Viruses are obligate parasites that use the host cellular machinery to produce progeny virions. The host responds to this invading pathogen by induction of the immune system; however, the virus employs a variety of strategies to overcome these attacks. The complexity of the virus-host interaction is of great interest to researchers with aims to both characterize the relationship and target steps of the viral life cycle to hinder infection. Many targeted tactics employ single protein analysis; however, approaches that examine the whole set of virus/host interactions are available. Transcriptional alterations within host cells have been determined for many virus- host interactions by micro-array techniques; however little is known about the effects on cellular proteins. This study uses a quantitative mass spectrometric-based method, SILAC, to study differences in a host cell's proteome with infection by a virus. Mammalian reoviruses and herpes simplex viruses are prototypical viruses commonly studied to determine virus life cycle and interactions with hosts. Using three strains of reoviruses and one HSV1 strain, cells were infected to identify differentially regulated proteins at different times. Thousands of proteins were identified for each virus type, some up or down regulated after infection. Biological functions and network analyses were performed using online networking tools. These pathway analyses indicated numerous processes including cell death and inflammatory response are affected by T1L reovirus infection. Comparing reovirus strains revealed a greater overall proteomic change in host function when infected with the more pathogenic T3DC strain. For the HSV infection, host proteins altered during the different immediate early, earlyand late phases of infection helped characterize the host-virus interaction parallel to the virus life cycle. Overall, my study has characterized proteomic changes in different virus infection systems, identifying numerous novel cellular functional pathways and specific proteins altered during virus infections, specifically the secretogranin II protein that had opposite types of regulation in reoviruses and HSV and was examined for its effects on virus replication. Further studies on the novel proteomic characteristics may provide greater understanding to the complex virus-host interactome, leading to possible antiviral targets. / October 2015

Le syndrome de choc de dengue (DSS) est une complication potentiellement mortelle dela dengue, première arbovirose humaine et problème majeur de santé publiquemondial. Il survient chez une fraction des patients, et résulte d’une fuite plasmatiquemassive, non prédictible, dont la physiopathologie est mal connue. Le décryptage de laréponse de l’hôte est donc essentiel pour améliorer le pronostic et le traitement despatients. Ce travail de thèse a abordé les mécanismes de la fuite plasmatique du DSS dedeux façons : versant immunitaire et versant endothélial. D’une part, nous avons comparéen ex vivo les profils transcriptionnels sanguins de patients présentant différentes formescliniques de dengue, afin d’identifier des mécanismes contribuant à la survenue du DSS.Cette étude a révélé l’activation chez les patients en DSS, de signatures proinflammatoiresà l’interface entre immunité innée et métabolisme lipidique, représentantde nouveaux bio-marqueurs potentiels du DSS. D’autre part, les études in vitro desinteractions entre un virus de dengue et deux lignées de cellules endothélialesmicrovasculaires humaines (CEM), a révélé des différences d’intensité de réponseantivirale, ainsi que des différences dans l’expression de protéines impliquées dans laperméabilité, selon l’origine des territoires endothéliaux. Ces résultats suggèrent que levirus contribue directement au dysfonctionnement endothélial, au côté de mécanismesindirects médiés par des facteurs de l’hôte. Les deux types d’approches mises en oeuvreont ainsi établi de nouvelles données sur la physiopathologie du DSS, qui pourraient àterme trouver des applications dans la prise en charge des malades. / Dengue Shock Syndrome (DSS) is a life-threatening form of dengue infection, which is thefirst arboviral disease worldwide and a major public health problem. This severecomplication happens in a fraction of patients, and is the consequence of anunpredictable massive plasma leakage. The pathophysiology underlying DSS is stillunknown. Deciphering the host response to dengue infection is essential to improve boththe prognosis and the therapeutic management of dengue patients. This thesis workintended to study the mechanisms involved in DSS’ plasma leakage at both immunity (exvivo study) and endothelium (in vitro study) levels. First, in a ex vivo study, we comparedwhole blood cells’ transcriptional profiles of patients suffering from different clinicalpresentations of dengue disease, in order to identify mechanisms contributing to DSSoutcome. This study revealed the activation of pro-inflammatory signatures at theinterface of innate immunity and lipid metabolism, in DSS patients. Those signatures maybe new bio-markers of DSS. Second, in vitro studies of the consequences of a directinteraction between a dengue virus and human microvascular endothelial cells (MEC),revealed differences in antiviral response intensities and in the expression of proteinsinvolved in the endothelial permeability, depending on the endothelial origin of theMEC. Those results suggest that the virus directly contributes to the endotheliumdysfunction, together with indirect mechanisms triggered by soluble and cellular factors.Our investigations have produced new data on the pathophysiology of DSS that couldhave applications to the monitoring and treatment of the patients.

Orientador:Jorge Vega / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-12T04:10:59Z (GMT). No. of bitstreams: 1 Messias_Ueliton_D.pdf: 899792 bytes, checksum: c79dd05dd46e901a7add3fa66d7afc3f (MD5) Previous issue date: 2008 / Resumo: As plantas defendem-se continuamente contra ataques de bactérias, vírus, fungos, invertebrados e de outras plantas. O estresse oxidativo é um tipo de resposta fisiológica da planta após o reconhecimento do patógeno, podendo resultar em sintomas nas plantas dependendo da sensibilidade do hospedeiro, e também relacionada à mecanismos de defesa. Foram analisadas plantas de soja cultivares BRS132 (muito sensível) e IAC17 (pouco sensível) infectadas pelo Cowpea mild mottle virus (CPMMV) e plantas de feijoeiro cultivar BT2 infectado pelo Cowpea aphid-borne virus (CABMV). O trabalho teve como objetivos avaliar a concentração de peróxido de hidrogênio, analisar a resposta antioxidante das plantas à infecção viral quanto às variações na atividade da superóxido dismutase, catalase, ascorbato peroxidase, guaiacol peroxidase e siringaldazina peroxidase e verificar as localizações dos vírus e das peroxidases em diferentes tecidos das plantas. O CPMMV induziu uma doença aguda, com sintomas severos e culminando com a morte da planta de soja 'BRS132'. Na soja 'IAC17', o vírus induziu uma doença crônica com mosaico leve a partir da segunda folha trifoliolada. As concentrações de peróxido de hidrogênio e as atividades da catalase, ascorbato peroxidase, guaiacol peroxidase e siringaldazina peroxidase foram maiores nas plantas infectadas, tanto na soja 'BRS132' como na soja 'IAC17', em relação às plantas sadias. O CPMMV foi localizado nos tecidos do pecíolo e do caule, na soja 'BRS132' nas regiões periféricas e medula, e na soja 'IAC17' principalmente nas regiões periféricas. No feijoeiro cultivar BT2, o CABMV induziu resposta aguda na primeira folha foram maiores nas plantas infectadas, exceto a atividade da superóxido dismutase, que apresentou valores similares nas plantas infectadas e nas sadias. O CABMV foi localizado nas regiões periféricas e medula dos tecidos do pecíolo, e no caule a invasão foi limitada à região periférica. As peroxidases e a siringaldazina peroxidase foram localizadas nas mesmas regiões do pecíolo onde foram detectados o CPMMV e o CABMV. No feijoeiro 'BT2', a infecção viral induziu uma resposta semelhante à observada na soja 'BRS132', com algumas diferenças relacionadas ao fato de que neste caso, a infecção pelo CABMV não resultou na morte da planta de feijoeiro. Também se observou aumento expressivo de atividade da siringaldazina peroxidase no 7º dia após inoculação, diferente da soja 'BRS132' em que este aumento ocorreu mais tarde. A invasão generalizada dos vírus no pecíolo foi semelhante em feijoeiro 'BT2' e soja 'BRS132', principalmente nos dias em que começou a ocorrer abscisão dos folíolos. Já a invasão do caule foi generalizada em soja 'BRS132' e limitada à região periférica em feijoeiro. Possivelmente, o aumento precoce de atividade da siringaldazina peroxidase em feijoeiro, já no 7º dia após inoculação, limitou a invasão do vírus aos tecidos periféricos do caule. Isto explicaria o fato de o feijoeiro 'BT2' não sofrer morte da gema apical e da planta. trifoliolada, apresentando sintomas de mosaico, maior rugosidade, lesões necróticas nas nervuras e folíolos ''fechados''. Nesta cultivar, todos os parâmetros avaliados. / Abstract: Plants defend themselves from attacks of bacteria, fungi, viruses, invertebrate and other plants. Oxidative stress is a kind of physiological response of the plant related to defense mechanisms after recognition the pathogen, which may result in disease symptoms depending on host sensitivity. In this work, plants of two varieties of soybean infected by Cowpea Mild Mottle Virus (CPMMV), one highly sensitive (BRS132) and other with low sensitivity (IAC17) to the virus, were analyzed. Also, responses of bean plants (cv. Black Turtle 2, BT2) to Cowpea Aphid-Borne Mosaic Virus (CABMV) were examined. The parameters assessed included peroxide content (as hydrogen peroxide, H2O2), and activity of the following enzymes: superoxide dismutase, catalase, ascorbate peroxidase, guayacol peroxidase and syringadazine peroxidase. Distribution of virus and peroxidases in different tissues was also examined. In soybean cv BRS132, CPPMV induced an acute disease with severe symptoms finally resulting in plant death. In the less sensitive soybean cv IAC17, CPMMV induced a chronic disease with mild mosaic which was only visible in the second trifoliate and later leaves. Peroxide content and activity of guayacol and syringaldazine peroxidases were higher in infected plants of both cultivars. The virus was immuno-localized in stem and petiole cross sections, appearing in peripheral tissues and pith in cv BRS132, whereas in cv IAC17 it was localized mainly in the peripheral portion. In bean cv BT2, CABMV induced a acute response in the first trifoliate leaf, which presented a rough mosaic, necrotic lesions in veins and a "wilted" aspect. In this species all the assessed parameters showed higher values in the infected plants. Only the activity of superoxide dismutase was similar in healthy and infected plants. The vírus was localized in the pith and peripheral tissues of bean petioles, and only in the peripheral region of stems. Peroxidase and syringaldazine peroxidase activities were localized in the same tissues of the petiole where the CPMMV was localized in soybean plants and CABMV in bean plants. The response to CABMV observed in bean cv BT2 was similar to the response of soybean BRS132 to CPMMV, with some differences, since in bean the virus infection did not induce plant death. A significant rise in syringadazine activity was detected seven days after inoculation (DAI) in beans, while in soybean this increase occurred one day later. Both species also showed similar pattern of invasion of petiole tissues by the virus, mainly at the moment of abscission of leaflets. However, the virus invasion of stem was generalized in soybean BRS132 and contrastingly, limited to the peripheral tissues in bean. One possibility is that the early increase in syringaldazine activity observed in bean 7 DAI is indicative of some type of restriction to the spread of the virus, limiting it to the stem peripheral tissues. Probably this restricted spread of the virus in the stem underlies the survival of the apical meristem in bean cv BT2 in contrast to the death of the meristem in soybean cv BRS132. / Doutorado / Doutor em Biologia Vegetal