosis and the characterization of these vírus became more effective. Beside vírus detection by PCR, isolation of vírus in specific cellular cultures to increase the amount of pathogens have been used in severallaboratories. Studies on prevalence of respÍratory vírus in elderly are rare. The objective of this research was to evaluate the occurrence of Influenza, HRSV and of the hMPV and the risk factors involved in the diseases caused by these vÍruses. Nasopharyngeal swabs were collected and used for inoculation in cells culture and dÍrect analysis by RT-PCR. The results from RT-PCR of Influenza Vírus and RSV were negative. We also tested the samples with GeneScan, that is a technique based on fluorescent primers specific to the studied vírus. Results for Flu virus and HRSV were the same ofRT-PCR, but 1 MPV sample was positive. 55,32% ofthe 47 studied elderly were vaccinated against Influenza; 14,9% had tabagism habits and 70,2% were women. These risk factors might had influenced in the absence of positive samples for the Influenza vírus andHRSV. / Mestre

Tesi realitzada a l'Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) / Los receptores de la familia SLAM se encuentran expresados diferencialmente en la superficie de los leucocitos y juegan un papel importante en la inmunidad innata y adaptativa. En este trabajo se demuestra que el citomegalovirus (CMV) murino reduce específicamente la expresión de determinados miembros SLAM en la superficie de los macrófagos infectados. Mediante el escrutinio de un panel de mutantes de deleción del CMV murino se ha identificado m154 como el producto viral que modula la expresión del receptor SLAM CD48, el cual constituye el ligando de alta afinidad de CD244, una molécula involucrada en la regulación funcional de las células NK y T citotóxicas. Se muestra que m154 es una proteína tipo mucina, que se expresa con una cinética temprana y se localiza en la superficie de la célula infectada. La proteína viral actúa a través de un mecanismo que implica la degradación de CD48, siendo capaz de interferir con la respuesta citotóxica de las células NK desencadenada por la infección de los macrófagos por el CMV murino. Finalmente, demostramos en el modelo murino, que m154 le proporciona al virus protección in vivo frente al ataque de las células NK. Estos hallazgos contribuyen a un mejor entendimiento de la patogénesis del CMV y proveen un nuevo ejemplo de evasión de la inmunidad innata del huésped desarrollado por CMV. / The signalling lymphocyte-activation molecules (SLAM) family of receptors encompasses a number of proteins expressed on the surface of leukocytes that play critical roles in both innate and adaptive immunity upon engagement through homotypic or heterotypic interactions amongst them. In this study, we show that murine cytomegalovirus (MCMV) drastically downregulates several SLAM receptors during the course of the infection of macrophages, in a manner dependent on viral gene expression. By screening a battery of MCMV deletion mutants, we have identified m154 as an immunoevasin that effectively reduces the cell surface expression of the SLAM family member CD48, a high affinity ligand for natural killer (NK) and cytotoxic T cell receptor CD244. m154 is a mucin-like protein, expressed with early kinetics, that can be localized predominantly at the cell surface of the infected cell. During infection, m154 leads to proteolytic degradation of CD48, primarily via a proteasomal dependent mechanism. This viral protein interferes with the NK cell cytotoxicity triggered by MCMV infected macrophages. In addition, we demonstrate that mutant MCMVs lacking expression of m154 result in an attenuated phenotype in vivo which can be substantially restored after NK cell depletion of mice. Thus, we present here a novel tactic of cytomegalovirus to subvert detection by NK cells during acute infection, based on the modulation of a SLAM family member.

Tesi realitzada a l'Institut de Biologia Molecular de Barcelona (IBMB-CSIC) i a l'Institut de Recerca Biomèdica de Barcelona (IRBB) / Picornaviridae are among the most diverse and oldest known viral families that include many important pathogens of humans and animals. They are small, icosahedral (+)ssRNA viruses, causing a variety of diseases, such as encephalitis, and poliomyelitis. Vaccines are available for poliovirus, hepatitis A virus and foot-and mouth disease virus, but no effective prophylaxis is implemented for other picornaviruses. Thus far, anti-viral research has focused on the capsid, whereas inhibitors targeting non-structural proteins (i.e. proteases, helicases, polymerases) have remained largely unaddressed. The project was focused on structural and biochemical characterization of the enterovirus-B93 (EVB93) 3C protease alone and in complex with several covalent inhibitors. The second objective was to identify the first non-covalent potent inhibitors of the EV-B93 3C protease and their further biochemical, antiviral, and structural evaluation. This work studied the in-vitro proteolytic activity of the EV-B93 3C protease, alone and in the presence of two known covalent inhibitors - rupintrivir and compound 1, as well as three low molecular weight covalent inhibitors - NZO, NZN and DB5_60. The crystal structures of the EV-B93 3C protease alone and in complex with rupintrivir, compound 1, and NZN molecule were solved at high resolution (1.57, 1.50, 1.32, and 1.73 Å, respectively). The structures revealed that the protein adapts a chymotrypsinlike fold similarly to other picornavirus 3C proteases and possesses His-40, Glu-71 and Cys-147 as a catalytic triad. The STD NMR-based fragment screening was performed to select non-covalent binders of the EV-B93 3C protease. Validation and profiling of the most promising non-covalent hits were done using thermal shift assay (TSA), surface plasmon resonance (SPR), and proteolytic activity assay. 44 analogs of the most potent molecule were evaluated in the in-vitro proteolytic activity assay. The most active compound displayed IC50 value of 5 flM. Further chemical optimization was performed resulting in more efficient inhibitor with similar IC50 value. Selected analogs were tested in the in-vitro proteolytic assay against analogous 3C proteases from the following viruses: human rhinovirus-A49, enterovirusD68, aichivirus A, porcine sapelovirus, and equine rhinitis B virus. All compounds exhibited good inhibitory activity against three of the tested proteases. Furthermore, in a cell-based proteolytic assay and an antiviral assay the compounds did not exhibit either proteolytic or antiviral activity, which may be explained by several factors such as lack of cell permeability, low solubility and/or high toxicity. Extensive co-crystallization and soaking trials were performed to obtain crystal structures of noncovalent complexes of the EV-B93 3C protease with the most potent compounds. Regrettably, no additional electron density was identified in the proteolytic active site. Bioinformatics docking simulations suggested potential binding mode of the optimized compound. These pointed to the presumed pockets occupied by the compound that interact with the two conserved residues from the catalytic triad. Since the most potent compound is a relatively large and rigid molecule, it is unable to bind to the protease without its previous rearrangement, which is unfavorable in the crystalline state of the protein. This observation may explain the inability of the non-covalent molecules to co-crystallize with EV-B93 3C protease. The results obtained in this study may aid the design of potent, noncovalent antivirals targeting enteroviral 3C proteases. / Los Picornaviridae son una de las familias de virus más diversas y conocidas desde hace más tiempo. Esta familia incluye importantes agentes patógenos que afectan a humanos y a animales. Los Picornaviridae son virus pequeños, icosaédricos, de ARN de cadena sencilla de sentido positivo y causan una gran variedad de enfermedades, tales como encefalitis y poliomielitis. Se dispone de vacunas para el poliovirus, el virus de la hepatitis A y el virus de la fiebre aftosa, pero no se ha implementado ninguna profilaxis efectiva para otros picornavirus. Hasta ahora, la investigación antiviral se ha centrado en la cápside, mientras que los inhibidores dirigidos a proteínas no estructurales (como las proteasas, las helicasas y las polimerasas) están todavía por explorar. Este proyecto se centró en la caracterización estructural y bioquímica de la proteasa 3C de enterovirus B93 (EV-B93) y de los complejos de esta proteasa con varios inhibidores covalentes. El segundo objetivo fue conseguir inhibidores no covalentes de la proteasa EV-B93 3C y realizar una caracterización bioquímica, antiviral y estructural de los complejos de EV-B93 3C con estos inhibidores.