Les recherches menées sur les cellules dendritiques tolérogènes au cours des 20 dernières années ont abouti à leur application thérapeutique dans plusieurs essais cliniques. Parmi ces essais, notre équipe est pionnière en transplantation rénale en réalisant de la thérapie cellulaire à l’aide des cellules dendritiques tolérogènes autologues générées avec une faible dose de GM-CSF (ATDC). Nos études précliniques ont démontré que ces cellules sont capables d’augmenter la survie de différentes allogreffes chez les rongeurs et n'induisent pas d'effets indésirables chez les primates. Lors de ma thèse, j’ai montré que les ATDC humaines présentent une faible expression de molécules co-stimulatrices, ne maturent pas et inhibent la prolifération des cellules T. De plus, les ATDC présentent un phénotype, un profil transcriptomique et un métabolisme particuliers qui les dissocient de d’autres cellules myéloïdes. Afin de déterminer les mécanismes suppressifs de ces cellules, j’ai réalisé différents tests démontrant que les ATDC sont capables d’inhiber la prolifération des cellules T CD4+, d’altérer la production d'IFN et IL- 17A et d’induire la différenciation CD4+CD25+FoxP3hiTreg par des mécanismes indépendants du contact cellulaire. L'analyse du surnageant des ATDC (ATDC-SN) a révélé que ces cellules produisent une forte concentration de lactate,qui est en partie responsable de leur effet immunosuppresseur. Cette étude a permis de démontrer que la sécrétion d’acide lactique est un nouveau mécanisme des ATDCs et ouvre une nouvelle perspective de thérapie cellulaire associée à la production de petites molécules.. / The research focused on tolerogenic dendritic cells during the last 20 years has culminated on their therapeutic application in several clinical trials. Among these trials, our team is currently conducting the first trial in the context of kidney transplantation using tolerogenic dendritic cells generated with low dose of GM-CSF (ATDC). We previously reported that Tol-BMDC generated with this protocol prolong thesurvival of different allografts in rodent models and do not induce adverse effects in nonhuman primates. In this work I demonstrated that ATDC derived from human monocytes, similarly to their bone marrow equivalent, display a low expression of costimulatory molecules, do not maturate and impair T-cell proliferation. Interestingly, ATDC display a particular phenotype, transcriptomic profile and metabolism comparing to other myeloid cells. In order to determine the suppressive mechanisms of these cells, I performed different assays demonstrating that ATDC impaired CD4+T-cells proliferation and IFN and IL-17A production, and induced de novo CD4+CD25+FoxP3hiTreg by contactindependent mechanisms. The analysis of ATDC supernatant (ATDC-SN) revealed a high concentration of lactate. I demonstrated that this lactate production is in part responsible of ATDC immunosuppressive effects. This study allowed to demonstrate that lactic acid secretion is a novel mechanisms displayed by ATDC and opens a new perspective of cell therapy based on the production of small molecules.

No description available.

No description available.

Protein-protein interactions (PPIs) across the cell envelope of Gram-negative bacteria are critical for mediating signal transduction pathways that underpin cellular homeostasis. The Ton and Tol Pal systems are two conserved, ancestrally related protein networks that are also required for bacterial pathogenesis. Both Ton and Tol-Pal traverse the periplasm to effect different functions at the outer membrane (OM). Tol-Pal is composed of a homologous complex of three inner membrane proteins, TolQ-TolR-TolA (linked to proton motive force) and two additional periplasmic proteins TolB and Pal. The physiological role of the Tol-Pal system is to stabilise the OM, however the mechanism involved is unknown. TolA is however known to form a crucial protein-protein interaction via its C-terminus with the disordered N-terminus of TolB. Prior to this thesis, determination of the molecular features underlying a protein-protein complex between TolA and an endogenous binding partner TolB had never been accomplished. In this work, I describe the first structure comprising the TolA-TolB complex from Gram negative bacteria. The structure of this complex was determined from Pseudomonas aeruginosa by solution NMR spectroscopy. I determined the interaction between P. aeruginosa TolA and a TolB N terminal peptide to be relatively weak using fluorescence anisotropy. I found that TolB interacts with TolA through an analogous mechanism to that seen in TonB-dependent transporters. Based on these studies and bioinformatics analyses, I hypothesize that the evolutionary resilience of the Tol-Pal system to external pressures is contingent on the preservation of the TolA-TolB interface. Structure-based mutations within the TolA-TolB complex were also evaluated for their effect on in vivo function of the Tol-Pal complex and impact on complex formation in vitro. Taken together, the results demonstrate that protein networks which transduce energy to the OM through PMF-dependent systems in bacterial cells appear to follow a common β-strand augmentation mechanism.

Body fluid identification is important in the field of forensic science as it can provide valuable information to an investigation. An accurate method for detecting blood at a crime scene or on evidence is beneficial to an analyst or investigator. A piece of evidence may be any house-hold object or material; therefore, a test must be able to accurately detect blood on a variety of substrates. The most common preliminary testing method for blood is based on the peroxidase-like activity of hemoglobin. Tests such as phenolphthalein (Kastle-Meyer), Ortho-Tolidine (O-Tol), and Luminol utilize this method. The sensitivity of presumptive blood tests was evaluated using a series of diluted bloodstains on six fabrics: fleece, felt, linen, denim, flannel, and terrycloth. In addition to a direct testing method, two indirect methods were tested utilizing a piece of dry filter paper or a moistened cotton swab. The last portion of this study compared commercial field kits to the laboratory-prepared reagents. This study yielded overall sensitivities for Kastle-Meyer, O-Tol, and Luminol of 1:1000, 1:5000, and 1:10000, respectively. The direct testing resulted in a slightly lower sensitivity with fleece versus the other fabrics. Fleece also resulted in slower and weaker reactions compared to thinner fabrics such as denim, linen, and terrycloth. This suggests that highly absorbent fabrics, such as fleece, can have a negative effect on the sensitivity of catalytic color tests such as Kastle-Meyer and O-Tol. The indirect testing methods utilizing a moistened swab or a dry filter paper were less sensitive compared to direct testing methods. The field kits tested in this study mimic the methods of a moistened swab technique, and the results demonstrated that the field kits were about the same sensitivity or less sensitive compared to the indirect testing methods.

No description available.

Orientadores: Anete Pereira de Souza, Ricardo Aparício / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T00:47:17Z (GMT). No. of bitstreams: 1 Santos_CleltonAparecidodos_D.pdf: 17722787 bytes, checksum: bfe46a8c0582a66be16a3f8b35fc9ada (MD5) Previous issue date: 2013 / Resumo: Xylella fastidiosa é uma bactéria responsável por inúmeras doenças de plantas em culturas economicamente importantes ao redor do mundo, incluindo a clorose variegada dos citros. Após a infecção de seu hospedeiro, as células de X. fastidiosa é apta a formarem uma estrutura de biofilme que bloqueia os vasos xilemáticos, levando a uma condição de estresse hídrico na planta hospedeira e desencadeando o desenvolvimento da doença. Tendo como estímulo a relevância econômica da citricultura para o Brasil e, visando reduzir os prejuízos provocados pelos problemas fitossanitários que acometem esta cultura, foi realizado um consórcio de pesquisa com o intuito de se conhecer completamente o genoma da linhagem 9a5c de X. fastidiosa. Inúmeras proteínas associadas com patogenicidade, adaptação e sobrevivência bacteriana foram identificadas, incluindo XfDsbC (proteína disulfeto isomerase), Xf5'-Nt (5'-nucelotidase), XfTolB (proteína de translocação B) e XfPal (lipoproteína associada ao peptidoglicano) que foram caracterizadas neste estudo. Empregando ferramentas de caracterização de proteínas, aspectos funcionais e estruturais destas quatro proteínas alvos foram avaliados. Dentre os resultados destaca-se a imunodetecção de XfDsbC, Xf5'-Nt, XfTolB e XfPal durante as diferentes fases de formação e desenvolvimento do biofilme de X. fastidiosa, que é tido como o principal mecanismo de patogenicidade deste fitopatógeno, confirmando a predição inicial de tais proteínas como associadas à patogenicidade bacteriana. Adicionalmente, resultados funcionais e estruturais revelaram detalhes finos do papel biológico desempenhado por cada uma das proteínas estudadas. Juntos, os resultados apresentados neste trabalho contribuem para o melhor entendimento de patogenicidade bacteriana, especialmente com respeito ao fitopatógeno X. fastidiosa / Abstract: Xylella fastidiosa is a plant pathogen bacterium responsible for numerous economically important crops diseases around the world, including the citrus variegated chlorosis. Following the host infection, the X. fastidiosa cells are able to form a biofilm structure which block the xylem vessels, leading to a hydric stress condition in the host plant and triggers the disease development. Given the economic relevance of citriculture for Brazil and in order to reduce the damage caused by phytosanitary problems that affect the citrus production, a research consortium was established with the aim to elucidate the complete genome sequence of the X. fastidiosa 9a5c strain. Numerous proteins associated with bacterial pathogenicity, adaptation and survival have been identified, including XfDsbC (protein disulfide isomerase), Xf5'-Nt (5'-nucleotidase), XfTolB (protein translocation B) and XfPal (peptidoglycan-associated lipoprotein) which were characterized in this study. Using tools for protein characterization, structural and functional aspects of these four protein targets were evaluated. Among the results, we highlight the immunodetection of XfDsbC, Xf5'-Nt, XfTolB and XfPal during the different stages of X. fastidiosa biofilm formation and development which is considered the primary mechanism of pathogenicity of this pathogen. These findings, confirming the initial prediction that relates such proteins as associated with bacterial pathogenicity. Additionally, structural and functional results revealed accurate details of the biological role played by each protein studied. Taken together, the findings presented in this study contribute to a better understanding of bacterial pathogenesis, especially with regard to the plant pathogen X. fastidiosa / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular

L’objectif prioritaire de ce travail était de provoquer un état de tolérance immunologique à des allogreffes cardiaques et cutanées chez des souris injectées avec des cellules souches hématopoïétiques (CSH) transduites avec le gène de l’interleukine 10. Un deuxième but était d’améliorer la survie des greffons cutanés en utilisant des cellules dendritiques immatures tolérogène. Le foie fœtal de souris contient en moyenne 2% de cellules souches capables de se différencier dans toutes les lignées hématolymphoïdes. De plus, leur relativement faible expression des antigènes du CMH fait d’elles un matériel biologique parfois susceptible de s’adapter à un environnement allogénique. L’IL-10 est une cytokine anti-inflamatoires. Produite par les lymphocytes Th2 principalement, elle inhibe la production de cytokines pro-inflamatoires telle l’IL-2. Elle empêche aussi la fonction de présentation des antigènes des CPA. Les cellules dendritiques (DC) dérivent de CSH, elles jouent un rôle central dans l’immunité et sont capables d’interagir avec les cellules du système immunitaire inné et adaptatif. Elles sont essentielles à la mise en place d’une réponse régulatrice ou tolérogène, ceci en fonction des informations fournies par le microenvironnement cellulaire. Les résultats montrent d’une part que les CSH fœtales, de souris C57 BL/6 transduites avec le gène de l’IL-10 et injectées plusieurs fois à des souris allogéniques (BALB/c), induisent une prolongation de survie du greffon cardiaque de même souche. Cette survie est de 86.25+13.8 jours versus 11.5+0.6 jours pour les groupes contrôles. Les DC tolérogènes (tol-DC) de souris DBA1 traitées avec le TNFα sont injectées à des souris allogéniques (BALB/c). Il en résulte une prolongation de survie du greffon cutané de même souche que les tol-DC : 15 jours vs 7.5 jours pour les contrôles. Seuls les animaux transplantés avec des tol-DC présentent un état de tolérance autorisant la prolongation de la survie de greffonsallogéniques / The main objective of this work was to induce a state of immunological tolerance to cardiac and skin allografts in mice injected with hematopoietic stem cells (HSCs) transduced with the gene for interleukin 10 (IL-10). A second goal was to improve the survival of skin grafts using immature dendritic cells well known for their tolerogenic function. Mouse fetal liver contains 2% of stem cells on average that can differentiate into all blood-lymphoid lineages. In addition, their relatively low antigen expression of major histocompatibility complex (MHC) makes them a biological material sometimes capable to adapt to an allogeneic environment. IL-10 is a cytokine with anti-inflammatory properties. Mainly produced by Th2 lymphocytes cells, IL-10 inhibits the production of pro-inflammatory cytokines such as IL-2. It prevents antigen presenting function of APCs. Dendritic cells (DC) derived from HSCs and play a central role in immunity. They are able to interact with cells of the innate and adaptive immune system. They are essential to the establishment of a regulatory or tolerogenic response, this based on the information provided by the cellular microenvironment. Results firstly show that fetal HSC of C57 BL/6 mice transduced with IL-10 gene and injected several times to allogeneic mice (BALB/c) sublethally irradiated induce a prolongation of heart transplant survival of the same strain. This survival is of 86.25+13.8 days in comparison with 11.5+0.6 days for control groups. Tolerogenic dendritic cells (tol-DC) of DBA1 mice treated with TNFα are injected into allogeneic mice (BALB/c) sublethally irradiated. This results in a prolongation of skin graft survival of same strain as tol-DC: 15 days compared to 7.5 days for the control groups. Only tol-DC transplanted animals have a tolerance state allowing prolonged survival of allogeneic skin grafts

Les colicines sont des toxines protéiques sécrétées par Escherichia coli ou des espèces apparentées. Leur mécanisme d'action se décompose en plusieurs étapes impliquant des domaines structurellement distincts de la toxine : le domaine central interagit avec un récepteur spécifique de membrane externe, le domaine N-terminal est transloqué à travers la membrane externe via un translocateur, puis transite dans le périplasme et le domaine C-terminal porte l’activité létale. L’étape de transit des colicines dites du groupe A implique l'interaction du domaine N-terminal de la colicine avec les protéines du système Tol. Ce système est formé de cinq protéines : TolQ, TolR, TolA, TolB et Pal. Le système TonB, composé des protéines TonB, ExbB et ExbD, est quant à lui parasité par les colicines dites du groupe B. La combinaison de techniques in vivo et in vitro, nous a permis de mettre en évidence pour la première fois l’interaction d’une colicine avec la protéine TolQ. Nous avons également montré que le clivage protéolytique de la protéine TolA, une protéine clé du système Tol, contrôle les interactions séquentielles engagées entre les colicines du groupe A et les composants de leur machinerie d’import chez E. coli. La colicine interagit avec TolB, puis TolA et finalement avec TolR et/ou TolQ. Nous avons également pu attribuer un rôle à la protéase FtsH dans ce mécanisme de dégradation. Parallèlement, nous avons entrepris de caractériser la colicine TonB-dépendante appelée colicine M (ColM), la seule colicine connue à ce jour capable de perturber la synthèse de peptidoglycane et dont l’activité nécessite la présence de la protéine périplasmique FkpA, un chaperon possédant une activité peptidyl-prolyl isomérase. Nous avons proposé une nouvelle approche pour étudier la ColM et délimiter plus précisément ses domaines afin d’identifier la séquence minimale requise pour sa toxicité. Nous avons montré que dans E. coli, la production périplasmique de la ColM (sp-ColM) est toxique et que son activité dépend de FkpA. Le domaine minimal requis pour cette toxicité correspond aux 153 derniers acides aminés C-terminaux deColM. Contrairement à la ColM entière, la toxicité de ce domaine C-terminal dans le périplasme d’ E. coline requiert pas FkpA.L’ensemble des données montrent que les colicines sont capables de parasiter des systèmes bactériens pour pénétrer dans la cellule et aussi de détourner la fonction physiologique de certaines protéines pour atteindre leurs cibles. / Colicins are toxin proteins secreted by Escherichia coli or related bacteria species. The actionmechanism of the colicins can be divided into several steps that involve distinct structural domains: thebinding of its central domain to an outer membrane specific receptor, the translocation of its N-terminaldomain through the outer membrane, the transit of this same domain through the periplasm and the lethalactivity carried by the C-terminal domain. The transit step of the group A colicins requires the interactionof colicin N-terminal domain with the Tol system which is composed of five proteins: TolQ, TolR, TolA,TolB and Pal. The TonB system, composed of TonB, ExbB and ExbD, is parasitized by the group Bcolicins. Using a combination of in vitro and in vivo experiments, we identified for the first time aninteraction between a colicin and the TolQ protein. We have also shown that the proteolytic cleavage ofthe TolA protein, a key protein of the Tol system, controls the sequential interactions of the group Acolicins with the components of their import machinery in E. Coli and we assigned a role to FtsH proteasein this degradation mechanism. We defined that the colicin interacts first with the TolB protein, then withTolA, and finally with TolR and/or TolQ.In parallel, we undertook the characterization of the TonB-dependent colicin M (ColM), the only colicinknown to be able to disrupt the peptidoglycan synthesis and that requires for its toxic activity the presenceof FkpA, a chaperone and peptidyl propyl isomerase protein located in the periplasm. We proposed a newapproach to investigate the in vivo activity of ColM designed to identify the different domains of ColMand the minimal sequence that retains toxic activity. We have shown that in E. coli, the periplasmicproduction of ColM is toxic and that its activity is FkpA dependent. The minimal domain required fortoxicity corresponds to the C-terminal last 153 amino acids of ColM. Unlike the full-length protein, thisdomain produced in the E. coli periplasm does not require FkpA for toxic activity.All these data show that colicins are able to parasitize bacterial systems to enter the cell and also to divertthe physiological function of certain proteins to achieve their targets

Le système Tol-Pal est un complexe de l’enveloppe d’Escherichia coli composé de CINQ protéines. Les protéines ToIQ, ToIR, TolA forment un complexe dans la membrane interne; la lipoprotéine Pal interagit avec le peptidoglycane avec la protéine périplasmique TolB. Ce système est conservé chez la plupart des bactéries à Gram négatif. Il joue un rôle important dans le maintien de la stabilité de l’enveloppe et dans l’étape tardive de la division cellulaire. Une interaction entre TolA et Pal relie les membranes interne et externe, et dépend des protéines ToIQ, TolR et de la force proton motrice (PMF). Les protéines ToIQ-R-A formeraient un moteur moléculaire utilisant la PMF afin de relier membranes interne, externe et le peptidoglycane. Mon travail a consisté à étudier l’organisation des segments transmembranaires (STs) de TolQ et TolR au sein de la membrane interne d’E. coli en utilisant l’approche expérimentale du « cysteine scanning ». Ainsi, nous avons pu identifier les résidus impliqués dans les interactions entre les STs et améliorer la connaissance de l’organisation moléculaire de ce système. Nous avons aussi démontré la dimérisation du ST de TolRet l’importance de la dynamique dans le fonctionnement de ce moteur. Le système Tol est parasité par certaines toxines (comme les colicines) et par des phages filamenteux. Les colicines sont produites par des souches d’Escherichia coli et active contre les entérobactéries. Je me suis aussi intéressé à l’organisation structurale de la protéine d’immunité de la colicine A. La colicine A forme un canal ionique dans la membrane interne pour tuer la bactérie cible. Les cellules produisant la Colicine A synthétisent également une protéine d’immunité (Cai) qui les protègent de l’action de la colicine A. Par une approche combinant « cysteine scanning » classique et un « anti-cysteinescanning », nous avons pu apporter des informations nouvelles sur l’organisation des quatre STs deCai. Nous avons montré que Cai forme un dimère dans la membrane et que ce dimère se dissocie au contact de sa cible, la colicine A. / The Tol-Pal system is a protein complex of the Escherichia coli cell envelope. It consists offive proteins. The ToIQ, TolR, TolA proteins form a complex in the inner membrane, the lipoproteinPal interacts with the peptidoglycan and with the periplasmic protein TolB. This system is conservedin most Gram-negative bacteria. It plays an important role in maintaining the integrity of the outermembrane and in the late stage of cell division. The interaction between Pal and TolA connects innerand outer membranes and depends on ToIQ, TolR and the proton motive force (pmf). The ToIQ-R-Aproteins are suspected to form a molecular motor using pmf to connect the inner and outermembranes and the peptidoglycan. The first aim of my work was to study the organization of thetransmembrane helices (TMHs) of E. coli TolQ and TolR using the cysteine scanning approaches. Weidentified residues involved in the interactions between the TMHs and improved the knowledge ofthe molecular organization of this system. We have also demonstrated the dimerization of the TMHof TolR and the importance of its dynamic movement in the system. The second aim of my work wasto analyze the structural organization of the immunity protein to colicin A. The colicins are producedby certain strains of E. coli and are active against other Enterobacteriaceae. The colicin A forms anion channel in the bacterial inner membrane which kill the bacteria. It hijacks the Tol system to enterin the cell. Cells producing colicin A also synthesize the colicin A immunity protein (Cai) whichprotects the producing cells against the action of colicin A. The approaches combining "cysteinescanning" and "anti-cysteine-scanning”, we found that Cai form a dimer in the membrane whichdissociates upon contact with its target, the colicin A