This thesis considers a novel problem of obtaining global channel state information (CSI) at every node in an ad-hoc wireless network. A class of protocols for dissemination and estimation are developed which attempt to minimize the staleness of the estimates throughout the network. This thesis also provides an optimal protocol for CSI dissemination in networks with complete graph topology and a near optimal protocol in networks having incomplete graph topology. In networks with complete graph topology, the protocol for CSI dissemination is shown to have a resemblance to finding Eulerian tours in complete graphs. For networks having incomplete graph topology, a lower bound on maximum staleness is given and a near optimal algorithm based on finding minimum connected dominating sets and proper scheduling is described in this thesis.

This thesis considers a novel problem of obtaining global channel state information (CSI) at every node in an ad-hoc wireless network. A class of protocols for dissemination and estimation are developed which attempt to minimize the staleness of the estimates throughout the network. This thesis also provides an optimal protocol for CSI dissemination in networks with complete graph topology and a near optimal protocol in networks having incomplete graph topology. In networks with complete graph topology, the protocol for CSI dissemination is shown to have a resemblance to finding Eulerian tours in complete graphs. For networks having incomplete graph topology, a lower bound on maximum staleness is given and a near optimal algorithm based on finding minimum connected dominating sets and proper scheduling is described in this thesis.

E. coli is the most frequently isolated Gram negative pathogen from bacteremia in cancer patients and is repeatedly recovered from many other extraintestinal illnesses. These infections are commonly endogenous in nature and interfere with the treatment of cancer resulting in increased healthcare costs, morbidity, and mortality rates. Cancer and the treatments related to cancer cause alterations in the microbiome of the gut and other organs. Despite this point, there is a serious lack of knowledge about the genetic types of E. coli infecting cancer patients. This gap results in vague prevention strategies and limited treatment options for cancer patients. Multi Locus Sequence Typing (MLST) was used to successfully genotype 105 sequentially collected E. coli isolates from patients admitted to H. Lee Moffitt Cancer Center (HLMCC, Tampa, FL) with confirmed extraintestinal infections between 2010 and 2012. In total, 24 distinct genotypes (STs) have been identified in this dataset using EcMLST (STEC Reference Center). Of these, ST34 constituted 39% of the isolates and may represent a disseminating clone at HLMCC. Furthermore, 17 isolates not found in the EcMLST database have been identified. Importantly, phylogenetic analysis of DNA sequence data for MLMCC E. coli revealed only 22% of HLMCC E. coli clustered with ECOR reference strains commonly attributed to the B2 phylogroup of extraintestinal pathogenic E. coli (ExPEC). Four HLMCC E. coli belonging to ST171 and attributed to life-threatening blood infections clustered with Shiga toxin (Stx) producing E. coli (STEC) strain TW06296. HLMCC E. coli belonging to ST34 clustered with enteroaggregative E. coli (EAEC) strain TW10263. Importantly, these non-B2 phylogroup strains demonstrated more pathogenic potential than HLMCC E. coli clustered with B2 ExPEC,which included a higher incidence of bacteremia and sepsis, as well as resistance to first-line antibiotics. Upon further investigation, ST34 may equate to ST131 by another MLST database. These findings suggest that isolates previously characterized as commensal and intestinal pathogenic E. coli have an increased ability to cause infection outside of the gastrointestinal tract in cancer patients and that selective pressures are contributing to increased antibiotic resistance. These findings may change the approach to clinical management of E. coli infections at cancer centers.

Vibrio parahaemolyticus is a leading cause of seafood-borne illness with a newly emerged pandemic strain. Previous studies compared the pandemic and non-pandemic strains to understand the evolution of the pandemic strain but no definitive explanation for its emergence has been discovered. This study investigated the molecular characteristics of the pandemic strain and growth characteristics at different temperatures. The hypothesis tested was that pandemic strains of V. parahaemolyticus have modifications to their proteome that give a selective advantage over the other V. parahaemolyticus strains at temperatures normally encountered in the environment. Molecular typing techniques; automated ribotyping, pandemic specific PCR and multilocus sequence typing (MLST), were compared to determine the best method for pandemic strain determination. MLST was the best method because it was the most informative and accurate. Furthermore, nine Florida outbreak strains were identified as pandemic. Using representatives of both strains, growth curves were produced at four temperatures. The five pandemic strains had a significantly faster growth rate at 12°C than five non-pandemic strains. Temperature specific proteomic comparisons were completed using liquid chromatography followed by tandem mass spectroscopy. The proteome differences between these two groups at 12°C included three proteins (DnaA, DnaJ-related protein and DnaK-related protein) with functions related to cold stress. DnaA was expressed in the non-pandemic strain and not the pandemic strain, while the reverse was true for DnaJ-related and DnaK-related proteins. Western blot analysis and LC-MS/MS analysis on additional strains did not support the initial LC-MS/MS results. Growth studies using expression recombinants were employed to investigate these proteins on growth at 12°C. The overexpression of DnaA and DnaJ-related proteins did not significantly alter the growth rates compared to the control strain, but the overexpression recombinant strains DnaK-5 has a significantly slower growth rate than the control strain, the opposite direction as expected. The pandemic strain grows faster at lower temperatures, but the reason has not been determined. A theory is offered in which the pandemic growth advantage related to regulation of cold stress, leading to a shorter lag phase and faster growth rate after acclimation to the lower temperatures. Further experiments to investigate this theory are discussed.

The amphibian pathogen, Batrachochytrium dendrobatidis (Bd), has been the cause of mass declines of amphibian populations worldwide (Berger et al. 1998). This pathogen has been shown to infect approximately 387 different amphibian species and causes declines in approximately 200 species (Skerratt et al. 2009). The total impact on amphibian biodiversity as well as their ecosystems has yet to be determined but it has already been suspected in some species extinctions (Schloegel et al. 2006). The distribution of this amphibian pathogen has been described by two competing hypotheses, the novel and endemic pathogen hypotheses. The endemic pathogen hypothesis states that the pathogen has always been a part of the ecosystem and has only recently become pathogenic due to environmental factors. The novel pathogen hypothesis states that the pathogen has just recently been introduced and has encountered a naïve host which has resulted in population declines (Rachowicz et al. 2005). Research into these two hypotheses has been very active yet the results have still been conflicted (Pounds et al. 2006; James et al. 2009). In our study we assess two relatively under surveyed locations for the presence of Bd, both in Peninsular Malaysia and British Columbia (BC). The results of the amphibian survey showed that Bd was currently ubiquitous throughout the province of BC. This was coupled with a population genetic evaluation of two Bd strains in British Columbia which led us to conclude that they were a part of a novel pathogen which may have been introduced through the amphibian trade possibly from the east coast of Canada. During the first two years of surveying for the presence of Bd in Peninsular Malaysia we found no evidence of the pathogen. In the third and final year of the survey we did discover low prevalence of the pathogen, which was supported by a recently published report of initial Bd detection in Peninsular Malaysia (Savage et al. 2011). We were not able to definitively state which of the competing hypotheses (NPH vs EPH) was correct for either collection region. Our population genetic results for two isolates collected from Bullfrogs on Vancouver Island suggest that Bd may have been introduced via the animal trade however the endemicity for the rest of the province remains unresolved. In peninsular Malaysia Bd may represent a novel pathogen or it could exist as an endemic pathogen with a low prevalence. / Graduate

Orientador: Maria de Lourdes Ribeiro de Souza da Cunha / Resumo: Introdução: o isolamento de estafilococos coagulase negativos (ECN) de pacientes em Unidades de Terapia Intensiva Neonatal (UTIN) pode representar, muitas vezes, uma contaminação ao invés de bacteremia. O padrão ouro para o diagnóstico de sepse neonatal é a positividade de duas hemoculturas coletadas num intervalo de 48 horas associada às manifestações clínicas sugestivas de sepse. A resistência aos antimicrobianos e a formação de biofilme são fatores seletivos na persistência de cepas de S. epidermidis e S. haemolyticus. A técnica de Eletroforese em Gel de Campo Pulsado (PFGE), altamente discriminatória, é frequentemente usada para a investigação de surtos. A tipagem por Multilocus Sequence Typing (MLST) se tornou o método de escolha no estudo epidemiológico em longo prazo. Objetivos: esse estudo teve como objetivos caracterizar o perfil das infecções de corrente sanguínea por Staphylococcus spp. em RNs internados na UTIN, os fatores de risco para infecção, perfil de virulência e resistência antimicrobiana dos estafilococos isolados, bem como a presença de clones prevalentes na unidade. Resultados: foram isolados 72 Staphylococcus spp. de hemoculturas de 54 recém-nascidos de Março de 2014 a Agosto de 2015. Foram confirmados 37 episódios de Infecção da Corrente Sanguínea (ICS) causados por Staphylococcus spp., sendo 27 associados a espécie S. epidemidis, 3 S. haemolyticus, 2 S. capitis e 5 S. aureus. A maioria dos Recém-nascidos (RNs) possuía extremo baixo peso, nasceu com me... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Abstract Introduction: The isolation of coagulase negative staphylococci (CoNS) of Neonatal Intensive Care Unit (NICU) patients can often represent contamination instead of bacteremia. The golden standard for the diagnosis of sepsis is the positivity of two blood cultures, collected within a 48-hour gap, associated to clinic manifestations suggestive of sepsis. Resistance to antimicrobials and biofilm formation are selective factors to S. epidermidis and S. haemoyticus strains to persist. Pulsed Field Gel Electrophoresis (PFGE), a highly discriminatory technique, is often used in investigating outbreaks. Multilocus Sequence Typing (MLST) has become the preferred method when it comes long-term epidemiologic studies. Objectives: this research aimed to characterize the profile of infections caused by Staphylococcus spp. at NICU, the virulence profile and antimicrobial resistance of isolated staphylococci, as well as the presence of clusters prevalent in the unit. Results: 72 Staphyloccocus spp. have been isolated from blood cultures taken from 54 newborns from March 2014 to August 2015. Thirty-seven bloodstream infection (BSI) episodes caused by Staphylococcus spp. were confirmed, 27 of which are associated to S. epidemidis, 3 to S. haemolyticus, 2 to S. capitis and 5 to S. aureus. Most newborns presented extremely low weight, were born before the 31st week of pregnancy (72.2%), used catheter and mechanical ventilation. From the 72 samples of Staphylococcus spp. under analysis, ... (Complete abstract click electronic access below) / Doutor

Submitted by Pós graduação Farmácia (ppgfar@ufba.br) on 2017-05-25T21:38:30Z No. of bitstreams: 1 RIOS RSR-UFBA.pdf: 2890961 bytes, checksum: fae1486048636d899ff4db4f5903144f (MD5) / Approved for entry into archive by Patricia Barroso (pbarroso@ufba.br) on 2017-05-29T17:23:59Z (GMT) No. of bitstreams: 1 RIOS RSR-UFBA.pdf: 2890961 bytes, checksum: fae1486048636d899ff4db4f5903144f (MD5) / Made available in DSpace on 2017-05-29T17:23:59Z (GMT). No. of bitstreams: 1 RIOS RSR-UFBA.pdf: 2890961 bytes, checksum: fae1486048636d899ff4db4f5903144f (MD5) / CAPES / Streptococcus pneumoniae é um dos principais agentes causadores de doenças invasivas graves como: pneumonia, meningite e septicemia. Estima-se o óbito em cerca de 2,0 milhões de crianças em países em desenvolvimento. O padrão ouro para diagnóstico é a cultura, mas a sua sensibilidade é baixa e por isso o aprimoramento no diagnóstico dos casos continua um desafio. O objetivo do presente estudo foi avaliar o uso de técnicas moleculares diretamente em líquor visando detectar e caracterizar genotipicamente Streptococcus pneumoniae em casos de meningite. Um estudo prospectivo de corte transversal foi realizado no Hospital Couto Maia, no período entre abril de 2006 a dezembro de 2010. As amostras de líquor foram selecionadas de acordo com critérios de inclusão como celularidade superior a 100 células/mm3 e/ou Glicorraquia inferior a 40 mg/dl e/ou Proteinorraquia superior a 65 mg/dl, e foram submetidas a análises de rotina do hospital, e posteriormente testadas por técnica de PCR em tempo real para detecção de S. pneumoniae, usando como alvo o gene lytA e a reação de PCR-Multiplex para a dedução do sorotipo capsular. A caracterização genotípica foi realizada através da técnica de Multilocus Sequence Typing (MLST). Foram analisadas 1141 amostras de líquor provenientes de pacientes com suspeita clínica de meningite bacteriana. Destas, 92 amostras foram positivas para S. pneumoniae por PCR em tempo real. Das quais, 68 amostras (73,9%) foram cultura positiva e 24 (26,1%) cultura negativa. Os sorotipos de 21 dos 24 casos cultura negativa foram deduzidos. Cerca de 46% dos sorotipos encontrados nos casos cultura negativa, foram não vacinais. Dentre os sorotipos vacinais mais frequentes estavam o 6A/B/C com 6 casos (25%), 14 com 3 casos (12,5%), 23F com 2 (8,3%), 18A/B/C com 1 caso (4,2%) e 4 com 1 caso (4,2%). Do total de 24 casos cultura negativa, três casos (12,5%) não apresentaram positividade para qualquer dos 40 sorotipos testados, embora tenham apresentado positividade para o gene cpsA, permanecendo então como não determinados. Seis amostras (25%) tiveram genótipo caracterizado por MLST, sendo definidos os STs 750, 6403 e 2814. Este estudo conclui que as técnicas moleculares de PCR em tempo real e Multiplex PCR podem ser bastante úteis quando aplicadas diretamente em espécimes clínicos, possibilitando uma melhor detecção e diagnóstico dos casos, mesmo quando todas as outras técnicas diagnósticas convencionais não são capazes de fazê-lo, além de proporcionar um ganho de informações epidemiológicas que não seriam possíveis de serem obtidos por métodos convencionais, devido à ausência de positividade das culturas bacteriológicas. / Streptococcus pneumoniae is a major cause of severe invasive diseases such as pneumonia, meningitis and bacteremia. It is estimated death in about 2.0 million children in developing countries. The gold standard for diagnosis of this agent is the culture, but its sensitivity is low and therefore the accurate diagnosis of cases of pneumococcal meningitis remains a challenge. The aim of this study was to evaluate the use of molecular techniques to detect directly in CSF samples, and genotype Streptococcus pneumoniae in meningitis cases. A prospective cross-sectional study was conducted at Hospital Couto Maia, in the period from April 2006 to December 2010. The CSF samples were selected according to inclusion criteria as cellularity above 100 cells/mm3 and / or CSF glucose less than 40 mg/dl and / or protein levels over than 65 mg / dl, and were submitted to routine hospital and subsequently tested by PCR in real time for the detection of S. pneumoniae, using targeting the lytA gene and PCR-Multiplex for the deduction of capsular serotype. The genotypic characterization was performed by Multilocus Sequence Typing technique (MLST). One amount of 1141 CSF samples were collected from patients with suspected bacterial meningitis and submitted for analysis. Of these, 92 samples were positive for S. pneumoniae by real-time PCR. Of which, 68 samples (73.9%) were culture positive and 24 (26.1%) culture negative. The serotypes of 21 of the 24 culture-negative cases were found. About 46% of the serotypes found in culture-negative cases were non vaccine types. Among the most common vaccine serotypes were the 6A/B/C with 6 cases (25%), 14 with 3 cases (12.5%), 23F with 2 (8.3%), 18A/B/C with one case (4.2%) and 4 with 1 case (4.2%). Of the total of 24 culture-negative cases, three cases (12.5%) showed positivity for any of the 40 serotypes tested, although they presented positive result for the gene CPSA amplification and remain as not determined. Six samples (25%) had the genotype characterized by MLST, the STs being set 750, 6403 and 2814. This study concludes that the molecular techniques of real-time PCR and multiplex PCR can be useful when applied directly on clinical specimens, enabling better detection and diagnosis of cases, even when all other conventional diagnostic techniques are not able to do it, and provide a gain of epidemiological information that would not be possible to obtain by conventional methods, due to the absence of positive bacterial cultures.

Le complexe d'espèces Fusarium solani regroupe des champignons phytopathogènes également impliqués en pathologie humaine dans des infections parfois profondes et souvent de mauvais pronostic. Dans un premier temps, une méthode de MLST, s'appuyant sur 5 gènes de ménage a donc été développée. Validée sur 51 isolats épidémiologiquement distincts, cette méthode stable et reproductible présente un pouvoir discriminant de 99,1 %. Après comparaison à la technique de référence utilisée en phylogénie, un schéma consensus à 8 loci a été proposé. Dans un second temps, une étude de la sensibilité de ce pathogène à l'amphotéricine B et au voriconazole a été menée par deux techniques d'évaluation des CMI : microdilution CLSI M38-A2 et bandelettes E-test. Devant le paradoxe entre une sensibilité diminuée in vitro au voriconazole et la recommandation de cette molécule pour le traitement curatif de la fusariose humaine, des mécanismes de résistance ont été exploré. L'hypothèse d'un phénomène d'efflux n'a pas été retenue alors que celle d'une modification de la cible, la 14 alpha stérol déméthylase, peut être envisagée après la description de différentes mutations pour les isoformes CYP51A, B et C / Fusarium solani species complex includes phytopathogenic fungi also involved in human infections with poor prognosis. Firstly, MLST method, based on five housekeeping genes has been developed. This method has been validated on 51 isolates epidemiologically distinct, and has been shown to be stable and reproducible and provides a discriminating power of 99.1%. After comparison with the reference technique used in phylogeny, a consensus method with 8 loci has been proposed. Secondly, a study of the susceptibility to amphotericin B and voriconazole has been conducted with two MIC determination methods : CLSI M38-A2 microdilution and E-test. The paradox between decreased susceptibility to voriconazole in vitro and recommendation of this molecule for the curative treatment of Fusarium infections has lead to the exploration of resistance mechanisms. The hypothesis of an efflux phenomenon has not been retained whereas a change in the target, the sterol 14 alpha demethylase may be considered following the description of different mutations on proteins CYP51A, B and C

Nous nous intéressons à la production de peptides bioactifs dans des laits fermentés par la bactérie lactique Streptococcus thermophilus. Pour ce faire, il est nécessaire que cette bactérie en internalise le moins possible lors de sa croissance. Il était donc important de caractériser le système protéolytique S. thermophilus. Tout d’abord, les relations phylogéniques liant 30 souches de S. thermophilus ont été recherchées par MLST. Ensuite, un système de transport de type ABC qui semble fonctionnel a été identifié chez la souche LMD-9 et appelé OTS. Une étude de la variabilité des systèmes de transport Ami et OTS des 30 souches de S. thermophilus a été réalisée. Enfin, l’hydrolyse des caséines par la protéase PrtS de S. thermophilus a été étudiée. Cette protéase habituellement ancrée à la paroi de la bactérie est retrouvée chez la souche 4F44 également sous forme libre. La séquence protéique de PrtS4F44, différente de celle de PrtS de la souche LMD 9 (PrtSLMD-9), n’est pas la cause de la libération partielle de PrtS4F44. La sortase A, acteur de l’ancrage de PrtS à la paroi de la bactérie, présente chez la souche 4F44 (srtA4F44) un allèle différent de celui de la souche LMD-9 (srtALMD-9). En effet, PrtSLMD-9 se trouve libérée lorsque srtALMD-9 est remplacée par srtA4F44 dans la souche LMD-9 montrant ainsi que SrtA4F44 est déficiente, entrainant par conséquent un défaut d’ancrage de PrtS4F44 et sa libération partielle dans le milieu extracellulaire. L’hydrolyse des caséinates bovines totales par la forme libre de PrtS4F44 a permis d’obtenir des peptides bioactifs qui pourront être utilisés pour la fonctionnalisation de produits laitiers fermentés / We are interested in the production of bioactive peptides in fermented milk by the lactic acid bacterium Streptococcus thermophilus. For this, it requires that the bacterium internalize them as few as possible during its growth. Therefore, it was important to characterize the proteolytic system of S. thermophilus. First, phylogenetic relationships linking 30 S. thermophilus strains have been searched by MLST. Secondly, an ABC-type transport system which seems to be functional was identified in the LMD-9 strain and named OTS. A study of the variability of Ami and OTS transport systems of the 30 strains of S. thermophilus was performed. Finally, the hydrolysis of caseins by proteinase PrtS of S. thermophilus was studied. This proteinase usually anchored to the wall of the bacterium was also found in a free form in strain 4F44. The protein sequence of PrtS4F44, different from the one of PrtS in the LMD-9 strain (PrtSLMD-9), is not the cause of the partial release of PrtS4F44. Sortase A, the actor of the anchoring of PrtS to the wall of the bacteria, presents different alleles between the strain 4F44 (srtA4F44) and the LMD-9 strain (srtALMD-9). Indeed, PrtSLMD-9 is released when srtALMD-9 is replaced by srtA4F44 in the strain LMD-9 showing that SrtA4F44 is deficient, causing consequently a default of PrtS4F44 anchoring and its partial release into the extracellular medium. Additionally, hydrolysis of bovine caseinates was performed using the free form PrtS4F44 and allowed the production of bioactive peptides that can be used for the functionalization of fermented dairy products

Streptococcus agalactiae est la première cause d’infections néonatales, et est aussi un pathogène émergent chez l’adulte immunodéprimé. L’objectif de ce travail de thèse a été de caractériser l’opéron métabolique fru2 de S. agalactiae (i) en étudiant sa phylogénie, (ii) en identifiant ses inducteurs, et (iii) en élaborant son schéma de régulation. Cet opéron est composé de 7 gènes qui codent un activateur transcriptionnel de la famille DeoR-like (Fru2R), un transporteur PTS (PTSFru2), et trois enzymes qui sont potentiellement impliquées dans la voie non oxydative des pentoses phosphates. Nous avons mis en évidence que cet opéron avait été acquis au cours de l’évolution, et n’était présent que chez les souches de complexes clonaux responsables d’infections chez l’adulte immunodéprimé et la personne âgée. Nous avons ensuite montré que certains milieux complexes, sources de carbone, et liquides biologiques humains permettaient l’activation de cet opéron. Ensuite, nous avons caractérisé le rôle et fonctionnement de la protéine Fru2R (i) en montrant son rôle d’activateur transcriptionnel, (ii) en identifiant les acides aminés essentiels à son activité, et (iii) en démontrant sa capacité à se fixer au niveau de la région promotrice de fru2. / Streptococcus agalactiae, commonly known as group B streptococcus, is a leading cause of neonatal morbidity and mortality. It is also an emergent pathogen in immunocompromised and elderly adults. The objective of this study was to characterize the phylogeny, the induction and the regulation of the S. agalactiae fru2 operon. This operon encodes a PTS transporter of the fructose-mannitol family, a transcriptional activator of the DeoR-like family, an allulose-6 phosphate-3-epimerase, a transaldolase and a transketolase. Our results, concerning the phylogeny, indicate that fru2 was acquired during the evolution of S. agalactiae. Then, we highlighted that the fru2 promoter was active in complex medium, in chemically defined medium with various carbon sources and in human biological fluids. Then, we demonstrated that the Fru2R protein (i) was a transcriptional activator, (ii) contains amino acids which are essential for the activity of the Fru2R and fru2 promoter, and (iii) interacts with the fru2 intergenic region. Then, we demonstrated the role of the PTSFru2 proteins of S. agalactiae A909 fru2.