Une approche in vitro a consisté à étudier la formation de biofilm de souches d’origine du genre Lactobacillus d’intérêt probiotique. Nous nous sommes également attachés à évaluer l’impact de conditions de stress mimant l’environnement intestinal sur la formation du biofilm pour l’ensemble de ces souches. Les effets antagonistes des surnageants de cultures en biofilm ou en planctonique contre des agents pathogènes alimentaires ont été appréhendés. Non seulement toutes les souches testées forment des biofilms mais ce mode de croissance génère un effet antagoniste accentué pour certaines d’entre elles. Parmi les critères de sélection des bactéries à intérêt probiotique, les effets immunomodulateurs des probiotiques sont souvent recherchés. L. casei ATCC334 connue pour ses effets anti-inflammatoires a été retenue pour notre étude. A l’aide du modèle de lignée cellulaire THP-1 et en présence de LPS, le surnageant de culture de L. casei ATCC334 cultivée en biofilm s’est avéré présenter un effet anti-inflammatoire bien supérieur à celui des cultures planctoniques. Une approche utilisant des techniques biochimique et immunologique a permis d’identifier un des principes actifs responsable de l’effet anti-inflammatoire de cette souche. L’utilisation du modèle poisson zèbre a permis de montrer la colonisation de l’intestin des larves et de confirmer le rôle anti-inflammatoire de L. casei ATCC334 avec une diminution de la production des interleukines pro-inflammatoires et une augmentation de la production d'IL-10. Le recrutement des macrophages fluorescents mesuré en cytométrie de flux est également atténué chez la larve nourrie auparavant par le probotique en présence d’un agent inflammatoire. Le résultat majeur de cette étude est l’identification de la protéine GroEL qui contribue de façon significative à l’effet anti-inflammatoire de la souche L. casei ATCC334 lorsque qu’elle est cultivée en biofilm. / An in vitro approach was used to study biofilm formation by bacterial strains with probiotic properties and belonging to the Lactobacillus genus. We also evaluated the impact of stress conditions mimicking the intestinal environment on biofilm formation for all of these strains. The antagonistic effects of supernatants from cultures in biofilm or planktonic conditions against food-borne pathogens were apprehended. This growth mode generates an antagonistic effect accentuated for some of them. Among the selection criteria of interest probiotic bacteria, the immunomodulatory effects of probiotics are often sought. L. casei ATCC334 known for its anti-inflammatory effects was selected for our study. Using the model cell line THP-1 and in the presence of LPS, the culture supernatant of L. casei ATCC334 grown in biofilm was found to have an anti-inflammatory effect much greater than planktonic cultures. An approach using immunological and biochemical techniques has allowed the identification of the active substances responsible for the anti-inflammatory effect of this strain. Using the zebrafish model, we showed the colonization of the gut of the larvae and confirmed the anti-inflammatory role of L. casei ATCC334 with a decreased production of pro-inflammatory interleukins, and increased IL-10 production. Recruitment of fluorescent macrophages measured by flow cytometry was also mitigated in larvae fed previously by probotic in the presence of an inflammatory agent. The major result of this study is the identification of the GroEL protein that contributes significantly to anti-inflammatory effect of the strain L. casei ATCC334 when it is grown in biofilm.

abstract: This work aims to characterize protein-nanoparticle interactions through the application of experimental techniques to aid in controlled nanoparticle production for various applications from manufacturing through medical to defense. It includes multiple steps to obtain purified and characterized protein and then the production of nanoparticles using the protein. This application of protein requires extremely pure homogenous solution of the protein that was achieved using numerous protein separation techniques which were experimented with. Crystallization conditions, protein separation methods and protein characterization methods were all investigated along with the protein-nanoparticle interaction studies. The main protein of study here is GroEL and the inorganic nanoparticle used is platinum. Some studies on MBP producing gold nanoparticles from an ionic gold precursor were also conducted to get a better perspective on nanoparticle formation. Protein purification methods, crystallization conditions, Car-9 tag testing and protein characterization methods were all investigated along with the focus of this work. It was concluded that more Car9 studies need to be carried out before being used as in the form of a loop in the protein. The nanoparticle experiments were successful and platinum nanoparticles were successfully synthesized using GroEL. The direction of further research in protein-nanoparticle studies are outlined towards the end of the thesis. / Dissertation/Thesis / Masters Thesis Chemical Engineering 2019

Indiana University-Purdue University Indianapolis (IUPUI)

Introduction : L’inflammation intestinale observée dans les maladies inflammatoires chroniques de l’intestin (MICI) est essentiellement située au niveau du côlon et peut sur le long terme provoquer l’apparition du cancer colorectal (CCR). Cette inflammation est étroitement régulée par les cellules immunitaires de la muqueuse intestinale qui sont majoritairement représentées par les macrophages. Ces derniers jouent un rôle clé dans la pathogenèse des MICI et contribuent au maintien de l’inflammation intestinale. A l’heure actuelle, les traitements disponibles proposés pour traiter ces pathologies visent surtout à diminuer l’inflammation. Les travaux menés au cours de cette thèse, s’inscrivent dans ce contexte. En effet, des expériences précédentes réalisées par notre laboratoire avaient montré que des surnageants de biofilms du probiotique Lactobacillus induisaient une réponse anti-inflammatoire chez les macrophages humains. Il avait également été remarqué que l’immunodéplétion de GroEL (une protéine de choc thermique) du surnageant bactérien abrogeait de manière dose-dépendante ces propriétés anti-inflammatoires. Méthodes : Par conséquent, au cours de ce travail de thèse nous nous sommes intéressés aux effets bénéfiques que pourrait exercer GroEL sur l’inflammation intestinale via son action au niveau des macrophages. Pour ce faire, nous avons étudié les effets de GroEL provenant de la souche Lactobacillus reuteri sur des modèles in vitro (macrophages humains), ex vivo (explants tissulaires de côlon) et in vivo (modèles murins d’inflammation intestinale et de polypose). Résultats : Nous avons démontré que la protéine GroEL provenant de la souche L. reuteri prévient la polarisation des macrophages vers un phénotype M1 (pro-inflammatoire) tout en favorisant la polarisation des macrophages vers un phénotype M2 (anti-inflammatoire). De façon similaire, le traitement des macrophages par cette protéine a permis de diminuer l’inflammation et le stress oxydant induits par le LPS. Ces expériences ont été confirmées sur des explants tissulaires de côlon, où le traitement par GroEL a permis de réduire l’inflammation, l’apoptose et le stress oxydant provoqués par le LPS. Par ailleurs, l’effet anti-inflammatoire de GroEL de L. reuteri a pu être démontré sur un modèle murin d’inflammation intestinale induite par le DSS. Dans ce modèle, l’administration intra-rectale de GroEL de L. reuteri a permis de prévenir le raccourcissement du côlon, la dégradation de la muqueuse intestinale et la perte de poids provoqués par le DSS. Enfin, nous avons également remarqué que GroEL de L. reuteri pouvait agir comme un agent prophylactique étant donné que les souris APCMin/+ traitées avec cette protéine présentaient une diminution significative du nombre de polypes sporadiques situés au niveau du côlon. Conclusion : La protéine GroEL de L. reuteri, par ses propriétés anti-inflammatoires, pourrait être un complément thérapeutique intéressant aux traitements disponibles dans la prise en charge des MICI et la prévention du CCR.Mots clés : GroEL, inflammation du côlon, probiotiques, MICI et CCR. / Introduction : Intestinal inflammation observed in inflammatory bowel disease (IBD) is mainly located in the colon and could lead to the onset of colorectal cancer (CRC). This inflammation is tightly regulated by the immune cells of the intestinal mucosa which are predominantly represented by macrophages. These cells play a key role in the pathogenesis of IBD and contribute to the maintenance of intestinal inflammation. Currently, available treatments proposed to treat these pathologies are aimed primarily at reducing inflammation. The work carried out during this thesis is part of this context. Indeed, previous experiments performed by our laboratory showed that supernatants of probiotic Lactobacillus biofilms induced an anti-inflammatory response in human macrophages. It was also noted that this effect was abrogated upon immunodepletion of the stress protein GroEL (a heat shock protein). Methods : Therefore, during this thesis work we have been interested in the beneficial effects that GroEL could exert on intestinal inflammation via its action on human macrophages. In order to do this, we used GroEL from the Lactobacillus reuteri strain and its effect in vitro (in human macrophages isolated from buffy coats), ex vivo in colon human explants and in vivo in two different models was analyzed (murine models of intestinal inflammation and polyposis). Results : We found that Lactobacillus reuteri GroEL inhibited the polarization of macrophages towards a M1-like phenotype (pro-inflammatory) while facilitating the polarization of macrophages towards an M2-like phenotype (anti-inflammatory). As a consequence, macrophages treated with L. reuteri GroEL presented a decrease in inflammation and oxidative stress induced by LPS. This was confirmed in colon tissues from biopsies, where L. reuteri GroEL was able to decrease inflammation, apoptosis and oxidative stress induced by LPS. Besides, the beneficial anti-inflammatory effect of Lactobacillus GroEL was demonstrated in a mice model of DSS-induced intestinal inflammation. Rectal administration of L. reuteri GroEL prevented colon shortening and degradation of intestinal mucosa. Further, it abrogated the drastic weight loss induced by DSS. We also showed the potential role of GroEL as a prophylactic agent since APCMin/+ mice treated with L. reuteri GroEL exhibited a significant decrease in the number of sporadic colonic polyps. Conclusion : L. reuteri GroEL protein, by its anti-inflammatory properties, could be an interesting adjunct therapy to the available treatments in the care of IBD and the prevention of CRC.Keywords : GroEL, colonic inflammation, probiotics, IBD and CRC.

Malaria is caused by a protozoan parasite belonging to the genus Plasmodia. Plasmodium falciparum is responsible for the fatal form of human malaria. Spread of drug resistant parasites warrants for sound biological understanding of the parasite at both cellular and biochemical level. Heat shock proteins are highly conserved group of proteins required for correct folding, transport, and degradation of substrate proteins in vivo. Hsp60 is found in eubacteria, mitochondria, and chloroplasts, where in cooperation with Hsp10, it participates in protein folding. Keeping in mind the central importance of chaperones in biological processes, our lab has been interested in examining roles of heat shock proteins in malarial parasite during its asexual growth in human erythrocytes. During its life cycle, the parasite continually shuttles between a cold-blooded insect vector with the body temperature of 27°C and a warm-blooded human host with the body temperature of 37°C and parasite experiences episodes of heat shock periodically. Therefore malaria parasite serves as good model to study heat shock protein functions. Like all biological systems, the malaria parasite expresses several chaperones including proteins of the Hsp40, Hsp60, Hsp70, Hsp90 and Hsp100 families. Towards this we have systematically characterized different families of stress proteins Hsp40, Hsp60, Hsp70, Hsp90 as well as Hsp100. In addition to cloning their genes we have studied their expression, localization, abundance, complexes and their biological roles. Earlier studies from our lab showed PfHsp90 is essential for parasite growth and survival in human erythrocytes. Our present study attempts to study heat shock protein 60 of the malarial parasite (PfHsp60). In this connection we have been successful to clone and express PfHsp60 gene from Plasmodium falciparum in E. coli and to raise antibodies specific to PfHsp60. We have examined its expression and import in the mitochondrion of malarial parasite during its asexual growth in human erythrocytes. Analysis of the total parasite lysates resolved by two-dimensional gel electrophoresis followed by western blotting using specific antibodies showed PfHsp60 exhibits an isoelectric point corresponding to its signal uncleaved precursor (pI - 6.2). Mass spectrometric analysis of the spot corresponding to precursor PfHsp60 confirmed the presence of signal peptide region. Co-immunoprecipitation analysis of total parasite lysates with antibodies specific to PfHsp60 showed precursor PfHsp60 to be associated with PfHsp70 and PfHsp90. Co-immunoprecipitation from the mitochondrial and cytoplasmic fraction confirmed the position of mature PfHsp60. Indirect immunofluorescence analysis also showed presence of a pool of PfHsp60 in the cytoplasm of the parasite, in addition to its expected localization in the mitochondrion. Treatment of parasite infected erythrocytes with an inhibitor of Hsp90 disrupted its association with cytoplasmic chaperones and targeted precursor Pfhsp60 for intracellular degradation. On the other hand treatment with the mitochondrial import inhibitor further inhibited the import of precursor PfHsp60 into the mitochondrion and stabilized its interaction with cytosolic chaperones. Previous reports have shown that there are four fold accumulations of PfHsp60 transcripts in heat shocked parasites. However, the expression of PfHsp60 was not induced upon heat shock in the blood stages of P.falciparum. Biochemical data indicate that the mitochondrion is not the source of ATP in the parasite. Furthermore the genome does not seem to encode the critical subunits of Fo-F1 ATP synthase. Yet, the active mitochondrial electron transport chain serves for regeneration of ubiquinone required for pyrimidine biosynthesis. The active electron transport chain is critical for parasite survival. Recent study with the lab-grown 3D7 strain of malaria parasite concluded that mitochondria are not required for energy conversion. Transcriptome analysis of the parasite derived directly from blood samples of infected patients showed that genes encoding the proteins of mitochondrial biogenesis, oxidative phosphorylation, respiration and highlighted the mean expression level for PfHsp60 is dramatically up regulated in parasites. Gene up regulation doesn’t always translate to increase in protein function or metabolic up regulation. When we analyzed the total parasite lysates of field isolates resolved by two-dimensional gel electrophoresis also showed presence of the precursor form of Pfhsp60 in the cytoplasm of the parasite. Overall, our observations indicated accumulation of precursor PfHsp60 in the parasite cytoplasm suggesting an inefficient mitochondrial protein import in the malarial parasite. The defect in mitochondrial protein import is possibly reflective of the compromised energy state of the parasite mitochondrion. This fits with the model that has been reported in mutant strains of yeast, Saccharomyces cerevisiae lacking functional F o-F1-ATPase. These strains were found to grow very poorly under anaerobic conditions and are known to accumulate Hsp60 protein in the cytoplasm mainly its precursor form. Under optimal growth conditions most eukaryotes maintain close co-ordination between gene expression, translation and translocation efficiently. As a result, mitochondrial precursor proteins are usually not found to accumulate in the cytoplasm. To our knowledge this the first report suggesting an inefficient co-ordination in the synthesis and translocation of precursor PfHsp60 and possibly other proteins during asexual growth of malarial parasite in human erythrocytes under optimal growth conditions. Finally, expression of the PfHsp60 gene in E.coli resulted in its association with bacterial GroEL subunits co-fractionating with a size of 920 kDa, corresponding to the tetra decameric form. The observation indicated possible existence of a hybrid chaperonin complex consisting of subunits from ectopically expressed PfHsp60 and endogenous GroEL.

We recently reported results from a high-throughput screening effort that identified 235 inhibitors of the Escherichia coli GroEL/ES chaperonin system [Bioorg. Med. Chem. Lett. 2014, 24, 786]. As the GroEL/ES chaperonin system is essential for growth under all conditions, we reasoned that targeting GroEL/ES with small molecule inhibitors could be a viable antibacterial strategy. Extending from our initial screen, we report here the antibacterial activities of 22 GroEL/ES inhibitors against a panel of Gram-positive and Gram-negative bacteria, including E. coli, Bacillus subtilis, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae. GroEL/ES inhibitors were more effective at blocking the proliferation of Gram-positive bacteria, in particular S. aureus, where lead compounds exhibited antibiotic effects from the low-lM to mid-nM range. While several compounds inhibited the human HSP60/10 refolding cycle, some were able to selectively target the bacterial GroEL/ES system. Despite inhibiting HSP60/10, many compounds exhibited low to no cytotoxicity against human liver and kidney cell lines. Two lead candidates emerged from the panel, compounds 8 and 18, that exhibit >50-fold selectivity for inhibiting S. aureus growth compared to liver or kidney cell cytotoxicity. Compounds 8 and 18 inhibited drug-sensitive and methicillin-resistant S. aureus strains with potencies comparable to vancomycin, daptomycin, and streptomycin, and are promising candidates to explore for validating the GroEL/ES chaperonin system as a viable antibiotic target.

Bartonella, a group of Gram negative facultative intracellular bacteria, are known to cause diseases, such as Cat Scratch Disease, Trench Fever and Carrion’s Disease, that involve angiogenesis during the infective cycle. B. bacilliformis, the etiological agent of Carrion’s Disease, causes a bi-phasic infection resulting in the formation of blood-filled angiogenic proliferative cutaneous nodules called verruga peruana. The work presented here was undertaken to characterize the mechanism by which these nodules are produced. Previous work in our laboratory suggested that the Bartonella henselae genome contains a homologue to the virB operon, a set of genes coding for a Type IV Secretion System (TFSS) that has been implicated in the pathogenesis of other α-2-proteobacteria. We identified virB operons in two additional Bartonella pathogens, B. quintana and B. clarridgeiae. No corresponding operon sequences were detected in B. bacilliformis DNA, however. This finding suggests that virB gene products are not required for verruga peruana formation. To continue our search for factors involved in B. bacilliformis-induced angiogenesis, we conducted a microarray analysis of differential gene expression in infected and uninfected endothelial cells. The results suggest similarities between later stage (36 hours) B. bacilliformis infection and that of HHV-8, the causative agent of Kaposi’s Sarcoma, particularly in relation to the host immune response. Finally, our research focused on the secreted factors that B. bacilliformis produces during its host infective cycle. Our data suggest that the B. bacilliformis homologue to the molecular chaperone GroEL not only induces angiogenesis in endothelial cells, but also protects endothelial cell tubule from the degradation seen when these cells are in the presence of live B. bacilliformis. In summary, the induction of verruga peruana nodules via B. bacilliformis may be the result of multiple factors over the course of persistent infection. Early infection may cause vascular damage, which induces VEGF and hypoxia factors. As infection persists, bacterial secretion of a unique GroEL may result in continued angiogenesis and the ensuing activation of immune cells, producing a localized environment of continual incomplete angiogenesis in areas of cutaneous infection.

E. coli SecA is an essential component for protein translocaiton across membrane. SecA can be deleted from its N- and/or C-terminal ends without losing complementation activity. In this study, we determined the dispensity of both ends of SecA molecule. The minimal length at the SecA C-terminus is dependent on the length of the N-terminal region. SecA10-826 and SecA22-829 are the two minimal length SecAs. One more amino acid deleted at the C-terminal end completely abolished their complementation activity. A hydrophobic amino acid is required at the 826th amino acid in the minimal-length SecAs. Both SecA22-828 and SecA22-829 could form a dimer, and have decreased ATPase and protein translocation activities. The active truncated SecA mutants tended to have more soluble form than membrane-bound form, but were stably embedded in membrane. In contrast, the inactive truncated SecA mutants tended to have more membrane-bound form, but were not stable in membrane. Thus, the loss of complementation is not related to dimerization, ATPase and translocation activity but to certain extent related to their biased subcelluar localization and conformation in membrane. Isolated membranes of E coli strains were solubilized and fractionated by sucrose gradient fractionation. These membranes fractions were depleted of SecY and YidC, but contained SecD, SecF and GroEL. Proteoliposomes reconstituted from these fractionated membrane proteins were active in pOmpA translocation which required SecA and ATP. Membrane fractions from strain CK1801 in which the unc gene is deleted were reconstituted into liposomes and also showed translocation activities. Moreover, proteoliposomes reconstituted with Bacteriorodopsin alone were not active in translocation, while proteoliposomes reconstituted with Bacteriorodopsin and CK1801 membrane fractions showed elevated translocation efficiency. These data suggested that proton motive force is not obligatory for, but stimulatory to translocation of pOmA. Purified GroEL was reconstituted into lipsomes and the reconstituted proteoliposomes were active in pOmpA translocation although at lower efficiency. This translocation also required SecA and ATP. These results together suggested that translocation of pOmpA is active in the absence of SecY and YidC. In the absence of SecYEG, translocation of pOmpA requires SecA and ATP. GroEL, SecD and SecF may participate in the SecY-independent translocation.

Submitted by Bruno Rossitto de Marchi (bruno_dmarchi@hotmail.com) on 2018-08-08T18:14:08Z No. of bitstreams: 1 Bruno De Marchi Tese Definitivo.pdf: 5408910 bytes, checksum: 6c7b13538e592fdcfc85ae1e3eb12212 (MD5) / Approved for entry into archive by Maria Lucia Martins Frederico null (mlucia@fca.unesp.br) on 2018-08-08T18:50:48Z (GMT) No. of bitstreams: 1 de marchi_ br_dr_botfca.pdf: 5386545 bytes, checksum: 8a0580cd031695c504bc6270ee8317c9 (MD5) / Made available in DSpace on 2018-08-08T18:50:48Z (GMT). No. of bitstreams: 1 de marchi_ br_dr_botfca.pdf: 5386545 bytes, checksum: 8a0580cd031695c504bc6270ee8317c9 (MD5) Previous issue date: 2018-07-31 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A mosca-branca, Bemisia tabaci (Gennadius) é uma praga de distribuição global que afeta centenas de diferentes plantas hospedeiras incluindo grandes culturas, olerícolas e ornamentais. B. tabaci causa danos principalmente através da transmissão de vírus de plantas como os begomovirus, crinivirus, ipomovirus, torradovirus e carlavirus. Atualmente, B. tabaci é considerada um complexo de pelo menos 40 espécies crípticas que apresentam diversidade genética, biológica e diferentes composições de bactérias endossimbiontes facultativas. No Brasil, tanto espécies nativas quanto exóticas de mosca-branca são encontradas e ainda há uma escassez de dados genômicos destas populações e das bactérias endossimbiontes. Na África Oriental, altas populações de moscas-brancas estão disseminando diferentes vírus de plantas que causam epidemias na cultura da mandioca (Manihot esculenta) e com perdas na produtividade. A principal forma de manejo desses vírus na África é através da utilização de variedades tolerantes. Portanto, é essencial a identificação de novos genes de resistência para o desenvolvimento de variedades e um manejo eficiente das doenças. O sequenciamento de transcriptomas é uma ferramenta que possibilita uma análise genômica da mosca-branca, dos vírus transmitidos por ela, das bactérias endossimbiontes e das plantas hospedeiras dessa praga. Portanto, os dados genômicos obtidos dão suporte para o desenvolvimento de novas técnicas que podem se tornar futuras alternativas de controle de mosca-branca e dos vírus associados. No Capítulo 1, dados de transcriptomas foram obtidos das diferentes espécies de B. tabaci encontradas no Brasil, tendo sido possível a obtenção de genomas mitocondriais completos de espécies exóticas e nativas de mosca-branca, além de genomas parciais do endossimbionte facultativo Hamiltonella. A análise filogenética revelou que as diferenças genéticas presentes no gene mtCOI entre as espécies nativas e as espécies exóticas se estendem ao genoma mitocondrial e ao endossimbionte facultativo Hamiltonella. Além disso, foi possível verificar uma deleção de aminoácidos somente no gene GroEL de Hamiltonella que está presente em populações de moscas-brancas nativas. Esse gene é conhecido por estar associado a transmissão de vírus de plantas. No Capítulo 2, foram sequenciados transcriptomas de plantas de mandioca naturalmente infectadas com vírus coletadas no campo em diferentes países da África. A partir desses dados, foi avaliada a diversidade de genes do cloroplasto e a relação com diferentes espécies de vírus. Há uma baixa diversidade dentre as cultivares de mandioca atualmente plantadas na África e não foi possível verificar nenhuma relação entre os genes de cloroplastos avaliados e as espécies de vírus detectadas ocorrendo naturalmente no campo. Os dados obtidos reforçam a necessidade da introdução de novos materiais genéticos para aumentar a diversidade genética das cultivares de mandioca plantadas nesses países, a fim de melhorar as alternativas de manejo das epidemias de vírus. / The whitefly, Bemisia tabaci (Gennadius) is a global pest that affects hundreds of different plant hosts including vegetable, fiber and ornamental crops. B. tabaci causes damage mainly by the transmission of plant viruses such as begomoviruses, criniviruses, ipomoviruses, torradoviruses, and carlaviruses. Currently, B. tabaci is known as a complex of at least 40 putative cryptic species that shows genetic and biological diversity and a different composition of bacterial facultative endosymbionts. In Brazil, both exotic and indigenous species of whiteflies are found and there is still a lack of genomic data available among these populations and also their bacterial endosymbionts. In East Africa, high populations of whiteflies are transmitting different plant viruses that are causing epidemics in cassava crops (Manihot esculenta) and leading to great yield losses. Currently, the management of these viral diseases in Africa is carried out mainly by growing cassava tolerant varieties. Therefore, is essential to identify new target genes for the development of new varieties for an efficient management of viral diseases. Transcriptome sequencing is a tool that allows a genomic analysis of the whitefly, whitefly-transmitted viruses, bacterial endosymbionts and plant hosts. Therefore, the genomic data obtained gives support for the development of new technics that might aid for future management alternatives of whiteflies and their associated viruses. In Chapter 1, transcriptome data were obtained from different B. tabaci species found in Brazil which allowed to obtain complete mitochondrial genomes from different whitefly species and draft genomes of the facultative endosymbiont Hamiltonella. The phylogenetic analysis revealed that the genetic differences among exotic and native populations present in the mtCOI gene extends to the mitochondrial genome and to the facultative endosymbiont Hamiltonella. In addition, it was verified an amino acid deletion in the GroEL gene from Hamiltonella present only in native populations of whiteflies. This gene is known to be associated with the transmission of plant viruses. In Chapter 2, transcriptome data were obtained from virus-infected cassava plants collected in the field in different African countries. The data allowed to evaluate the diversity of chloroplast genes and their relationship with different virus species. The data revealed a low genetic diversity among cassava currently grow in East Africa. In addition, there was no direct relationship between the evaluated chloroplast genes and the virus species detected. The obtained data reinforce the need of introduction of new genetic accession to increase the genetic diversity of the currently grown cassava in Africa in order to improve the alternatives of management of viral diseases. / CNPq 131324/2015-5 / CNPq 200826/2015-8

Abstract Chlamydia pneumoniae is a common respiratory pathogen worldwide. It does not only cause acute respiratory infections, but is also associated with chronic inflammatory diseases, such as asthma and coronary heart disease (CHD). Chlamydial heat shock protein 60 (Hsp60) is associated with the development of immunopathological damage following C. trachomatis infections, but the role of Hsp60 in C. pneumoniae infections is unclear. A slightly elevated level of C-reactive protein (CRP), as a marker of systemic inflammation, predicts cardiovascular events, but its role in asthma has not been studied. The aim of this study was to develop an EIA method for the measurement of Hsp60 antibodies and for studying the host immune responses to C. pneumoniae and chlamydial and human Hsp60 proteins, CRP levels and their interactions in asthma and CHD. Elevated levels of serum IgA antibodies to the Hsp60 protein of C. pneumoniae were associated with asthma and decreased pulmonary function. CRP levels were also higher in the asthma patients than in the controls. The patients with moderate asthma had higher CRP levels than those with mild asthma. The patients with a CRP level over 2 mg/l had higher levels of serum IgA antibodies to C. pneumoniae and chlamydial Hsp60 than the patients with lower CRP levels. A prospective nested case-control study was carried out, to study the role of Hsp60 antibodies as coronary risk predictors, and their association with C. pneumoniae infection and inflammation. The participants were obtained from the Helsinki Heart Study: 241 myocardial infarctions or coronary deaths occurred during the 8.5-year period among dyslipidemic middle-aged men. An elevated level of human Hsp60 IgA antibodies in baseline serum predicted the occurrence of a coronary event several years later, especially when present simultaneously with a high C. pneumoniae IgA antibody level and an elevated CRP level. Further studies showed that only persistently, not transiently, elevated levels predicted coronary events. The risk associated with elevated antibody levels increased markedly in the presence of an elevated CRP level, and vice versa. In conclusion, these results suggest that chlamydial Hsp60 is involved in the association between C. pneumoniae infection and asthma, while autoimmunity to human Hsp60 is implicated in the association between C. pneumoniae infection and CHD. Inflammation evidently plays an important role in these associations. It can also be concluded that IgA antibodies, compared to IgG antibodies, against C. pneumoniae and Hsp60 are better markers of chronicity, especially when they are persistently elevated.