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Submitted by ANA CLAUDIA SILVA BRAGA null (anabragga@gmail.com) on 2017-08-31T16:15:53Z No. of bitstreams: 1 Tese Doutorado Ana Claudia Silva Braga.pdf: 4552779 bytes, checksum: 456de4a6fbfd60347292d8755d6c8c47 (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-09-01T14:16:37Z (GMT) No. of bitstreams: 1 braga_acs_dr_sjrp.pdf: 4552779 bytes, checksum: 456de4a6fbfd60347292d8755d6c8c47 (MD5) / Made available in DSpace on 2017-09-01T14:16:37Z (GMT). No. of bitstreams: 1 braga_acs_dr_sjrp.pdf: 4552779 bytes, checksum: 456de4a6fbfd60347292d8755d6c8c47 (MD5) Previous issue date: 2017-08-04 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O vírus da hepatite C (HCV) causa a doença da Hepatite C e estima-se que cerca de 3% da população mundial esteja infectada com o vírus. A infecção por HCV promove a alteração na expressão de várias proteínas celulares. Estudos têm demonstrado que muitas proteínas de choque térmico (HSPs) possuem um perfil de expressão alterado na presença do vírus e algumas HSPs interagem diretamente com proteínas do HCV. Assim, o presente estudo teve como objetivo avaliar in vitro os níveis de expressão de proteínas de choque térmico na presença e ausência de HCV. Com este propósito, células de hepatoma humano Huh7.5 e células Huh7.5 infectadas com o vírus (HCV JFH-1) foram submetidas à extração de RNA e síntese de cDNA. A expressão diferencial de 84 HSPs e chaperonas foi avaliada por qPCR Array. Os resultados demonstram que cinco genes apresentaram expressão aumentada (em Log2 2), enquanto outros cinco apresentaram expressão reduzida. Para validar estes resultados os 10 genes diferencialmente expressos foram testados por qPCR em três modelos celulares para o HCV: células contendo replicon subgenômico do HCV (SGR-JFH-1), células infectadas com JFH-1 (ambos do genótipo 2a) e células contendo o replicon subgenômico S52 (genótipo 3). O gene HSPB8 mostrou expressão aumentada nos três modelos testados, condizente com os resultados obtidos por qPCR Array. Em seguida, promovemos o silenciamento de HSPB8 e foi observado um aumento na replicação viral. Em contraste, quando aumentamos a expressão de HSPB8, o HCV teve uma diminuição na taxa de replicação. O mesmo procedimento foi adotado para o gene DNAJC5B, validado no modelo viral genótipo 3, e o HCV mostrou padrão de replicação semelhante ao observado para o gene anterior. Esses resultados sugerem que HSPB8 pode atuar como um fator intracelular contra a replicação do vírus da hepatite C e DNAJC5B apresenta a mesma função, mas específico para o genótipo 3. Também avaliamos interações diretas com proteínas do HCV e os resultados demonstraram uma interação física entre a proteína NS4B de HCV e HSPB8. Esses resultados podem contribuir para uma melhor compreensão dos mecanismos envolvidos na replicação do HCV. / Hepatitis C virus (HCV) causes Hepatitis C disease and it is estimated that about 3% of world population are infected with the virus. HCV infection promotes alteration in the expression of several cellular proteins. Studies have shown that many heat shock proteins (HSPs) have an altered expression profile in the presence of the virus and some HSPs interact directly with HCV proteins. Thus, the present study aimed to evaluate in vitro the expression levels of heat shock proteins in the presence and absence of HCV. With this purpose, human hepatoma Huh7.5 cells and Huh7.5 cells infected with the virus (HCV JFH-1) were subjected to RNA extraction and cDNA synthesis. The differential expression of 84 HSPs and chaperones was assessed by qPCR Array. The results demonstrate that five genes showed increased expression (over Log2 2), while five other presented reduced expression. To validate these results, the 10 differentially expressed genes were tested by real-time PCR in three different HCV cell culture models: subgenomic HCV replicon cells (SGR-JFH-1), JFH-1 infected cells (both genotype 2a) and subgenomic S52 cells (genotype 3). The HSPB8 gene showed increased expression in all of three tested models, consistent with qPCR Array results. Then we promoted the silencing of HSPB8 and observed an increase in viral replication. In contrast, when we increased an expression of HSPB8, HCV had a decrease in replication rate. The same procedure was adopted for the DNAJC5B, validated in the viral model genotype 3, and HCV showed replication pattern similar to that observed for the previous gene. These results suggest that HSPB8 may act as an intracellular factor against hepatitis C virus replication and DNAJC5B have the same function, but genotype 3 specific. We also evaluated direct interactions with HCV proteins and the results demonstrated a physical interaction between the HCV NS4B protein with HSPB8. These results can contribute for a better understanding of the mechanisms involved in HCV replication. / FAPESP: 2013/17253-9

Le syndrome du QT long congénital (LQTS) est une canalopathie génétique, à l’origine de syncopes et mort subite. Le dépistage génétique identifie des variantes génétiques dans 50-70% des cas, suggérant l’implication d’autres gènes. Nous avons recueilli les caractéristiques des patients avec un LQTS à l’ICM, et recruté 12 patients avec un génotype négatif pour le LQTS pour un séquençage à haut débit des exons afin d’identifier de nouvelles variantes causales. Nous avons développé une approche analytique par étapes : (1) les gènes connus du, (2) les gènes dans des loci identifiés par des études d’association sur le QT, et (3) les gènes montrant la même variante chez plusieurs patients. L’analyse génétique a identifié de nouvelles variantes dans: (1) KCNJ2, ANK2 et AKAP9, et (2) dans NOS1AP. (3) Deux patientes avec des phénotypes semblables présentent la même variante homozygote dans TECRL, un nouveau gène candidat dont le rôle est inconnu. / Long QT syndrome (LQTS) is a channelopathy, causing syncope and sudden death. Genetic testing of individuals identifies genetic variants in up to 50-70% of cases, suggesting that additional genes may be involved. We have identified 50 patients with a diagnosis of LQTS at MHI, and recruited 12 patients reported negative for clinical testing of mutations in LQTS for a whole-exome next generation DNA sequencing approach in order to identify new variants and candidate genes. We have developed a stepwise analytic approach that focuses on (1) the known LQTS genes, (2) the genes in loci identified in genome-wide association studies of QT-interval, and (3) the loci showing variants across multiple patients. Our approach identified new variants in (1) KCNJ2, ANK2 and AKAP9, and (2) in NOS1AP. We identify 2 patients with a very similar phenotype with a homozygous variant in TECRL, an novel candidate gene with an unknown role in LQTS.

Le semis précoce du tournesol, d’un à deux mois par rapport à la période habituelle (Avril dans le sud ouest de la France), a été envisagé pour esquiver les périodes de sécheresses estivales. Cette stratégie conduit à un abaissement des températures de l’ordre de 5 à 10°C durant les premières phases de développement de la culture. L’objectif de ce travail est donc d’identifier des génotypes de tournesol adaptés à des conditions de basses températures en début de cycle, et de fournir des outils pour la sélection de ces nouveaux idéotypes. Dans un premier temps le modèle de culture SUNFLO, développé pour l’analyse des interactions génotype x environnement chez le tournesol, a été utilisé pour identifier par simulation un idéotype pour le semis précoce. Cette étude a révélé que le type variétal valorisant le mieux de semis précoce présente une levée précoce et un cycle tardif. Dans un deuxième temps, la variabilité génétique d’une population de lignées recombinantes de tournesol a été une analysée pour des traits agro-morphologiques et physiologiques caractérisant le développement (vitesse de germination, phénologie) et la croissance à basse température (élongation de l’hypocotyle, production de biomasse, et traits physiologiques impliqués dans la tolérance au froid). L’analyse génétique de ces caractères a permis d’identifier les régions chromosomiques impliquées dans la variation de ces caractères (QTLs) ainsi que les marqueurs moléculaires associés à ces QTLs qui représentent des marqueurs d’intérêts pour la sélection. L’analyse des processus impliqués dans la levée (germination et élongation de l’hypocotyle) montre que la température de base pour l’élongation de l’hypocotyle présente un gain génétique significatif à basse température. Ce trait est sous le contrôle génétique de deux QTLs majeurs dont l’un, qui explique 40% de la variabilité phénotypique observée, est lié au marqueur SSR ORS1128. Le temps thermique du semis à la floraison est un caractère contrôlé par des QTLs spécifiques en conditions de semis précoces, parmi lesquels deux sont colocalisés avec des QTLs identifiés pour des traits relatifs à la levée. L’étude des traits physiologiques impliqués dans la réponse aux basses températures a révélé que le tournesol a un potentiel de sélection pour la tolérance au froid, notamment pour le potentiel osmotique. Le maintien des membranes plasmiques stables à basses températures est également un trait jouant un rôle important dans la tolérance au froid. Un QTL à effet majeur lié au marqueur SSR ORS331_2 a été identifié pour ce trait et pourrait être utilisé pour aider à la sélection de génotypes de tournesol adaptés au froid. / Early sowing to escape the drought during summer was studied in sunflower. Sowing one or two months earlier leads to reduce about 5 to 10°C during first stages of development compared with traditional sowing (April in south parts of France) in this species. The aim of this study is to identify sunflower genotypes adapted for low temperature and to identify tools for selecting them. Firstly the crop model SUNFLO, Which is developed to analyze “genotype x environment” interactions in sunflower, was used to identify by simulation favorable ideotypes for early sowing. Results show that they have to present early emergence and a late development cycle. Then, several experiments were undertaken to study genetic variability for agro-morphologic and physiologic traits under early sowing in sunflower. A population of 95 recombinant inbred lines and their two parents were used at low temperature in all experiments. Germination rate, hypocotyl elongation, biomass production and some physiological traits for cold tolerance were studied. Genetic analyses were performed and genomic regions (QTLs) involved in the variation of these traits as well as SSR markers associated with them were identified. Analysis of physiological processes related to emergence (germination and hypocotyl elongation) show that the base temperature of hypocotyl elongation presents a significant genetic gain at low temperature. This trait is controlled by two major QTLs and one of them explains 40% of the phenotypic variance and contains the SSR marker ORS1128. The thermal time from sowing to flowering is controlled by specific QTLs in early sowing and two of them are collocated with QTLs detected for emergence related-traits. The study of physiological traits implied with response to low temperature showed that sunflower present a high potential for cold tolerance variability, especially for the osmotic potential. The cell membrane stability at low temperature is also an important trait for cold tolerance. A major QTL associated with the SSR marker ORS331_2 was identified for this trait and should be used to select sunflower cold tolerant genotypes.

Hepatitis C virus (HCV) is a major global pathogen that infects an estimated 170 million people worldwide, and for which currently no vaccine is available. HCV is a highly diverse viral pathogen and exists as 6 major genotypes sharing only 75% sequence homology; developing a vaccine that is cross-reactive between genotypes is a major challenge. Defining immune responses that target different HCV genotypes will facilitate pan-genotypic T cell vaccine development. HCV genotype 3 (gt3) is now the most common infecting genotype in the United Kingdom and large parts of Asia; however, data regarding the T cell antigenic targets of this genotype is very limited. In this thesis, HCV gt3 specific T cell targets were defined in acute, chronic and spontaneously resolved infection: in chronic gt3 infection, T cell responses were low in magnitude and narrowly focused in specificity, similar to those previously reported for gt1; in contrast, resolved infection was associated with a higher magnitude and broader specificity of CD4+ and CD8+ T cell responses across the genome. Overall, T cell specificity in gt3 infection was markedly different to that previously described for gt1, confirming that sequence differences between genotypes result in distinct immunological profiles. Previous work from our laboratory demonstrated that, though T cell responses induced by a potent T cell vaccine containing HCV gt1b non-structural regions do target epitopes dominant in natural infection, induced T cells show limited cross-reactivity against other genotypes. In this thesis, it was assessed whether T cells primed in natural gt3 infection are able to recognize viral sequence variants at dominant epitopes, which would make these potential targets in cross-reactive vaccine design. For seven gt3-specific T cell epitopes identified here as dominant, major sequence variability was observed within and between genotypes, and limited T cell cross-reactivity observed against identified viral variants. This suggests that regions frequently targeted in natural infection may not serve as attractive targets for cross-reactive vaccine design. These results informed the subsequent design of a cross-reactive vaccine based on fragments of HCV that are conserved between genotypes. A generic algorithm was developed to define viral regions conserved between major HCV genotypes (for 1a/1b, 1/3a, 1-6), and these were joined to form immunogens between 819 and 1543 AA long. Possible artificial, non-HCV epitopes formed by junctions were identified using online epitope prediction servers, and abrogated through the insertion of 2-6 amino acid linkers. To address the concern that conserved regions may not be immunogenic, epitopes described in natural HCV infection were mapped on HCV sequences, showing that conserved segments are well populated with epitopes; additionally, strong binding peptides were predicted for conserved segments using online epitope prediction programs, suggesting potential in vivo immunogenicity. In conclusion, HCV T cell specificity is distinct between genotypes, with limited T cell cross-reactivity between viral variants. Leading from this result, vaccine immunogens were designed entirely based on conserved viral regions. This work paves the way for future studies of novel HCV immunogens based on conserved viral segments between genotypes.

In patients after allogeneic haematopoietic stem cell transplantation (HSCT) is a human cytomegalovirus (CMV) one of the most important viral pathogens. Its detailed characteristic could provide information about the impact of each CMV genotype on overall survival of the patient, and some serious complications, such as graft versus host disease (GvHD). This thesis deals with retrospective genetic analysis of samples from 1877 patients transplanted at the Clinic of Pediatric Hematology and Oncology, University Hospital Motol and the Institute of Hematology and Blood Transfusion since 2002. DNA from biological samples (especially whole blood) was isolated kit Qiagen DNA Blood Mini or Qiagen DNA Mini and samples were prospectively detected presence of CMV DNA. Samples were subsequently stored at -20 řC. Genotyping was performed using real-time PCR technologies to the genes of 2 structural proteins glycoprotein B, glycoprotein H and using sequence specific primers and probes. In 1343 samples (71.6%) from 390 patients there was only one strain of CMV; in 256 (13.6%) samples from 113 patients have detected mixed infection caused by two or more strains of CMV. The most common genotype demonstrated in "single" infection was in pediatric and adult patients gB1/gH2 detected in 118 (28.4%) patients. Most...

Le modèle étudié est l’agent phytopathogène vasculaire Ralstonia solanacearum, en portant une attention particulière aux souches de phylotype II. Cette bactérie d’origine tellurique est très diversifiée, tant au plan génétique que phénotypique. Sa classification en constante évolution témoigne d’une volonté de clarifier cette biodiversité inhabituellement forte, tout en cherchant à reconnaître les écotypes structurant ce complexe d’espèces, i.e., des groupes de souches partageant à la fois des traits génotypiques et biologiques spécifiques. Dans le cadre de ce pathosystème modèle, nous nous sommes attachés dans un premier temps à revisiter de façon précise les pathotypes au sein d’écotypes bien décrits dans la littérature, ou à en faire la description (phylotype III africain). Nous avons observé une forte convergence phénotypique entre les souches de phylotype III des hauts plateaux africains et les souches Brown rot de phylotype IIB-1, capables de flétrir la pomme de terre et d’autres Solanacées à température froide. L’adaptation de souches aussi diverses pour la tolérance au froid nous a conduits à dresser un bilan de la situation R. solanacearum en Europe et in extenso dans le bassin méditerranéen. Cette approche a permis d’apprécier les degrés de divergence significative dans le pouvoir pathogène (virulence et agressivité) sur Solanaceae au sein de souches quasi clonales unifiant l’écotype Brown rot, qui s’établissent aussi sous forme d’infections latentes dans les tissus vasculaires de bananiers (Musacées). Dans le même temps, le phénotype de souches pathogènes du bananier, unifiant l’écotype Moko, a aussi été revisité sur Solanaceae qu’elles parviennent à flétrir, y compris des ressources génétiques résistantes au flétrissement bactérien. L’ensemble de ces données expérimentales a permis de dégager les critères de sélection pour le choix de trois nouvelles souches du complexe d’espèces R. solanacearum, dont nous avons obtenu les séquences génomiques. Notre approche en génomique comparative a permis de décrire le premier pangénome chez cet agent pathogène : l’ensemble les gènes repérés de l’espèce. Ces données ont été exploitées par différentes approches bio-informatiques et permettent de concevoir une refonte pertinente du complexe d’espèces R. solanacearum en trois nouvelles espèces génomiques, regroupant les souches de phylotypes I (Asie) et III (Afrique) d’une part, puis les souches de phylotype II (Amérique), et enfin les souches de phylotype IV (Indonésie) d’autre part. Ce pangénome a ensuite été exploité en concevant et développant une puce à ADN, un outil permettant l’exploration à haut débit d’une grande quantité de souches. La densité des données expérimentales accumulées permet une démarche vers l’écologie moléculaire et de reconstituer certains pans du passé évolutif des souches de phylotype II chez R. solanacearum. Par ailleurs, l’analyse approfondie de ces données de génomique, associant phylogéographie et structuration des populations de l’écotype Brown rot, montre une double situation épidémiologique en Europe, recoupant des influences andines et africaines. De la même façon, l’écotype Moko présente trois structures génétiques distinctes. Ces données ont été analysées de manière à retracer les principaux flux de gènes dans les états ancestraux des phylotypes et de dégager la forte contribution de la partie mobile du génome, des gènes relatifs à l’adaptation environnementale et à la pathogénie, comme moteurs dans l’évolution de cet important organisme phytopathogène. / The studied model is the vascular plant pathogen Ralstonia solanacearum, with a particular focus on phylotype II strains. This telluric bacterium has a wide diversity, both on genotypic and phenotypic levels. Its evolving classification reflects the need to clarify its unusual biodiversity and seek to identify ecotype structure in this species complex, i.e., groups of strains with both genotypic and specific biological traits. Within the framework of this model pathosystem, we initially focused on deeply revisiting pathotypes among ecotypes, although well described in the literature, or describing new ecotypes (African phylotype III). We observed high phenotypic convergence between strains from phylotype III from the African highlands and Brown rot strains from phylotype IIB-1, both able to trigger wilt symptoms on potato and other Solanaceae at cold temperatures. Adaptation of diverse strains for cold tolerance led us to investigate the R. solanacearum situation in Europe and more specifically in the Mediterranean regions. This strategy allowed us to appreciate the significant divergence towards pathogenicity (virulence and aggressiveness) on Solanaceae within clonal-like structure of strains in the Brown rot ecotype, which also established latent interactions in the banana vascular system. In the mean time, phenotypes of banana pathogenic strains unifying the Moko ecotype, was also revisited on Solanaceae, and was able to trigger symptoms on both susceptible and resistant genetic resources to bacterial wilt. All these experimental data yielded selection criteria for choosing three new candidate strains in the R. solanacearum species complex for complete genome sequencing. Our genomic comparative approach allowed us to describe the first pangenome of this pathogen: all targeted identified genes of this species complex. These data were analyzed by various bioinformatic approaches and allowed us to design a complete reshaping of R. solanacearum species complex into three distinct genomic species, firstly clustering strains from phylotype I (Asia) with strains from phylotype III (Africa); strains from phylotype II (America); and lastly, strains from phylotype IV (Indonesia). This pangenome was then used for designing a DNA microarray, a high resolution tool that allowed us to explore a wide set of genomes. The density of accumulated data allowed for a molecular ecological approach to retrieve a certain amount of the evolutionary past of R. solanacearum phylotype II strains. Furthermore, a deeper analysis of these genomic data, combining phylogeography with population structure analysis of the Brown rot ecotype, revealed a dual epidemic situation in Europe, both across Andean and African influences. Similarly, the Moko ecotype presents three distinct genetic structures. These data were analyzed within the purpose of tracking the main gene flows in the ancestral states of phylotypes and to unravel the strong contribution of the mobile elements, genes related to environmental adaptation, and pathogenicity as a major driving force into the evolution of this successful plant pathogen.

Orientador: Fernando Sebastián Baldi Rey / Resumo: A compreensão de processos regulatórios e organização molecular dos organismos vivos progrediram consideravelmente na última década. As metodologias também evoluíram com o sequenciamento de DNA e RNA e de ferramentas genômicas permitindo a análise de centenas ou milhares de genes, proteínas ou metabólitos. O uso simultâneo dessas informações auxilia na obteção de informações relevantes sobre as variáveis que envolvem as variações fenotípicas de características de interesse. O objetivo do presente estudo foi integrar dados fenotípicos, genotípicos e transcriptômicos em busca de aprimoramento sobre os mecanismos genéticos e metabólicos que determinam o perfil de ácidos graxos na carne de bovinos Nelore, a fim de contribuir para o melhoramento da composição de ácidos graxos da carne. Foram utilizados machos da raça Nelore terminados em confinamento, abatidos com média de idade 24 meses. Amostras do músculo L. thoracis, entre a 12ª a 13ª costela foram coletadas para as análises de perfil de ácidos graxos, extração de RNA e de DNA. Os resultados foram apresentados nos capítulos 2 e 3. No capítulo 2, o objetivo foi identificar genes diferencialmente expressos pelo método RNA-seq e perfil de ácidos graxos no músculo L. thoracis com uso de componentes principais (principal components: PC). Foram selecionados dois grupos de 10 animais, os quais possuíam valores de PC1 e PC2 extremos (Alto x Baixo) para os grupos somatórios de ácidos graxos (AG): ácidos graxos saturados (AGS), ácidos g... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The understanding of regulatory processes and molecular organization of organisms has progressed considerably in the last 10 years.The methodologies also evolved with the sequencing of DNA, RNA and genomic tools allowing the analysis a lot of genes, proteins or metabolites. The simultaneous use of this information should help to obtain relevant information about the variables that result the phenotypic variations of traits of interest. The objective of the present study was to integrate phenotypic, genotypic and transcriptomic studies in order to clarify the genetic and metabolic mechanisms that determine the fatty acid profile in Nelore beef, in order to contribute to the improvement of the fatty acid composition of the meat. Nelore males were used in feedlot, coming from farms that integrate three breeding programs and slaughtered with an average of 24 months. Samples of the L. thoracis muscle between the 12th to 13th rib were collected for analysis of fatty acid profile, RNA and DNA extraction. The results were presented in chapters 2 and 3. In chapter 2, the objective was to identify genes differentially expressed by RNA-seq method and fatty acid profile in the L. thoracis muscle with the use of Principal Components (PC). Two groups of 10 animals were selected, which had PC1 and PC2 extreme values (High x Low) for the fatty acids (FA) groups: saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), omega 3 (n-3) and omega 6 (n-6... (Complete abstract click electronic access below) / Doutor

Nas últimas décadas observou-se um número crescente de relatos de infecções invasivas por Trichosporon em ambientes hospitalares, devido ao aumento da população suscetível e a melhoria dos métodos diagnósticos. Leveduras do gênero Trichosporon, depois de Candida, são as mais relacionadas à infecção fúngica invasiva em pacientes hematológicos, sendo Trichosporon asahii responsável por 90% dos casos. A identificação de espécies de Trichosporon é realizada através do sequenciamento da região IGS1 do DNA ribossomal, técnica considerada padrão-ouro. Através do estudo dos polimorfismos da região IGS1 do DNA ribossomal, diversos genótipos de T. asahii têm sido descritos, entretanto sem relação com a distribuição geográfica, perfil de suscetibilidade aos antifúngicos ou patogenicidade. O presente estudo teve como objetivo padronizar um método de análise por sequenciamento multilocus para a espécie T. asahii, definindo novos genes (loci) para melhor descrever a filogenia da espécie. Foram analisadas 21 cepas de T. asahii de diferentes origens (Brasil, Europa, Ásia) e genótipos (1,3,4,5,6,7). As sequências de genes estruturais (housekeeping genes) dos genomas de T. asahii (CBS2479 e CBS8904) disponíveis no GenBank foram alinhadas e analisadas in silico para o delineamento e avaliação dos novos primers. Após as reações de PCR e análise das sequências de DNA, quatro novos loci foram selecionados para a análise filogenética multilocus: phosphate carrier protein, topoisomerase 1 (TOP1), beta-1-tubulin, copper-exporting ATPase. As árvores filogenéticas demonstraram dois clados bem distintos, com altos valores de bootstraps. Além disso, os genótipos 1 e 3 foram alocados em clados diferentes. Nossos resultados sugerem uma reclassificação genética para a espécie T. asahii. Novos estudos, incluindo um maior número de cepas e outros marcadores genéticos, são necessários para melhor abordar a filogenia atual de T. asahii / In the last decades there has been a significant increase of the reported cases of invasive fungal infections by Trichosporon in hospital settings, related to the increase of the susceptible population and to the improvement of diagnostic methods. Trichosporon are the most frequent yeast related to invasive fungal infection in hematological patients after Candida, with Trichosporon asahii accounting for 90% of the cases. The gold standard method for Trichosporon species identification is the sequence analysis of the intergenic spacer region 1 (IGS1) from the ribosomal DNA. Based on the polymorphisms of the IGS region of ribosomal DNA, several T. asahii genotypes have been described, without relation with geographical distribution, antifungal susceptibility profile or pathogenicity. The objective of the study was to evaluate a multilocus sequencing method for the T. asahii species, defining new loci to better describe the phylogeny of the species. Twenty-one strains of T. asahii from different origins (Brazil, Europe, Asia) and genotypes (1,3,4,5,6,7) were analyzed. Housekeeping genes from T. asahii genomes (CBS2479 and CBS8904) available in GenBank were aligned and in silico analyses were carried out to design and evaluate the new primers. After PCR reactions and DNA sequence analysis, four new loci were selected for the multilocus plylogenetic analysis along with the IGS1 region from the rDNA: topoisomerase 1 (TOP1), phosphate carrier protein, beta-1-tubulin, copper-exporting ATPase. Phylogenetic trees revealed two well-distinct clades, with high bootstraps values. Moreover, IGS genotypes 1 and 3 strains were split into the different clades. Our results suggest a different genetic background for the species T. asahii. Further studies including more T. asahii strains and other genetic markers are necessary to better address the current phylogeny of T. asahii

Parvovirus B19 is a common human pathogen that typically infects erythroid progenitors and causes hematological problems such as anemia and aplastic crises. The clinical presentation depends mainly on the immunological status of the patient. PVB19 can cause serious clinical disorders in immunocompromised patients after transplantation. More than 1500 samples from 90 patients who passed the HSCT in 2015 were tested for the presence of PVB19 in this work. This work describes the incidence of the virus and two typical periods of onset of infection in patients after the transplantation. Although several sources report the negative effect of PVB19 infection on the survival of allogeneic graft patients, this work did not confirm this assertion. Also, the results of this work suggest that allogenic grafts are not the main source for transmission, but that it is likely to be reactivated after long-term persistent or latent PVB19 infections. PVB19 is divided into 3 genotypes. Genotype 1 is the most widespread, genotype 2 is very rare in Europe for the last 10 years, and genotype 3 occurs mainly in tropical localities. This work as the first describes the distribution of genotypes in the Czech Republic. More than 130 samples from 125 PVB19 positive patients, stored in the Motol University Hospital from 2004...