Orientador: Urara Kawazoe / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-15T09:56:42Z (GMT). No. of bitstreams: 1 Pinola_TalineBueno_M.pdf: 812699 bytes, checksum: 060ac3635a386505de2cf212ec4c9a59 (MD5) Previous issue date: 2009 / Resumo: Este estudo teve como objetivos obter o isolado precoce de Eimeria maxima "Ca" e posterior estudo comparativo da patogenicidade entre as aves (Gallus gallus) inoculadas com isolados: parental e precoce, com os seguintes critérios de avaliação: ganho de peso corporal, escore de lesão da mucosa intestinal e contagem de oocistos produzidos pelas aves infectadas. Para a obtenção do isolado precoce foram inoculados sucessivamente em aves sadias, os primeiros oocistos eliminados, a partir da geração parental. Os oocistos contidos nas fezes foram isolados e cultivados em solução de dicromato de potássio 2% para a esporulação. Através deste processo foram realizadas 25 passagens dos primeiros oocistos nas aves até a estabilização do período prépatente. Foi obtida uma redução de 36 horas na eliminação dos primeiros oocistos, de 124h30min para 88h30min. Os isolados parental e precoce foram comparados quanto a patogenicidade, através da inoculação de doses específicas em aves sadias. Nesse estudo foram utilizadas 75 aves distribuídas em cinco grupos: I e II para a produção de oocistos, III e IV para ganho de peso e escore de lesão e grupo V como controle negativo (aves sadias). Houve uma diminuição significativa da patogenicidade em aves infectadas com o isolado precoce quando comparadas a aves inoculadas com a geração parental. As aves infectadas com o isolado precoce obtiveram uma média de escore de lesão da mucosa do intestino estatisticamente inferior à parental. O valor médio do escore de lesão obtido pelo isolado precoce foi de 0,77 enquanto que a geração parental 1,67. As aves infectadas com o isolado precoce apresentaram uma redução significativa na produção de oocistos, quando comparadas à respectiva geração parental. Apresentaram, também, ganho de peso estatisticamente superior em relação à geração parental. Este isolado precoce é um excelente candidato à produção e comercialização de uma vacina viva atenuada. No entanto, testes em bateria e no campo sobre imunidade protetora desse isolado precoce devem ser realizados no futuro que poderá caracterizá-lo confiável para o uso comercial como vacina viva atenuada no controle da coccidiose aviária. / Abstract: The aim of this study was to obtain a precocious line of Eimeria maxima, "Ca" strain and the comparative pathogenicity study between precocious line and parental strain. The criteria used for this evaluation was: body weight gain, lesion score of intestinal mucosa and oocyst counting dropped by infected chickens. The precocious line was obtained by successive oral inoculation of parental E. maxima oocysts in chickens. First oocysts eliminated after inoculation were collected, isolated and cultivated in 2% dichromate potassium solution for their sporulation. The pre-patent period of first oocysts elimination diminished and stabilized after 25 passages in chickens. It was obtained a time reduction of 36 hours between precocious line and parental strain: 124h30' for parental strain and 88h30' for precocious line. The comparative evaluation of pathogenicity between precocious line and parental strain were performed using body weight gain, lesion score and oocyst production compared to non - infected chickens in groups of five chickens per cage with three replications. Parental strain infected in chickens showed loss of body weight while in chickens inoculated with precocious line showed weight gain of 75% when compared to non-infected control birds. The lesion score of intestine mucosa was 0.8 for chickens with precocious line and 1.7 for parental strain showing statistically significant difference. The precocious line showed also significant less oocyst production than parental strain in infected chickens. The precocious line of E. maxima obtained in the present study seems a good candidate for live attenuated vaccine against avian coccidiosis although there is a need for complementary study about protective immunity in battery and field tests. / Mestrado / Mestre em Parasitologia

La dengue est une maladie virale endémique dans les pays tropicaux. Bénigne dans sa forme classique (FC), elle peut être redoutable lors de complications associées à sa forme sévère (FS). Actuellement, l’établissement du pronostic d’évolution vers une dengue sévère est peu fiable. Afin d’identifier des marqueurs précoces d’évolution vers une forme sévère, une étude de caractérisation différentielle de plasmas FC et FS prélevés précocement, a été effectuée par différentes approches de protéomique. La protéine non-structurale NS1 est une des protéines virale associée à la pathogénicité. Une méthode de profilage des plasma utilisant la technologie SELDI-TOF/MS (Surface Enhanced Laser Desorption Ionization-Time of Flight/Mass Spectrometry) couplée à un anticorps monoclonal anti-NS1 a été utilisée afin d’identifier les ligands de cette protéine. La protéine NS1 a été détectée spécifiquement dans des plasmas de patients dengue. Aucun partenaire de la NS1 n’a pu être identifié. La méthode peut néanmoins être utilisée pour sérotyper les échantillons.Une analyse différentielle en ICPL (Isotope Coded Protein Labelling) des protéines plasmatiques de patients FC ou FS a permis d’identifier trois marqueurs potentiellement liés la sévérité de la maladie qui ont été validés en ELISA. La synthèse du virus mettant en jeu des partenaires cellulaires, le viroprotéome associé à chaque pathologie a été caractérisé par LC-MS/MS. Une méthode de purification du virus de la dengue a préalablement été mise au point sur un modèle in vitro. Cette méthode a ensuite été appliquée sur des pools de plasmas de patients prélevés en phase virémique. Des protéines virales ainsi que des protéines de l’hôte potentiellement associées aux virus ont été identifiées. Après analyse, une « empreinte » mettant en jeux des voies canoniques spécifiques a pu être déterminée pour les FS. Des ELISA ont été réalisés afin de valider le différentiel d’expression sur un choix de protéines.Au-delà de l’identification d’outils susceptibles d’aider les praticiens à poser un pronostic, ces études s’inscrivent dans la compréhension des mécanismes complexes qui sous-tendent le passage d’une FC à une FS. / Dengue is a endemic viral disease in tropical countries. If its classical form (CF) is benign, its severe form (SF) leads however to serious complications. Currently, the prognosis of severe dengue is unreliable. Differential proteomic studies on acute CF and FS plasma specimens were performed in order to identify early markers of progression to severe forms.The non-structural protein 1 (NS1) is a viral protein associated with pathogenicity. A method using SELDI-TOF/MS (Surface Enhanced Laser Desorption Ionization-Time of Flight/Mass Spectrometry) coupled to anti-NS1 monoclonal antibodies was developed in order to profile the proteins interacting with NS1 in plasma. Whereas NS1 protein was specifically detected in acute dengue plasma specimens, no NS1 ligand was identified. This method however allowed for sample serotyping.Plasma proteins of SF and CF patients were analyzed differentially using ICPL (Isotope Coded Protein Labeling). Three markers potentially related to disease severity were identified and validated by ELISA.As cellular partners are involved in virus biosynthesis, the viroproteome associated to each disease was characterized by LC-MS/MS. A method of dengue virus purification was first developed on an in vitro model. This method was then applied to pools of acute plasma of either CF or SF patients. We identified viral proteins as well as host proteins potentially associated with the viral particles. A footprint involving specific canonical pathways were subsequently identified in SF patients. Finally, a set of proteins found differentially expressed was validate by ELISA.Beyond identification of tools allowing assessment of dengue severity prognosis, these results give clues on the complex mechanisms underlying the transition from the CF to the SF.

Philosophiae Doctor - PhD (Biochemistry) / While the many completely sequenced genomes of bacterial pathogens contain all the determinants of the host-pathogen interaction, and also every possible drug target and recombinant vaccine candidate, computational tools for selecting suitable candidates for further experimental analyses are limited to date. The overall objective of my PhD project was to attempt to design reusable systems that employ the two most important features of bacterial evolution, horizontal gene transfer and adaptive mutation, for the identification of potentially novel virulence-associated factors and possible drug targets. In this dissertation, I report the development of two novel technologies that uncover novel virulence-associated factors and mechanisms employed by bacterial pathogens to effectively inhabit the host niche. More importantly, I illustrate that these technologies may present a reliable starting point for the development of screens for novel drug targets and vaccine candidates, significantly reducing the time for the development of novel therapeutic strategies. Our initial analyses of proteins predicted from the preliminary genomic sequences released by the Sanger Center indicated that a significant number appeared to be more similar to eukaryotic proteins than to their bacterial orthologs. In order determine whether acquisition of genetic material from eukaryotes has played a role in the evolution of pathogenic bacteria, we developed a system that detects genes in a bacterial genome that have been acquired by interkingdom horizontal gene transfer.. Initially, 19 eukaryotic genes were identified in the genome of Mycobacterium tuberculosis of which 2 were later found in the genome of Pseudomonas aeruginosa, along with two novel eukaryotic genes.Surprisingly, six of the M. tuberculosis genes and all four eukaryotic genes in P. aeruginosa may be involved in modulating the host immune response through altering the steroid balance and the production of pro-inflammatory lipids. We also compared the genome of the H37Rv M. tuberculosis strain to that of the CDC- 1551 strain that was sequenced by TIGR and found that the organisms were virtually identical with respect to their gene content, and hypothesized that the differences in virulence may be due to evolved differences in shared genes, rather than the absence/presence of unique genes. Using this observation as rationale, we developed a system that compares the orthologous gene complements of two strains of a bacterial species and mines for genes that have undergone adaptive evolution as a means to identify possibly novel virulence –associated genes. By applying this system to the genome sequences of two strains of Helicobacter pylori and Neisseria meningitidis, we identified 41 and 44 genes that are under positive selection in these organisms, respectively. As approximately 50% of the genes encode known or potential virulence factors, the remaining genes may also be implicated in virulence or pathoadaptation. Furthermore, 21 H. pylori genes, none of which are classic virulence factors or associated with a pathogenicity island, were tested for a role in colonization by gene knockout experiments. Of these, 61% were found to be either essential, or involved in effective stomach colonization in a mouse infection model. A significant amount of strong circumstantial and empirical evidence is thus presented that finding genes under positive selection is a reliable method of identifying novel virulence-associated genes and promising leads for drug targets. / South Africa

Fusarium oxysporum is a ubiquitous soil-borne fungus that includes pathogenic and non-pathogenic members. The pathogenic members are best known for causing Fusarium wilt diseases of many economically important agricultural crops. One such a crop is banana (Musa spp.), which is affected by a special form of the fungus known as F. oxysporum f.sp. cubense (Foc). Fusarium wilt was responsible for devastating losses of Gros Michel export bananas in Central America during the first half of the 20th century, and is now, once again, threatening world banana production that is primarily based on the sweet Cavendish varieties, both in the tropics and subtropics. To effectively manage Fusarium wilt, adequate knowledge of the pathogen, plant and environment is required. With this thesis I hope to contribute to the current knowledge available on the pathogen. Previous studies investigated the phenotypic and genotypic diversity, the spread and distribution, and the phylogeny of Foc. Some aspects related to the biology, physiology, diversity and pathogenicity of Foc, however, appeared to be unresolved. These aspects are important in order to develop a sustainable management strategy for Fusarium wilt to ensure continued banana production. Chapter 1 depicts a general review on F. oxysporum as the causal agent of Fusarium wilt of various fundamental crops, and gives a broad overview of the biology, taxonomy, physiology and pathogenicity of the pathogen. Through the application of modern molecular genetic techniques, a lot of progress has been made in the identification of genes and processes involved in the biology and pathogenesis of Fusarium wilt pathogens. The review concludes that some work, however, still needs to be conducted before topics such as race designation and pathogenesis in Foc are fully understood. Temperature, pH and nutrition are all factors contributing to the pathogenesis of F. oxysporum. The different factors can either favour or suppress the pathogen, or they can have a stimulating or inhibiting effect on the host plant. In Chapter 2 the pathogenicity and phenotypic characteristics of a genotypically uniform population of Foc was investigated. Physiological studies included determining the minimum, maximum and optimum temperatures and pH at which Foc grows in vitro, and what nitrogen sources stimulate and inhibit growth of Foc. Knowledge on these aspects could contribute to the management of the pathogen in the field. Differentiation among species of Fusarium can be problematic. To resolve questions related to the nomenclature in Fusarium, our research focus has shifted to the use of molecular tools for identification and determination of evolutionary relationships among and within species. In the past, phylogenetic studies on Foc were conducted using molecular tools such as sequencing, Restriction Fragment Length Polymorphisms, Random Amplified Polymorhic DNA and DNA Amplification Fingerprinting, with varying amounts of success. In Chapter 3 the usefulness of Amplified Fragment Length Polymorhism (AFLP) analysis to study diversity inFoc isolates was investigated. Of the 21 vegetative compatibility groups (VCGs) of Foc identified around the world, only VCG 0120 is found in South Africa. Chapter 4 aimed to identify an AFLP polymorphic DNA fragment unique to VCG 0120, and to develop a molecular marker of this fragment. Such a marker would be extremely valuable to distinguish between VCG 0120 and other isolates of F. oxysporum in terms of identification and confirmation of Fusarium wilt of banana in South Africa. Several pathogenicity-related genes have been identified in F. oxysporum. In Chapter 5, the presence of three pathogenicity-related genes (fmk1, pg1 and xyl3) in F. oxysporum isolates pathogenic and non-pathogenic to banana were verified by means of PCR amplification. The value of pathogenicity genes such as fmk1 and pg1 in comparative phylogenetic analysis was further substantiated. / Dissertation (MSc (Microbiology))--University of Pretoria, 2007. / Microbiology and Plant Pathology / unrestricted

In the western regions of North America, mountain pine beetle, Dendroctonus ponderosae Hopk., infestations take a tremendous toll of pines , especially lodgepole pine, Pinus contorta Dougl. var. latifolia Engelm.. Mass attack by the beetles is a devastating event for the trees. As well as girdling the tree, a massive inoculation of blue stain fungus "complex" (composed of several species of Ceratocystis, numerous yeasts and other mycelial fungi) is made beneath the bark. These fungi colonize and destroy the parenchyma tissue system of the host sapwood, primarily the ray parenchyma and resin duct epithelium. A blue stain is produced in the sapwood as a consequence of destruction of the sapwood parenchyma. The stain develops inward through the sapwood, and the transpiration stream is cut off. As more and more sapwood is stained, foliar water stress begins to increase. Foliage however, remains green and apparently healthy for up to 10 months after inoculation. When spring bud break begins the year following beetle attack, terminal buds of blue-stained trees begin to expand, then abort. Soon after, the needles of these trees fade to a reddish brown color. Transpiration stream disruption was not caused by penetration of tracheids by fungal hyphae; tyloses were not observed; nor was microconidial blockage of bordered pits seen. Though resin duct epithelium was eventually destroyed, little resin soaking was observed in the initial blue stained regions. Many bordered pits of tracheids in stained regions appeared to be aspirated, suggesting introduction of embolisms.

Yes / Lichenicolous (lichen-dwelling) fungi have been extensively researched taxonomically over many years, and phylogenetically in recent years, but the biology of the relationship between the invading fungus and the lichen host has received limited attention, as has the effects on the chemistry of the host, being difficult to examine in situ. Raman spectroscopy is an established method for the characterization of chemicals in situ, and this technique is applied to a lichenicolous fungus here for the first time. Xanthoriicola physciae occurs in the apothecia of Xanthoria parietina, producing conidia at the hymenium surface. Raman spectroscopy of apothecial sections revealed that parietin and carotenoids were destroyed in infected apothecia. Those compounds protect healthy tissues of the lichen from extreme insolation and their removal may contribute to the deterioration of the apothecia. Scytonemin was also detected, but was most probably derived from associated cyanobacteria. This work shows that Raman spectroscopy has potential for investigating changes in the chemistry of a lichen by an invading lichenicolous fungus. / This work was completed while D.L.H. was in receipt of an award from the Ministerio de Economica y Competitividad of Spain (Proyectos CGL 2014-55542-P).

Yes / Background. The spirochete bacterium Treponema pallidum ssp. pallidum is the etiological agent of syphilis, a chronic multistage disease. Little is known about the global T. pallidum proteome, therefore mass spectrometry studies are needed to bring insights into pathogenicity and protein expression profiles during infection. Methodology/Principal Findings. To better understand the T. pallidum proteome profile during infection, we studied T. pallidum ssp. pallidum DAL-1 strain bacteria isolated from rabbits using complementary mass spectrometry techniques, including multidimensional peptide separation and protein identification via matrix-assisted laser desorption ionization-time of flight (MALDI-TOF/TOF) and electrospray ionization (ESI-LTQ-Orbitrap) tandem mass spectrometry. A total of 6033 peptides were detected, corresponding to 557 unique T. pallidum proteins at a high level of confidence, representing 54% of the predicted proteome. A previous gel-based T. pallidum MS proteome study detected 58 of these proteins. One hundred fourteen of the detected proteins were previously annotated as hypothetical or uncharacterized proteins; this is the first account of 106 of these proteins at the protein level. Detected proteins were characterized according to their predicted biological function and localization; half were allocated into a wide range of functional categories. Proteins annotated as potential membrane proteins and proteins with unclear functional annotations were subjected to an additional bioinformatics pipeline analysis to facilitate further characterization. A total of 116 potential membrane proteins were identified, of which 16 have evidence supporting outer membrane localization. We found 8/12 proteins related to the paralogous tpr gene family: TprB, TprC/D, TprE, TprG, TprH, TprI and TprJ. Protein abundance was semi-quantified using label-free spectral counting methods. A low correlation (r = 0.26) was found between previous microarray signal data and protein abundance. Conclusions. This is the most comprehensive description of the global T. pallidum proteome to date. These data provide valuable insights into in vivo T. pallidum protein expression, paving the way for improved understanding of the pathogenicity of this enigmatic organism. / This work was supported by the grants from the Flanders Research Foundation, SOFI-B Grant to CRK, http://www.fwo.be/, a Public Health Service Grant from the National Institutes of Health to CEC, (grant # AI-051334), https://www.nih.gov/ and a grant from the Grant Agency of the Czech Republic to DS and MS (P302/12/0574, GP14-29596P), https:// gacr.cz/.

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