CD8 T cells play a critical role in the clearance of pathogenic bacteria, viruses, and protozoan parasites. Upon encountering their cognate antigen through either infection or vaccination, naïve CD8 T cells undergo robust proliferative expansion, which is followed by contraction and the formation of a memory population. Memory CD8 T cells are long-lived, and because they persist in increased numbers and possess enhanced functional abilities compared to naïve CD8 T cells, they are able to provide the host with increased protection following re-infection. Because of these properties, vaccines designed to elicit memory CD8 T cells have the potential to reduce health care burdens related to infection with pathogens including human immuno deficiency virus (HIV), malaria, influenza, and hepatitis virus. However, stimulating protective CD8 T cell responses against these pathogens through vaccination has proven challenging. Therefore, a better understanding of the properties of memory CD8 T cells generated following vaccination, and the characteristics of memory CD8 T cells best suited for providing protection against diverse pathogens is needed. While memory CD8 T cells can be maintained for as long as the life of the host, evidence suggests that their properties change with time after infection. Because CD8 T cell-mediated protection is based upon both the numbers and quality or functional abilities of memory cells present at the time of re-infection, changes in memory CD8 T cell function over time could impact their ability to provide protection upon re-infection. Therefore, a better understanding of how memory CD8 T cells change with time after infection is needed. As part of the studies presented in this thesis, I found that the phenotype and function of memory CD8 T cells including localization, interleukin (IL)-2 cytokine production, responsiveness to homeostatic cytokines, metabolic capabilities, and proliferation and secondary memory generation potential change with time after infection. Interestingly functional changes could not be completely explained by changes in subset composition that occur with time, as changes over time were also seen in defined CD62Lhi subsets. Importantly, functional changes of memory CD8 T cells that occurred with time led to an increased ability to provide protection against a chronic viral infection. These data improve our knowledge of the capabilities of memory CD8 T cells generated following infection, and suggests that the outcome of vaccination strategies designed to elicit protective memory CD8 T cells using single or prime-boost immunizations will depend upon the timing between antigen encounters. Following re-infection, memory CD8 T cells become activated and produce effector cytokines and cytolytic molecules that aid the host in clearing invading microbes. Activation can be triggered not only through cognate antigen recognition, but also by antigen-independent cytokine driven signals. However, our knowledge of how antigen-dependent and –independent signals contribute to CD8 T cell activation and protection following infection is incomplete. In the second part of my thesis, I show that the ability of memory CD8 T cells to become activated in response to inflammation decreases with time after infection, that antigen and inflammation act synergistically to induce activation of memory CD8 T cells, that the presence of cognate antigen enhances activation of memory CD8 T cells that contribute to clearance of infection, and that bystander memory CD8 T cell responses following unrelated bacterial infection do not provide the host with a protective benefit. Together, the data in this thesis further our understanding of memory CD8 T cells generated following infection and/or vaccination, and the properties of memory CD8 T cells important for providing protection upon re-infection with invading pathogens.

Submitted by (edna.saturno@ufrpe.br) on 2016-10-07T13:54:35Z No. of bitstreams: 1 Fernanda Silva de Meirelles.pdf: 599414 bytes, checksum: be09ab548e388263d8d38ab7cabb0cf5 (MD5) / Made available in DSpace on 2016-10-07T13:54:35Z (GMT). No. of bitstreams: 1 Fernanda Silva de Meirelles.pdf: 599414 bytes, checksum: be09ab548e388263d8d38ab7cabb0cf5 (MD5) Previous issue date: 2010-02-26 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / In Brazil, it is considered that the annual production of tilapias overcomes 100 thousand tons, mainly due to the productive potential of the Northeast area that answered for 41% of the total production of 70.000 tons in 2004, generating income of US$ 200.000.000,00, due to your climatic conditions, technology readiness and consumption market, regional and national in expansion. The intensification of cultivations has as consequence the increase of organic matter, that it favors the multiplication of microorganisms, making possible him/it supplies of diseases in the occurrence of adverse situations to the fish. Tilapia (Oerochromis niloticus) of cultivations of the state of Pernambuco they were appraised with objective of determining the frequency of bacterial diseases. For so much, samples of several farms were analyzed, in the several aspects, with relevance for the bacterial agents. The tilapia were examined with relationship to the streptococcus presence, víbrios, coliform, pseudomonas and aeromonas. Clinical exams and autopsy were accomplished, for microscopic analysis of the lesions and better definition of the probable agent etiological. The analyses were accomplished at the Laboratory of Sanity of Aquatic Animals (LASAq) of the Rural Federal University of Pernambuco (UFRPE) and in the Center of Development and Diffusion of Technology in Aquaculture of the State University of Bahia (UNEB). The principal pathological agents were víbrios and aeromonas, that are pathogens responsible opportunists for significant losses, being identified the following species in 46 isolated: V. natriegens (1/46), V. metschnikovii (2/46), V. halioticoli (1/46), V. fischeri (2/46), V. mimicus (23/46), V. diabolicus (1/46), V. furnissi (1/46), V. cholerae o1 (1/46), V. scophthalmi (4/46), V. proteolyticus (3/46), V. argarivorans (1/46), V. ordalii (2/46) and Vibrio spp. ( 3/46). The isolated vibrios presented larger sensibility to the antimicrobianos enrofloxacina (100%) and florfenicol (98,18%) and in decreasing order, gentamicina (90,91%), cotrimoxazol (sulfametoxazol+trimetroprim) (76,36%), tetraciclin (67,27%), eritromicin (30,91%) and amoxilina (3,64%). The isolated ones tested they came 100% resistant the penicillin. In this study, 96,4% (53/55) of the víbrios they presented index multiple resistance to antibiotics (MAR), superior to 0,22, characterizing multiple resistance. In 35 isolated the identified aeromonas were: A.caviae (1/35), A. shubertii (11/35), A. media (4/35), A. popoffii (1/35), A. sobria (3/35), A. encheleia (4/35), A. veronii (4/35) e A. jandaei (4/35) Forty were tested isolated of aeromonas with relationship to the sensibility to 08 antimicrobial, of these they showed larger sensibility the florfenicol (100,0%), enrofloxacina(95,0%), gentamicina (95,0%) and in decreasing order, cotrimoxazol (sulfametoxazol+trimetroprima) (67,5%), tetraciclina (65,0%). There was smaller sensibility the eritromicina (20,0%), penicillin (5,0%) and the amoxilina (2,5%), confirming the resistance existence. The index of multiple resistance to antibiotics (MAR) for the isolated of aeromonas it varied in an interval from 0,12 to 0,62, of these 95% (38/40) they came superior to 0,22 characterizing multiple resistance. Most of the species of isolated víbrios is not considered pathogenic for the fish (environmental), but nevertheless they can represent risk for the health of the tilapia for subject of opportunism and of the consumer, mainly if consumed raw. The isolated and identified movable aeromonas in this study are commonly considered as environmental, however many of the analyzed fish presented compatible symptomatology with the illness caused by these agents opportunists. / No Brasil, estima-se que a produção anual de tilápias supere 100 mil toneladas, principalmente devido ao potencial produtivo da região Nordeste que em 2004 respondeu por 41 % da produção total de 70.000 toneladas, gerando renda de US$ 200.000.000,00, decorrente de suas condições climáticas, disponibilidade de tecnologia e mercado de consumo, regional e nacional em expansão. A intensificação de cultivos tem como conseqüência o aumento de matéria orgânica, que favorece a multiplicação de microrganismos, possibilitando o surto de doenças na ocorrência de situações adversas aos peixes. Tilápias (Oerochromis niloticus) de cultivos do estado de Pernambuco foram avaliadas com objetivo de determinar a freqüência de doenças bacterianas. Para tanto, analisaram-se amostras de diversas fazendas, nos diversos aspectos, com relevância para os agentes bacterianos. As tilápias foram examinadas quanto à presença de estreptococos, víbrios, coliformes, pseudomonas e aeromonas. Foram realizados exames clínicos e necropsias, para análise microscópica das lesões e melhor definição do provável agente etiológico. As análises foram realizadas no Laboratório de Sanidade de Animais Aquáticos (LASAq) da Universidade Federal Rural de Pernambuco (UFRPE) e no Centro de Desenvolvimento e Difusão de Tecnologia em Aqüicultura da Universidade Estadual da Bahia (UNEB). Os principais agentes patológicos foram víbrios e aeromonas, que são patógenos oportunistas responsáveis por perdas significantes, sendo identificadas as seguintes espécies em 46 isolados: Vibrio natriegens (1/46), V. metschnikovii (2/46), V. halioticoli (1/46), V. fischeri (2/46), V. mimicus (23/46), V. diabolicus (1/46), V. furnissi (1/46), V. cholerae O1 (1/46), V. scophthalmi (4/46), V. proteolyticus (3/46), V. argarivorans (1/46), V. ordalii (2/46) e Vibrio spp.(3/46). Os vibrios isolados apresentaram maior sensibilidade aos antimicrobianos enrofloxacina (100%) e florfenicol (98,18%) e em ordem decrescente, gentamicina (90,91%), cotrimoxazol (sulfametoxazol+trimetroprima) (76,36%), tetraciclina (67,27%), eritromicina (30,91%) e amoxilina (3,64%). Os isolados testados apresentaram-se 100% resistentes a penicilina. Neste estudo, 96,4% (53/55) dos víbrios apresentaram índice múltipla resistência a antibióticos (MAR), superior a 0,22, caracterizando múltipla resistência. Em 35 isolados as aeromonas identificadas foram: Aeromonas caviae (1/35), A. shubertii (11/35), A. media (4/35), A. popoffii (1/35), A. sobria (3/35), A. encheleia (4/35), A. veronii (4/35) e A. jandaei (4/35). Foram testados 40 isolados de aeromonas quanto à sensibilidade a oito antimicrobianos, tendo mostrado maior sensibilidade a florfenicol (100,0%), enrofloxacina(95,0%), gentamicina (95,0%), cotrimoxazol (sulfametoxazol+trimetroprima) (67,5%), tetraciclina (65,0%). Houve menor sensibilidade a eritromicina (20,0%), penicilina (5,0%) e a amoxilina (2,5%), confirmando a existência de resistência. O índice MAR para os isolados de aeromonas variou de 0,12 a 0,62, dos quais 95% (38/40) apresentaram índices superiores a 0,22, caracterizando multiresistência. A maioria das espécies de víbrios isoladas não é considerada patogênica para os peixes (ambientais), mas ainda assim podem representar risco para a saúde das tilápias por questão de oportunismo e do consumidor, principalmente se consumido cru. As aeromonas móveis isoladas e identificadas neste estudo são comumente consideradas como ambientais, porém muitos dos peixes analisados apresentaram sintomatologia compatível com a enfermidade causada por estes agentes oportunistas.

O colapso traqueal é uma obstrução parcial ou total da traqueia caracterizado pelo achatamento dorsoventral dos anéis cartilaginosos e pela frouxidão da membrana traqueal dorsal. Acomete principalmente cães de raças pequenas, de meia idade a idosos, embora também possa ocorrer em cães jovens. O diagnóstico é feito com base nos sinais clínicos e exames complementares. A traqueobroncoscopia permite avaliar o diâmetro da traqueia e dos segmentos brônquicos, principalmente quando as radiografias e fluoroscopia não forem conclusivas e ainda permite a coleta de amostras para citologia, histopatologia e culturas. O objetivo deste estudo foi correlacionar a infecção traqueal com a inflamação da traqueia em cães com colapso de traqueia. A pesquisa foi realizada no HOVET da FMVZ-USP e no Hospital Veterinário Clinivet em Curitiba. A amostra foi constituída por 28 cães, sendo 12 com colapso de traqueia e 16 hígidos para o grupo controle, que propiciou parâmetros de normalidade em relação ao grupo colapso traqueal. Para a coleta de dados utilizou-se a traqueobroncoscopia, com a qual visualizamos a traqueia e graduamos o colapso, colhemos material para cultura bacteriana e citologia. Após a análise dos resultados foi observado diferença estatística significativa nos cães com inflamação e colapso de traqueia. Não foi observado correlação entre a infecção bacteriana e a inflamação na traqueia. Com um teste de dissimilaridade verificou-se que a população bacteriana da orofaringe foi semelhante a da traqueia nos cães do mesmo grupo. Portanto, concluímos que cães com colapso de traqueia tendem a ter a traqueia inflamada, porém não apresentam infecção bacteriana. A composição das bactérias na traqueia pode ser devido à aspiração do conteúdo da orofaringe. / The Tracheal collapse is a partial or total obstruction of the trachea, featured by dorsoventral flattening of the cartilaginous rings and by the laxity of the dorsal tracheal membrane. It mainly affects small breeds, middle-aged and older dogs, although it can also occur in young dogs. The diagnosis is made based on clinical signs and additional exams. The trachealbronchoscopy allows evaluating the trachea diameter and bronchial segments, especially when radiographic and fluoroscopy is not conclusive and still allows the collection of samples for cytology, histopathology and cultures. The objective of this study was correlating the tracheal infection with the tracheal inflammation in dogs with tracheal collapse. The research was conducted in the HOVET FMVZ-USP and Clinivet Veterinary Hospital in Curitiba. The sample consisted of 28 dogs, including 12 with collapsing trachea and 16 healthy subjects in the control group, which allowed normal parameters in relation to the group tracheal collapse. For data collection was used the trachealbronchoscopy, in which was visualized the trachea and the grade of the tracheal collapse was recorded. We also collected samples for cytology and bacterial culture. After analyzing the results we found statistically significant difference in dogs with tracheal collapse and inflammation of the trachea. There was no correlation between bacterial infection and inflammation in the trachea. With dissimilarity test was observed that the bacterial population of the pharynx was similar to the trachea in dogs of the same group. n this study, therefore, concluded that dogs with collapsing trachea tend to have the inflamed trachea, but it does not have bacterial infection. The composition of the bacteria in the trachea may be due to aspiration of pharynx\'s contents.

Les nourrissons âgés de moins de trois mois ont la particularité d’être à relativement haut risque d’infections bactériennes sévères (IBS), majoritairement représentées par les infections urinaires et en particulier celles plus invasives (IBI) que sont les méningites et les bactériémies. On ne dispose actuellement pas d’outil suffisamment fiable et d’un rapport coût-bénéfice bien évalué pour différencier les nourrissons fébriles porteurs d’une affection virale banale de ceux porteurs d’une infection bactérienne.Le travail doctoral avait pour objectifs d’évaluer l’épidémiologie et les pratiques de prise en charge françaises des infections bactériennes de l’enfant fébrile âgé de moins de trois mois admis aux urgences pédiatriques ainsi que des outils diagnostiques, tels que la bandelette urinaire et la procalcitonine dans cette population. Plus de 2000 nourrissons ont été inclus dans une étude prospective observationnelle multicentrique (PRONOUR) sur une période de trente mois d’octobre 2008 à Mars 2011.Nous avons dans un premier temps décrit les modalités de prise en charge de ces jeunes nourrissons fébriles et montré que les pratiques étaient hétérogènes entre les centres participants et variaient par rapport aux recommandations existantes.Nous avons dans un second temps, étudié les pratiques de dépistage des infections urinaires, IBS la plus fréquente dans cette tranche d’âge, et en particulier les performances de la bandelette urinaire. La majorité des urines étaient prélevées par poche collectrice et la bandelette urinaire avait une sensibilité pour la détection d’infection urinaire comparable à celle de l’analyse par microscopie avec cette méthode de recueil.Puis, nous avons réévalué les performances des algorithmes décisionnels existants pour la détection des enfants à faible risque d’IBS dans une nouvelle population (PRONOUR). Nous avons montré qu’ils avaient une valeur prédictive négative satisfaisante comme précédemment décrit, mais une faible valeur prédictive positive pour la distinction des enfants porteurs ou non d’une IBS.Enfin, les performances de la procalcitonine (PCT) dans la détection des IBS et IBI ont été calculées et comparées avec celles d’autres marqueurs inflammatoires usuels. La capacité discriminative de la PCT était excellente pour le diagnostique d’IBI et meilleure que celles des autres marqueurs inflammatoires. Pour la détection d’une IBS, la PCT avait des performances similaires à celles de la C-réactive protein.La prise en charge des nourrissons fébriles âgés de moins de trois mois est hétérogène et pourrait être améliorée par de nouveaux outils prédictifs La prise en charge des nourrissons fébriles âgés de moins de trois mois est hétérogène et pourrait être améliorée par de nouveaux outils prédictifs tels que l’utilisation de la procalcitonine et de la bandelette urinaire dans cette tranche d’âge. / The prevalence of severe bacterial infections (SBI),mainly represented by urinary tract infections is relatively high in infants less than three months of age and particularly those more invasive (IBI) that are meningitis and bacteremia. Current strategies to distinguish young infants with SBIs from those with viral infections are not absolutely reliable and their cost-effectiveness and the associated iatrogenic morbidity have not been extensively evaluated.The purposes of the study were to characterize the spectrum of disease, clinical outcomes and management of febrile infants aged three months or younger admitted to pediatric emergency department in France and to evaluate the performances of diagnostics tests that are urinary dipstick test and procalcitonin assay in this population. A prospective multicenter cohort study was conducted in 15 French pediatric emergency departments over a period of 30 months between October 2008 and March 2011(PRONOUR). More than 2000 infants were enrolled.First, we have described the management of these young febrile infants. We have showed that practices were heterogeneous between the participating centers and varied from the current guidelines.We have analyzed screening strategies of urinary tract infection, the most common SBI in this age group, and in particular we aimed to assess the test performances of urine dipstick test. Most of urine specimens were collected by bag. Dipstick tests on bag urine samples detected urinary tract infections in infants aged 7 to 92 days similarly to microscopy.Then, we have re-evaluated the performances of current strategies for identifying infants at low risk for SBI in a new population (PRONOUR). We have showed that current protocols maintained their good previously reported negative predictive values but have low positive predictive values to detect young infants with SBIs.Finally, the performances parameters of procalcitonine (PCT) for detecting SBI and IBI in this population were calculated and compared with usual biomarkers. Procalcitonin has better diagnostic accuracy than CRP for detecting IBI. The two tests perform similarly for identifying SBI in febrile infants aged 7 to 91 days.The management of febrile infants less than three months of age varied between centers should be improved by new predictive tests. The performance of PCT testing should encourage the development of decision-making rules incorporating PCT and urinary dipstick test.

The aim of this study was to assess the prevalence and predictive factors of secondary infectionsin patients with coronavirus disease 2019 (COVID-19) and compare with influenza. A retrospective cohort study which included COVID-19 and influenza patients with samples processed at Karolinska University Hospital Laboratory between 1st March 2020 to 1st January2021 and 1st January 2015 to 1st January 2021 respectively. Blood, urine and respiratory culture results from 7 days before and 7 days after the primary diagnosis collected from laboratory information system. Chi-square and Mann-Whitney U test used for descriptive comparison. Predictive factors of secondary infections analyzed using logistic regression. Data includes 16,354 patients:7470 COVID-19 and 8884 influenza. Secondary infections were significantly fewer in COVID-19 patients (26.6%) compared to influenza patients (30%) p<0.01. Lower proportion of episodes with growth (EWG) in blood culture of COVID-19 patients (1.8%) compared to influenza (2.9%) p<0.001. Lower proportion of EWG in respiratory tract cultures of COVID-19 patients (11.1%) compared to influenza patients (24.5%) p<0.001. Higher proportionof EWG in urinary tract cultures of COVID-19 patients (24.5%) compared to influenza (20.1%)p<0.001. Staphylococcus aureus were common bacteria in blood and respiratory tract in both cohorts. Escherichia coli were the most common bacteria in urine in both cohorts. Fungi were least common with unspecified yeast being the most frequent. Likelihood of secondary infection lower in males compared to females AOR 0.70 (95%CI 0.66-0.76)), lower in other clinicalsettings AOR 0.65 (95%CI 0.56-0.76) and increased with age in both COVID-19 and influenza patients (AOR 1.03(95%CI 1.02-1.04)). Higher probability of secondary infections in young influenza patients compared to young COVID-19 patients. A lower prevalence of secondary infections in blood, respiratory tract cultures of COVID-19 patients compared to influenza. Olderage, female sex, intensive care were predictive factors for secondary infections. Findings important for planning of treatment protocols.

Les enzymes de la famille Mur (MurA-MurG) sont essentielles pour la survie bactérienne, car elles catalysent les étapes cytoplasmiques de la biosynthèse du peptidoglycane, la principale composante de la paroi cellulaire. En outre, les Murs métabolisent des molécules qui sont absentes chez les eucaryotes, et ces enzymes sont structurellement et biochimiquement tractables. Cependant, malgré le fait que nombreux inhibiteurs anti-Mur ont été développés, un nombre tres réduit de ces molécules ont montré une activité antibactérienne prometteuse, ce qui a incité l'hypothèse selon laquelle, dans le cytoplasme bactérien, les enzymes Mur peuvent exister dans un complexe où les sites actifs sont à proximité, bloquant donc l'accès de petites molécules venant de l'extérieur. Cette hypothèse est soutenue par l'observation selon laquelle, dans de nombreux organismes, les gènes codant pour les enzymes Mur sont présents dans un seul opéron, souvent dans le même ordre; en outre, souvent des paires de gènes sont fusionnées pour générer un seul polypeptide, préconisant la possibilité que des complexes entre ces enzymes pourraient être formés dès qu'ils sont synthétisés. Nous avons obtenu les premières informations structurales et fonctionnelles sur la forme fusionnée MurE-MurF, présente dans le pathogène humain Bordetella pertussis, et nous avons montré qu'elle interagit avec la glycosyltransférase périphérique MurG, ce qui suggère la présence d'un complexe enzymatique ternaire. De façon intéressante, nous avons constaté que MurG de B. pertussis est capable de s'associer avec elle-même et de former différentes espèces oligomériques. Cette découverte pourrait renforcer le rôle de MurG en tant que protéine agissant comme une plateform capable d'ancrer d'autres enzymes Mur à la face interne de la membrane cytoplasmique bactérienne. Nos resultats pourront également être explorés pour comprendre le rôle potentiel de MurG en tant que régulateur de l'activité des enzymes de synthèse du PG. Ces résultats passionnants ouvriront le chemin vers la compréhension du mecanisme d’interaction des enzymes Mur dans le cytoplasme bactérien et pourraient permettre l'emploi éventuel des Murs comme cibles de facto pour développer de nouveaux antibiotiques. / Enzymes of the Mur family (MurA-MurG) are essential for bacteria, since they catalyse the cytoplasmic steps of peptidoglycan biosynthesis, the major component of bacterial cell wall; they metabolize molecules that do not exist in eukaryotes, and are structurally and biochemically tractable. However, despite the fact that many anti-Mur inhibitors have been developed, few of these molecules have shown promising antibacterial activity, which has prompted the hypothesis that within the bacterial cytoplasm Mur enzymes may exist in a complex where the active sites are in closed proximity, blocking small molecule access from the outside. This suggestion is supported by the observation that in many organisms, genes encoding Mur enzymes are present in a single operon, often in the same order, and often pairs of genes are fused to generate a single polypeptide, advocating the possibility that complexes between these enzymes could be formed as soon as they are synthesized. We have obtained the first structural and functional information on the MurE-MurF fused form, present in the human pathogen Bordetella pertussis, and shown that it interacts with the peripheral glycosyltransferase MurG, suggesting the presence of a ternary enzymatic complex. Interestingly, we have found that B. pertussis MurG is able to self-associate and form different oligomeric species. This finding could strengthen the hypothesis of MurG as a scaffold protein capable of anchoring other Murs to the inner face of bacterial inner membrane, but could be also further explored to understand its potential role as a regulator of the activity of PG synthesis enzymes. These exciting results will open the path towards the understanding of how Mur enzymes interact within the bacterial cytoplasm, and could permit the eventual employment of Mur enzymes as de facto targets for novel antibiotic development.

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