La découverte d'un contexte génétique chez les streptocoques – codant un petit peptide hydrophobe (SHP) et un régulateur transcriptionnel appartenant à la famille Rgg –, suivi de l'étude d'un de ces loci chez S. thermophilus LMD-9, a conduit à l'hypothèse que les protéines régulatrices Rgg en association avec une phéromone putative SHP pourraient intervenir dans un mécanisme de type quorum-sensing (QS) chez les bactéries à Gram positif. La première partie de ma thèse a consisté à confirmer cette hypothèse sur le locus shp/rgg1358 de S. thermophilus LMD-9, espèce contenant le plus grand nombre de systèmes SHP/Rgg dans son génome. Pour ceci, les étapes impliquées dans un mécanisme de QS ont été étudiées : la sécrétion, la maturation et la détection à une concentration seuil de la phéromone, sa réimportation à l'intérieur de la cellule, son interaction avec un régulateur transcriptionnel et enfin l'interaction de la protéine régulatrice à l'ADN. Par l'utilisation d'approches génétiques et biochimiques, nous avons démontré l'existence d'un nouveau mécanisme de QS impliquant pour la première fois un régulateur transcriptionnel Rgg et une phéromone SHP, importée à l'intérieur de la cellule par le transporteur d'oligopeptides AmiCDEF. Le rôle de la protéase membranaire, Eep, a également été démontré dans la maturation de la phéromone, dont la forme mature a été déterminée par spectrométrie de masse et validée in vivo. Dans un second temps, nous avons exploré la fonctionnalité de ce nouveau mécanisme sur d'autres loci shp/rgg, dans le but d’étudier l'existence d’éventuels phénomènes de cross-talk entre les bactéries. L'étude de nouveaux loci, en système hétérologue chez S. thermophilus LMD-9, a permis d'étendre la fonctionnalité du mécanisme à deux systèmes SHP/Rgg de streptocoques pathogènes, à savoir S. agalactiae et S. mutans. En parallèle à ce travail de caractérisation, l'identification des régulons des systèmes SHP/Rgg a été entreprise. La construction d'un arbre phylogénétique des protéines Rgg-like a permis d'identifier 68 systèmes SHP/Rgg, que nous avons classés en trois groupes. L'analyse des régions promotrices des gènes shp a conduit à l'identification d'un site putatif de liaison des protéines Rgg à l'ADN spécifiques de chaque groupe SHP/Rgg. Une approche in silico a ensuite été menée afin de rechercher, dans les génomes séquencés de streptocoques, les gènes cibles putatifs. Alors que des cibles proximales ont été détectées pour les groupes II et III, des cibles distales ont été identifiées dans les groupes I et II. Actuellement, la validation de certaines cibles est en cours au laboratoire. A l'avenir, ce travail pourrait permettre le développement de petits peptides permettant d'optimiser l'utilisation de S. thermophilus en industries laitières et de réduire la virulence des streptocoques pathogènes. / The discovery of a genetic context – encoding a small hydrophobic peptide (SHP) and a transcriptional regulator belonging to the Rgg family (in nearly all streptococcal genomes) –, following by the study of one of this loci in S. thermophilus LMD-9, led to the hypothesis that the regulatory proteins Rgg in association with a putative pheromone SHP could define a novel quorum-sensing (QS) regulatory mechanism in Gram-positive bacteria. The first part of my PhD consisted to validate this hypothesis. For this purpose, we analyzed the SHP/Rgg system in all the steps that are commonly involved in QS mechanisms: (i) secretion of the putative pheromone, (ii) maturation of the pheromone, (iii) capture of the pheromone from the external environment at a threshold concentration, (iv) importation of the pheromone inside the cell and (v) interaction of the transcriptional regulator to the promoter regions of targeted genes. Experimentally, we focused on the so-called shp/rgg1358 locus of S. thermophilus LMD-9, which is the streptococcal species containing the largest number of shp/rgg pairs in its genome. By using genetic and biochemistry approaches, we uncovered a new QS mechanism that involves the pheromone SHP, the oligopeptide transporter AmiCDEF for the uptake of the pheromone and the transcriptional regulator Rgg for the control of target gene expression. Furthermore, we showed that the membrane protease Eep participates in the production of the mature pheromone, which has been identified by mass spectrometry. Once characterized, the second part of my PhD was to explore the functionality of this new QS system in other streptococcal strain or species, in order to determine if cross-reactivity phenomenon between streptococci can occur. By using heterologous expression in S. thermophilus LMD-9, we extended the functionality of the SHP/Rgg system to two pathogenic streptococcal species, i.e. S. agalactiae and S. mutans. The last part of my PhD consisted in identifying the regulon of all SHP/Rgg systems. Following the construction of a phylogenetic tree of the Rgg-like proteins in low GC Gram-positive bacteria, we identified 68 SHP/Rgg systems that we classified in three groups. Analyzing the promoter regions of all shp genes led to the identification of a putative Rgg DNA binding site specific to each SHP/Rgg group. An in silico approach was used to scan all sequenced streptococcal genomes for the three identified patterns. Whereas proximal target genes were detected for groups II and III, distal target genes were found in groups I and II. In addition, we uncovered that putative Rgg DNA binding sites can be localized in coding or non-coding region. Currently, validations are in progress. To sum-up, my PhD studies provided evidences that the Rgg proteins in association with small peptide pheromones define a new QS mechanism that seems to regulate the expression of distal and proximal genes in a species-dependent manner. Important insights should be obtained concerning a putative crosstalk among streptococci that involves the SHP/Rgg QS system. My studies may constitute a basis for the development of small peptides to optimize the use of S. thermophilus in dairy factories and reduce the virulence of pathogenic streptococci.

Au cours de chimiothérapies, les cellules cancéreuses parviennent fréquemment à échapper aux effets toxiques des médicaments en développant des mécanismes de chimiorésistance qui résultent souvent de la présence d’un système d’efflux de ces médicaments. Cette chimiorésistance est corrélée à un phénomène appelé « phénotype MDR » pour (MultiDrug Resistance) et associé à la surexpression d’ATPases membranaires appartenant aux transporteurs ABC (ATP Binding Cassette). Le transporteur ABCG2 fait partie de cette grande famille de protéines. Un alignement de séquence a permis l’identification chez ABCG2 une séquence spécifique (LSGGE) très semblable à la séquence signature (VSGGE) de tous les transporteurs ABC. La mutation ponctuelle des résidus de cette séquence en alanine a produit une perte importante de fonction des mutants L352A et S353A, observée au niveau du transport et de l’activité ATPasique. Des relations structure-activité établies à partir de différents composés de la famille des flavonoïdes ont permis d’identifier MBLI 97, boéravinone G, MHT et ABI comme des composés puissants et spécifiques, capables d’abolir la résistance à de multiples drogues et chimiosensibiliser la croissance cellulaire. Le ciblage de séquences spécifiques et l'utilisation d'inhibiteurs spécifiques de ces transporteurs constituent des stratégies destinées à contrer la chimiorésistance et augmenter l’efficacité des traitements chimiothérapeutiques. / During chemotherapy, cancer cells frequently succeed to escape the toxic effects of drugs by developing mechanisms of chemoresistance which often result from the presence of an efflux system of these drugs. Such a chemoresistance is correlated to the MDR (MultiDrug Resistance) phenotype and associated to overexpression of membrane ATPases belonging to the ABC (ATP-Binding Cassette) transporters. The ABCG2 transporter belongs to this large family of proteins. Sequence alignment allowed the identification of a specific (LSGGE) sequence in ABCG2, which is quite similar to the canonical sequence signature (VSGGE) of all ABC transporters. Point mutation of these residues into alanine produced a loss of function in L352A and S353A mutants, as observed in transport and on ATPase activity. Structure-activity relationships drawn from some compounds among the family of flavonoids allowed the identification of MBLI 97, boeravinone G, MHT and ABI as potent and ABCG2-specific inhibitors, able to revert multidrug resistance and chemosensitize cell growth. The study of specific sequences and use of specific inhibitors of these transporters constitute strategies to abolish cancer cell chemoresistance and to increase the efficiency of chemotherapeutic treatments.

This study aims to explore effectiveness within intermodal transportation of semi-trailers based on the variables cost and time. Intermodal transport can be explained as a transportation of a carrier, from the origin to the final destination, using two or more modes of transport (Crainic & Kim, 2007 p.467). This study focuses on the two modes of transport rail and road. The rail transportation has big advantages towards other modes of transport since it has less impact on the environment, given that the energy is produced in Sweden (Trafikverket 2017) but there are more benefits to be utilized. The rail transportation is generally more profitable on distances greater than 500 km, thus it can transport high volumes of goods over long distances for a low cost. The fixed costs that´s added in the terminals and the rail transports low degree of flexibility compared to the road transport is the primary reasons why rail transportation isn´t profitable on shorter distances. This study has made a market research to see where the final destinations for the imported semi-trailers are located in Sweden and Norway. This, to see where there are potential to develop and create new rail shuttles from Port of Gothenburg to dry ports across the Swedish and Norwegian inland for transportation of semi-trailers. Interviews have been conducted with carriers to create a more correct view of the current situation. Study results show that the greatest volumes of the transported semi-trailers ends up in the areas around Helsingborg and Stockholm, with the region around Gothenburg excluded. Dry ports positioned in this area show the best potential for development of new rail shuttles. The results from conducted interviews with carriers express that time is the most important aspect for their operation and their clients, that is also the reason why they don´t use the railway for transportation of semi-trailers. Carriers also consider the trains to be unreliable and the railway operation in Port of Gothenburg has so far not been operating well enough for them. The result show that 38 percent of the imported semi-trailers have their final destination in the area around Gothenburg and naturally it´s not possible for the railway to compete with the road transport at such a short distance. In order for the railway to compete with the road transport, rail shuttles to regions with high flows of semi-trailers must be developed to not loose in time and flexibility. Thus, the railway transportation has other opportunities than just transportation. In dry ports there are opportunities for storage of goods, which is generally cheaper than in the port and simultaneously create time gains in the final transportation, thus the goods are closer to the customer. Port of Gothenburg can compete with other ports, which are geographically closer to a certain customer, through more rail shuttles with more frequent departures and then take more market shares within Nordic transportation. / Studien syftar till att undersöka transporteffektiviteten för intermodala transporter av semitrailrar med utgångspunkt från variablerna kostnad och tid. Intermodala transporter kan förklaras som en transport av en lastbärare från dess ursprung till dess slutdestination med hjälp av två eller flera olika transportsätt (Crainic & Kim, 2007 s.467) och denna studie fokuserar på de två transportsätten järnväg och väg. Just järnvägstransporten har en stor fördel gentemot andra transportsätt då det är ett mer miljövänligt alternativ, förutsatt att det är eldrivna tåg samt att elen är producerad i Sverige (Trafikverket 2017) men det finns även fler fördelar som kan utnyttjas. Järnvägstransporten är generellt lönsammare på avstånd längre än 500 km, då det kan transportera stora volymer över långa avstånd till en låg kostnad. Varför det inte är lönsamt på kortare sträckor beror framför allt på de fasta avgifter som tillkommer vid terminalerna och järnvägens relativt låga flexibilitet gentemot vägtransporten. Studien har gjort en marknadsundersökning för att se var de importerade semitrailrarna har sin slutdestination inom Sverige och Norge. Det för att se var det finns potential för att utveckla och skapa nya järnvägspendlar från Göteborgs Hamn till torrhamnar för transport av semitrailrar via järnväg. Intervjuer har utförts med speditörer i anslutning till hamnen för att skapa en så bra bild som möjligt av nuläget. Resultatet visar att de största volymerna i flödet av semitrailrar går till Helsingborg och Stockholm med omnejd, med Göteborg exkluderat. Det är de två områdena som visar potential för att eventuellt skapa järnvägspendlar till torrhamnar. Vid intervjuerna som utförts uttrycker speditörerna att tid är den viktigaste aspekten för deras verksamhet och kunder. Det är även anledningen till att de inte använder sig av järnvägstransport för semitrailrar, då tågen är för opålitliga och verksamheten för järnvägstransport kring Göteborgs Hamn inte har fungerat för dem hitintills. Resultatet visar även att drygt 38 procent av de importerade semitrailrarna har sin slutdestination i Göteborgsområdet och där kan naturligtvis inte järnvägstransporten konkurrera med vägtransporten på grund av det korta avståndet. För att järnvägstransporten ska kunna konkurrera med vägtransporten måste det skapas pendlar till de regionerna med större flöden för att inte förlora tid och flexibilitet gentemot vägtransporten. Dock så ger järnvägstransporten andra möjligheter än bara transport av gods. I torrhamnarna finns möjligheter för lagring av gods, som generellt är billigare än lagring i hamnen och ger samtidigt tidsvinster när den slutliga transporten till kund ska utföras, då godset är närmare kunden. Göteborgs Hamn kan konkurrera med andra hamnar som geografisk ligger närmare en viss kund, genom att fler järnvägspendlar med frekventare avgångar kan skapas och på så sätt ta marknadsandelar inom nordisk transport.

Idag transporteras kylda livsmedel, i Sverige, inom ett temperaturintervall på +2 °C - +8 °C. I vilken temperatur som animaliska livsmedel ska hanteras i regleras i lagstiftningen och varierar mellan +2 °C - +7 °C. Många kylskåp, både hos butik och konsument, är inställda på+8 °C. Till följd av detta är den stora majoriteten livsmedel, på förpackningen, markerade med en förvaringstemperatur på +8 °C. På senare år har diskussioner förts angående en sänkt temperatur i kylkedjan. Anledningen till detta är att överkomma matsvinnet. Forskning pekar på att ett livsmedels hållbarhet är starkt beroende av dess förvaringstemperatur. Hastigheten på mikrobiell tillväxt sjunker nämligen med lägre temperaturer. År 2020 träder en ny förordning om fluorerade växthusgaser (fgasförordningen)i kraft som förbjuder installation och påfyllning av kommersiell utrustning innehållande köldmedier med högre Global Warming Potential-faktor än 2500. Detta innebär att butiksledet i stor utsträckning kommer tvingas byta ut sina äldre kylsystem. Med nya kylsystem kan en lägre temperatur garanteras. För att vara väl förberedda ställer sig transportbranschen frågan vilka konsekvenser en sänkttemperatur kan tänkas föra med sig i deras del av kedjan. Problematiken grundar sig i att en del livsmedel kan ta skada av temperaturer nära fryspunkten. För att skydda dessa livsmedelkan transportören använda sig av produktskydd på kylkänsliga livsmedel. En annan beprövad metod är att dela in livsmedlen i flera temperaturzoner. Arbetets teoretiska bidrag är att det sammanställer tidigare forskning på områden inom livsmedelsegenskaper vid nedkylning, multitemperatursystem och produktskydd samt semistrukturerade intervjuer med experter inom sina respektive områden. Vidare undersöksäven kylkedjans uppbyggnad och dess aktörer. Perspektivet i rapporten utgår från ett av de större transportföretagen inom tempererad logistik i Sverige. Arbetets praktiska bidrag är baserat på en fallstudie som tillsammans med det teoretiska bidraget ligger till grund för en redogörelse över tänkbara konsekvenser. Analysen visar på att en del livsmedel, för att inte komma till skada och riskera kassation, måste skyddas. Antingen genom flera temperaturzoner eller genom produktskydd. Bådaalternativen kommer med för- och nackdelar. Att addera flera temperaturzoner för med sig investeringar i form av mellanväggar och kylsystem samt komplicerar livsmedelshanteringen. Flera temperaturzoner kan emellertid säkra ett livsmedels kvalitet. För produktskydden existerar det inget returlogistiksystem och ett system kan bli mycket kostnadsdrivande att upprätthålla. Slutsatsen är att en kombination av de båda lösningarna är att föredra men att branschöverskridande samarbete krävs för att samtliga parter ska gynnas. / Chilled foodstuffs are today transported within a temperature interval of +2 °C - +8 °C in Sweden. The correct temperatures in which foodstuffs with animal origin should be handled in are regulated by law and diverges between +2 °C - +7 °C. Many refrigeration systems in grocery stores and consumer’s homes are set at +8 °C. The great majority of foodstuffs are, as a result of this, on the packaging marked with a storage temperature of +8 °C. To be well prepared, the transport industry ask themselves which consequences might occur in case of a reduced temperature in the cold chain. The main reason for this is to overcome food waste. Research shows that foodstuffs durability is highly dependent on its storage temperature. The rate in which microbial growth occur drops with falling temperatures. In2020 a new EU regulation enters in to force which regulates the use of refrigerants with higher Global Warming Potential factor than 2500. The grocery stores have to, as a result of this, replace many of their older refrigeration systems. Regulating lower temperatures won’t be an issue with new modern equipment. As a result of this they can guarantee lower temperatures on foodstuff. To be prepared for what might come, the transport industry wants to know what consequences a lower temperature might bring along in their part of the cold chain. The main issue is that some foodstuff might be harmed in temperatures close to the freezing point. The use of protective coatings could offer shelter for cold sensitive products. One other proven method is to allocate foodstuff in different temperature zones. This thesis theoretical contribution compiles earlier research in areas such as foodstuffs characteristics when refrigerated, multi temperature systems, protective coatings and semi structured interviews with experts within their fields. It further examines the cold chain and the stakeholders within it. The focus of the thesis is thru the perspective of one of the major transport companies within cold chain logistics in Sweden. The practical contribution is based on a case-study together with the theoretical contribution which states consequences that possibly can occur along the transport company’s part of the cold chain. The analysis shows that, to minimize the risk of cassation, some foodstuffs need protection. Either thru multiple temperature zones or with protective covers. Both alternatives comes with advantages and disadvantages. To add temperature zones are associated with investments in the form of separable walls and cooling systems together with an increase in logistic complexity. Multi temperature systems can however secure the quality in a foodstuffs quality. The issue with protective covers is that there’s no return logistics system in place and such a system can be very expensive to uphold. The covers do however offer protection from outer temperature influence. The conclusions states that a combination of both solutions might be suitable. To accomplish this however requires collaboration over company and industry borders if all stakeholders are to be beneficial.

La schizophrénie est une pathologie sévère et fréquente. Elle constitue un problème majeur de santé publique. Les traitements disponibles présentent des problèmes de tolérance non négligeables et leur efficacité reste modérée. La recherche en pharmacogénétique des antipsychotiques a pour objectif d’aider les prescripteurs à choisir les traitements de façon plus rationnelle. Les carences méthodologiques des premières études réalisées expliquent sans doute le peu de résultat répliqué à ce jour. Dans une cohorte de patients schizophrènes caucasiens traités par olanzapine ou rispéridone et évaluée prospectivement pour l’efficacité et la tolérance du traitement, nous avons d’abord recherché des critères cliniques permettant de prédire la réponse au traitement. L’age précoce de début des troubles et la durée de la maladie sont des prédicteurs individuels de la mauvaise réponse au traitement. Nous avons également étudié l’implication de variants génétiques du transporteur de la noradrénaline dans l’efficacité des traitements. Nous avons observé l’implication de deux polymorphismes dans la décroissance des symptômes positifs sous traitement. L’analyse de l’impact du variant C825T de la GNB3 dans la prise de poids sous antipsychotique n’a pas retrouvé d’association significative. Enfin, nous avons étudié l’ensemble des variants alléliques du cytochrome P450 2D6 dans cette cohorte de patients schizophrènes comparée à des témoins. L’allèle CYP2D6*2 était associé à un effet protecteur vis à vis de la schizophrénie. Les associations retrouvées devraient aider à mieux comprendre les mécanismes physiopathologiques impliqués dans la schizophrénie et la réponse au traitement / Schizophrenia is a frequent and severe disease. It constitutes a major public health problem. All the available treatments however, have significant adverse side-effects and their efficacy remains moderate. The aim of pharmacogenetic research is to help practitioners to choose treatments in a more rational way. The methodological limits of the first published studies probably explain the lack of replication of such studies. In a prospective study of a sample of Caucasian schizophrenic patients treated with olanzapine or risperidone, clinical criteria were assessed as factors that may predict drug response. Early onset and duration of the disease, individually predicted an unfavourable drug response. We also studied genetic variants of the norepinephrine transporter to see how they may affect antipsychotic drug efficacy. Two polymorphisms were associated with a reduction in positive symptoms in treated schizophrenic patients. No association between the GBN3 C825T variant and weight gain in patients treated by antispychotic drugs was observed. Finally, we genotyped all the cytochrome P450 2D6 allelic variants in the same Caucasian schizophrenic sample and a Caucasian origin control cohort. The CYP2D6*2 allele was strongly associated with protection towards schizophrenia. The two observed associations may help to better understand the still unwell known physiopathological mechanisms implicated in schizophrenia aetiology and antipsychotic drug response

INTRODUÇÃO: A Asma Brônquica é caracterizada por obstrução ao fluxo aéreo, reversível ou não, e processo inflamatório pulmonar, caracterizado principalmente por eosinofilia. A persistência da inflamação pode induzir processo de reparo pulmonar associado à redução progressiva da função pulmonar. A recente descrição do sistema colinérgico anti-inflamatório, um mecanismo neural que suprime a resposta imune inata e controla a inflamação por inibição de citocinas proinflammatórias, e a detecção de alguns de seus componentes em células de vias aéreas sugerem uma importante participação deste sistema na fisiopatologia de doenças pulmonares. O principal mediador deste sistema é a acetilcolina (ACh), que é estocada em vesículas sinápticas pelo transportador vesicular de ACh (VAChT), proteína essencial para sua liberação. OBJETIVOS: Avaliar os efeitos da deficiência colinérgica por redução da VAChT nas alterações pulmonares observadas em modelo experimental de inflamação pulmonar induzida pela exposição crônica a ovoalbumina. METODOLOGIA: A redução colinérgica foi induzida pela modificação genética nos níveis de VAChT. Camundongos machos selvagens e mutantes foram submetidos ao protocolo de sensibilização subcutânea com ovoalbumina ou salina nos dias 0, 7 e 14. Após, foram submetidos a desafios inalatórios com ovalbumina a 1% ou inalações de salina por 20 minutos nos dias 26, 27 e 28. No dia 29, foi realizada a avaliação da mecânica pulmonar, da inflamação no lavado broncoalveolar e no tecido e análise histológica de remodelamento e expressão de MMP-9 e TIMP-1 por imunohistoquímica. Também foi quantificado por ELISA níveis de IL-4, IL-10 e de TNF-a no homogenato pulmonar. A análise estatística considerou um p<0,05 como significativo. RESULTADOS: Animais sensibilizados apresentaram hyperresponsividade brônquica, inflamação e edema peribrônquico e deposição de fibras colágenas e elásticas ao redor das vias aéreas comparado ao grupo salina (p<0,05). Além disso houve aumento de IL-4 no homogenato pulmonar e da expressão de MMP-9 e TIMP-1 nas células inflamatórias. Os animais mutantes, independente de serem ou não sensibilizados, apresentaram aumento de TNF-a no pulmão. Os animais mutantes que foram submetidos ao protocolo de sensibilização mostraram aumento da hiperresponsividade brônquica, do eosinófilos, do edema e da deposição de colágeno comparado aos animais selvagens que também foram sensibilizados com ovoalbumina. Estas alterações podem ser atribuídas ao aumento de IL-4 e de MMP-9/TIMP-1 que foi observado nos animais mutantes e sensibilizada em comparação com o os selvagens sensibilizados. Não houve diferença nos níveis de IL-10 nos grupos experimentais. Conclusão: A deficiência colinérgica piora a hiperresponsividade brônquica, a inflamação eosinofílica e o remodelamento, principalmente por interferir com a citocina pró-inflamatória IL-4 e na proporção de MMP-9 e TIMP-1. Estes dados sugerem que a via colinérgica antiinflamatória está envolvida na fisiopatogenia da asma e necessita ser mais investigada / BACKGROUND: Bronchial asthma is characterized by reversible or not airflow obstruction and pulmonary inflammation, mainly characterized by eosinophilia. The persistence of inflammation can induce lung repair process associated with progressive reduction in lung function. Recent evidence of the cholinergic anti-inflammatory system, a neural mechanism that suppresses the innate immune response and control inflammation by proinflammatory cytokines inhibition, and the detection of some of its components in airway cells suggest an important role of this system in pulmonary physiopathology. The main mediator of this system is acetylcholine (ACh), which is stored in synaptic vesicles by vesicular acetylcholine transporter (VAChT), an essential protein for ACh release. AIMS: To evaluate the effects of cholinergic deficiency by VAChT reduction on pulmonary alterations observed in an experimental model of pulmonary inflammation induced by chronic exposure to ovalbumin. METHODS: The cholinergic deficiency was induced by genetic modification on VAChT levels. Wild-type and mutant male mice were submitted to subcutaneous ovalbumin sensitization or saline protocol on days 0, 7 and 14. After, animals were submitted to inhalation challenge with ovalbumin 1% or saline for 20 minutes on days 26, 27 and 28. On day 29, we evaluated the pulmonary mechanics, inflammation in bronchoalveolar lavage and in airways, histological analysis of airway remodeling and the expression of MMP-9 and TIMP-1 by immunohistochemistry. It was also quantified by the levels of IL-4, IL-10 and TNF-a in lung homogenate. The statistical analysis were performed and a p<0.05 was considered significant. RESULTS: Sensitized animals presented bronchial hyperresponsividade, airway inflammation and edema and collagen and elastic fibers deposition of collagen and elastic fibers around the airways compared to saline group (p <0.05). Furthermore, there was an increase of IL-4 in lung homogenate and the expression of MMP-9 and TIMP-1 in inflammatory cells. The mutant animals, regardless the sensitization, showed an increase in lung content of TNF-a. The mutant and sensitized animals showed an increase in bronchial hyperresponsiveness, in eosinophils, edema and collagen deposition in airways compared to the wild type and sensitized animals. These changes can be attributed to increased IL-4 and MMP-9/TIMP-1 that were observed in mutant and sensitized animals. There was no difference in levels of IL-10 in the experimental groups. Conclusion: The cholinergic deficiency worsens bronchial hyperresponsiveness, eosinophilic inflammation, and airway remodeling mainly by interfering with the pro-inflammatory cytokine IL-4 and in MMP-9/TIMP-1 ratio. These data suggest that anti-inflammatory cholinergic pathway is involved in the asthma pathogenesis deserves further investigation

Glutamat ist der wichtigste exzitatorische Neurotransmitter im Zentralnervensystem der Säugetiere. Die präzise Kontrolle des extrazellulären Glutamatspiegels ist für eine normale synaptische Transmission wichtig und erforderlich, um die Neurone vor Exzitotoxizität zu schützen. Im Gehirn sorgen vor allem verschiedene hochaffine Na+-abhängige Glutamattransporter für diese Kontrolle. In der vorliegenden Arbeit wurde deshalb untersucht, welchen Einfluß die Inhibition der Glutamattransporter auf die exzitatorische synaptische Transmission in Schicht III, einer Region in der bei Alzheimer-Demenz, Schizophrenie und Epilepsie häufig Zellschädigungen und Zellverluste beobachtet werden, und Schicht V des medialen entorhinalen Kortex (mEC) hat. Extrazelluläre Messungen in den Schichten III und V der Ratte zeigten, daß die verwendeten Transport-Inhibitoren signifikant die negativen Feldpotentialkomponenten beider Schichten reduzierten. Schichtspezifische Unterschiede konnten dabei nicht festgestellt werden, was auf eine ähnliche Glutamatregulation in beiden Schichten schließen läßt. Für die anschließenden intrazellulären und patch-clamp Messungen wurden aus diesem Grund nur noch Neurone der Schicht III untersucht. Beide Transport-Inhibitoren (L-trans-2,4-PDC und DL-TBOA) reduzierten die Amplituden der pharmakologisch isolierbaren EPSPs/EPSCs ohne die Kinetik zu beeinflussen. Diese reduzierende Wirkung konnte durch trans-(±)-ACPD, einen Agonisten der Gruppe I und II metabotropen Glutamatrezeptoren (mGluRs), nachgeahmt werden. Die Vorinkubation der Hirnschnitte mit dem unspezifischen Gruppe I und II mGluR-Antagonisten MCPG verhinderte die durch trans-(±)-ACPD hervorgerufene Amplitudenreduktion und auch den reduzierenden Effekt der beiden Transport-Inhibitoren. In nachfolgenden Experimenten mit dem spezifischen Gruppe II mGluR-Antagonisten EGLU konnte dieser zwar die durch L-trans-2,4-PDC hervorgerufene Wirkung verhindern, nicht aber den durch DL-TBOA vermittelten Effekt, was auf eine Aktivierung von Gruppe I mGluRs hinweist. Zusätzlich führte die Applikation von DL-TBOA zu einer signifikanten Veränderung des Doppelpuls-Index, was auf einen präsynaptischen Wirkmechanismus hinweist. Die Applikation von L-trans-2,4-PDC hingegen hatte keinen Effekt auf den Doppelpuls-Index. Die Ergebnisse der vorliegenden Arbeit sprechen dafür, daß beide Transport-Inhibitoren die erregende synaptische Transmission über eine Aktivierung präsynaptischer metabotroper Glutamatrezeptoren der Gruppen I und II hemmen. Dabei konnte festgestellt werden, daß diese Hemmung unter Applikation von DL-TBOA die präsynaptische Transmitterausschüttung über einen negativen Rückkopplungsmechanismus durch Aktivierung von Gruppe I mGluRs vermindert, während L-trans-2,4-PDC seine Wirkung vor allem über eine Aktivierung der Gruppe II vermittelt. Dabei kann davon ausgegangen werden, daß L-trans-2,4-PDC in der benutzten Konzentration die mGluRs der Gruppe II direkt aktivieren kann und der Effekt nicht nur präsynaptisch vermittelt wird. / Glutamate is the primary excitatory neurotransmitter in the mammalian central nervous system. The precise control of extracellular glutamate is crucial for the maintenance of normal synaptic transmission and the prevention of excitotoxicity. High-affinity glutamate transporters ensure termination of glutamatergic neurotransmission and keep the synaptic glutamate concentration below excitotoxic levels. In layer III, a region that is especially prone to cell damage in Alzheimer's disease, schizophrenia and epilepsy, and layer V of the medial entorhinal cortex (mEC) effects of blocking glutamate uptake on excitatory synaptic transmission were studied. Extracellular recordings in rat brain slices revealed that application of glutamate uptake inhibitors significantly reduced stimulus-induced negative field potentials in both, layer III and V of the mEC. This effect showed no significant differences in both layers suggesting a similar glutamate regulation in layer III and V. Therefore, only layer III neurons of the mEC were used for the subsequent intracellular and patch-clamp recordings. Two competitive glutamate transporter antagonists, DL-TBOA and L-trans-2,4-PDC, reduced the amplitude of pharmacologically isolated EPSPs/EPSCs without changing the time course of the events. This effect was mimicked by trans-(±)-ACPD, an agonist of group I and II metabotropic glutamate receptors (mGluRs). The competitive group I and II mGluR antagonist MCPG blocked the depression of the EPSC amplitude induced by trans-(±)-ACPD and also masked the effect of either DL-TBOA or L-trans-2,4-PDC. Furthermore, EGLU, which selectively antagonizes group II mGluRs, masked the effect of L-trans-2,4-PDC but not that of DL-TBOA, indicating an involvement of group I mGluRs in the latter case. Finally, DL-TBOA significantly enhanced the paired-pulse index, suggesting a presynaptic mechanism for the depression of EPSP/EPSC amplitude, whereas application of L-trans-2,4-PDC had no significant effect on the paired-pulse behaviour. The present study shows that both transport inhibitors depress pharmacologically isolated EPSPs/EPSCs in layer III neurons of the mEC in combined entorhinal-hippocampal slices. This effect seems to be mediated via activation of different groups of mGluRs. The results suggest that DL-TBOA causes a negative feedback on glutamate release via indirect activation of presynaptic group I mGluRs, possibly due to an accumulation of glutamate, whereas application of L-trans-2,4-PDC most likely leads to an activation of presynaptic group II mGluRs reducing Ca2+-independent release. The latter might be due to a direct action of L-trans-2,4-PDC at these receptors. The present data suggest that blockade of glutamate transport in the mEC does not lead to an excessive accumulation of glutamate because of a counteractive autoinhibiting mechanism.

A cana-de-açúcar é uma cultura agrícola de grande importância econômica para o Brasil, e a expansão de seu cultivo para solos marginais requer uma maior utilização de fertilizantes à base de nitrogênio (N). Na maioria dos países produtores, a adubação nitrogenada se baseia em altas doses de aplicação, enquanto, no Brasil, o seu uso é relativamente baixo devido, em parte, ao processo de fixação biológica de nitrogênio (FBN) pela ação de bactérias diazotróficas. Além da FBN, as plantas adquirem fontes de N, como amônio e nitrato, por meio de transportadores de membranas localizados nas raízes. Há evidências que a associação com microrganismos pode favorecer as plantas por meio da regulação dos genes de transportadores de N. Desta forma, este trabalho teve como objetivo caracterizar o transporte de amônio e nitrato, avaliando a expressão gênica dos principais transportadores de N em cana-de-açúcar cultivada in vitro sob o efeito da associação com bactérias diazotróficas. Também foi descrita a comunidade bacteriana de plântulas in vitro, bem como o efeito da fertilização com N e da inoculação com bactérias diazotróficas em plantas maduras. Plântulas de \'SP70- 1143\' e \'Chunee\', que contrastam para FBN, foram empregadas em ensaios in vitro sob diversas concentrações e fontes de N em associação ou não com uma estirpe de Gluconacetobacter diazotrophicus ou um mistura de bactérias diazotróficas (G. diazotrophicus, Herbaspirillum seropedicae, H. rubrisubalbicans, Azospirillum amazonense e Burkholderia tropica). A caracterização do transporte de N por meio de ensaios de absorção de nitrato e amônio marcados (15N) revelou que a interação entre cana-de-açúcar x G. diazotrophicus induziu a expressão do gene do transportador de nitrato ScNRT2.1, o que levou a uma tendência no aumento no influxo de nitrato, assim como dos genes de transportadores de amônio ScAMT1.1 e ScAMT1.3, resultando em maiores influxos de amônio apenas para a cultivar \'SP70- 1143\'. Já a associação da cana-de-açúcar com a mistura de bactérias diazotróficas revelou que somente houve indução transcricional de ScAMT1.1, o que resultou na maior absorção de amônio em \'SP70-1143\'. Por sua vez, quando analisada a interação in vitro por 30 dias, a presença da bactéria, apesar de transiente, possivelmente favoreceu a expressão dos genes de transportadores de nitrato ScNRT1.1 e ScNRT2.1, e do transportador de amônio ScAMT1.1, resultando no maior acúmulo de 15N-nitrato de amônio nas plantas de \'SP70-1143\'. Foi detectada uma comunidade bacteriana associada a plântulas micropropagadas, a qual é distinta entre os genótipos \'SP70-1143\' e \'Chunee\' e se altera com a inoculação com G. diazotrophicus. Para as plantas cultivadas em campo, a comunidade bacteriana existente foi alterada pela fertilização de N, mas não pela inoculação com diazotróficas. Portanto, a inoculação com bactérias diazotróficas parece induzir a expressão dos principais genes transportadores de amônio e nitrato em plântulas do genótipo \'SP70-1143\' resultando na maior absorção de fontes inorgânicas de N. / Sugarcane has a large economic importance to Brazil, and it\'s the expansion of cultivation to marginal soils requires a larger application of nitrogen fertilizers (N) to maintain yield. In most producing countries, N fertilization is based on high application rates, whereas in Brazil N fertilization is relatively low, possibly due in part, to the process of biological nitrogen fixation (BNF). In addition, plants acquire inorganic N sources from the soil by membrane transporters that may be regulated by association with microorganisms. This study aimed to characterize the ammonium and nitrate transport evaluating the gene expression profile of the major transporters grown in vitro in association with diazotrophic bacteria. It was also described the bacterial community in micropropagated plants, as well as the effect of N fertilization or inoculation with nitrogen fixing bacteria in mature plants. \'SP70-1143\' and \'Chunee\' which contrasted to BNF, were used in in vitro experiments in several concentrations and N source, in association or not with a strain of Gluconacetobacter diazotrophicus or a bacteria mixture (G. diazotrophicus, Herbaspirillum seropedicae, H. rubrisubalbicans, Azospirillum amazonense and Burkholderia tropica). The characterization of the N transport by uptake assays with 15N-labeled ammonium and nitrate, revealed that the interaction between sugarcane x G. diazotrophicus induced, the nitrate transporter gene ScNRT2.1 expression, which lead to trend to increase nitrate influx, as well as the ammonium transporter genes ScAMT1.1 and ScAMT1.3, resulting in higher ammonium influx in \'SP70-1143\'. Sugarcane associated with the bacterial mixture revealed a transcriptional induction of ScAMT1.1 resulting in larger ammonium acquisition in \'SP70-1143\'. Further, the presence of bacteria in vitro for 30 days, although transient, possibly favored the expression of nitrate transporters ScNRT1.1 and ScNRT2.1, and the ammonium transporter ScAMT1.1, resulting in accumulation of 15N-ammonium nitrate in \'SP70-1143\'. A bacterial community associated with in vitro plants of \'SP70-1143\' and \'Chunee\' was detected with different composition between genotypes, and which changed with artificial inoculation. For plants grown in the field, the bacterial community was affected by N fertilization but not by inoculation with diazotrophic. These results indicate that the inoculation with diazotrophic bacteria appears to induce the expression of the major ammonium and nitrate transporters genes in \'SP70-1143\' plants resulting in higher uptake of inorganic N sources.

Les inhibiteurs sélectifs de recapture de la sérotonine (5-HT) (ISRS) bloquent directement le transporteur de la 5-HT (SERT) et stimulent indirectement de multiples auto- et hétérorécepteurs5-HT par l’augmentation de la concentration extracellulaire de 5-HT dans la fente synaptique. Cependant, le rôle des différents récepteurs ainsi que leur interaction dans les effets thérapeutiques des ISRS restent mal connus. Nous avons tenté de les identifier à l'aide de tests neurochimiques (microdialyse intracérébrale in vivo) et électrophysiologiques en utilisant une approche pharmacologique (utilisation de escitalopram, de ligands des récepteurs 5-HT1A/2A) et génétique (utilisation de souris knock-out [KO] SERT, 5-HT1A ou 5-HT2A). Les études neurochimiques et électrophysiologiques révèlent que les auto-(1A) et hétéro-(2A) récepteursagissent de concert pour maintenir une influence inhibitrice sur le système sérotoninergique, en particulier, en réponse au escitalopram : l'absence d'un récepteur est compensée par une régulation de l'autre. Enfin, les souris KO SERT constituent un nouveau modèle pour tester le mécanisme du escitalopram dans l’augmentation des concentrations de noradrénaline (NA). / Selective serotonin (5-HT) reuptake inhibitors (SSRIs) directly block the 5-HT transporter(SERT) and indirectly stimulate multiple 5-HT (auto- and hetero-) receptors by enhancing itsextracellular levels in the synaptic cleft, although the role of particular receptors as well asinteraction(s) among different receptors in the therapeutic effects of SSRIs is not fullyunderstood. We tried to highlight it using neurochemical (in vivo intracerebral microdialysis) andelectrophysiological tests with a pharmacological (using escitalopram, 5-HT1A/2A receptorsagonists and antagonists) and genetic (using SERT, 5-HT1A ou 5-HT2A receptor knock-out [KO]mice) approaches. Neurochemical and electrophysiological experiments indicated that 5-HT1Aauto- and 5-HT2A hetero-receptors act in concert to maintain an inhibitory influence on theserotonergic system, particularly in response of escitalopram to increased levels of endogenous 5-HT: the absence of one receptor being compensated by an up-regulation of the other. Finally,SERT knockout mice might be a new model to test the mechanism of escitalopram for anincrease of norephedrine (NE) level.

Les Légumineuses ont développé une interaction symbiotique avec des bactéries du sol, les rhizobia, qui fixent l’azote atmosphérique et le transfèrent à la plante sous forme assimilable.Cette interaction a lieu, au sein des nodosités, des organes racinaires où les bactéries intracellulaires se différencient en bactéroïdes. Chez Medicago truncatula, ces bactéroïdes correspondent à un stade de différentiation terminale corrélée à une endoréplication de leur génome, une augmentation de la taille des cellules, une modification des membranes et une faible capacité à se propager. Cette différentiation est induite par des facteurs de la plante appelés NCR (Nodule-specific Cysteine Rich). Les peptides NCRs ressemblent à des défensines, des peptides antimicrobiens ayant une activité antimicrobienne in vitro, tuant des bactéries. Ainsi, un élément clef dans la différenciation des bactéroïdes est la protéine bactérienne BacA, un transporteur membranaire qui confère une résistance contre l’activité antimicrobienne des peptides. Dans le cadre de ce travail de thèse, j’ai montré que l'expression des NCR est soumise à une régulation stricte et qu’ils sont activés dans trois vagues dans les cellules symbiotiques polyploïdes.Les mécanismes de contrôle par la plante sur les rhizobia intracellulaires demeurent à ce jourpeu connus et le seul modèle étudié, au début de ce travail de thèse, restait l'interaction entre M. truncatula et S. meliloti. Je me suis donc intéressée à la symbiose de certaines Légumineuses tropicales du genre Aeschynomene appartenant au clade des Dalbergoïdes où jemontre qu’ils utilisent une classe différente de peptides riches en cystéine (NCR-like) pour induire la différenciation des bactéroïdes. Ce mécanisme est analogue à celui décrit précédemment chez Medicago qui était jusqu'à présent supposé être limitée aux légumineuses appartenant au clade des IRLC. J’ai également montré que Bradyrhizobium, symbionte d’Aeschynomene possèdent un transporteur de type ABC homologues à BacA de Sinorhizobium nommé BclA. Ce gène permet l'importation d'une variété de peptides comprenant des peptides NCR. En l'absence de ce transporteur, les rhizobiums sont incapables de se différencier et de fixer l'azote.Cette étude a permis d'élargir nos connaissances sur l'évolution de la symbiose en montrant qu’au cours de l’évolution, deux clades de Légumineuses relativement éloignés (IRLC et Dalbergoïdes) aient convergé vers l’utilisation de peptides de l’immunité innée afin de contrôler leur symbionte bactérien et d’en tirer un bénéfice maximal au cours de l’interaction symbiotique. / The ability of legumes to acquire sufficient nitrogen from the symbiosis with Rhizobium relies on the intimate contact between the endosymbiotic, intracellular rhizobia, called bacteroids, and their host cells, the symbiotic nodule cells. A well-studied example is the symbiotic nitrogen fixing bacterium Sinorhizobium meliloti, which nodulates the legume Medicago truncatula. Nodules of M. truncatula produce an enormous diversity of peptides called NCRs which are similar to antimicrobial peptides (AMPs) of innate immune systems. These NCRs are involved in maintaining the homeostasis between the host cells in the nodules and the large bacterial population they contain. Although many NCRs are genuine AMPs which kill microbes in vitro, in nodule cells they do not kill the bacteria but induce them into the terminally differentiated bacteroid state involving cell elongation, genome amplification, membrane fragilization and loss of cell division capacity. Protection against the antimicrobial action of NCRs by the bacterial BacA protein is critical for bacteroid survival in the symbiotic cells and thus for symbiosis. As a part of my PhD thesis, I have shown that the differentiation of the symbiotic cells in M. truncatula is associated with a tremendous transcriptional reprogramming involving hundreds of genes, mainly NCR genes, which are only expressed in these cells. Although the extensive work on the model M. truncatula/S. meliloti, little is known how the plant controls its intracellular population and imposes its differentiation into a functional form, the bacteroids in other symbiotic systems.In my PhD work, I provide several independent pieces of evidence to show that tropical legumes of the Aeschynomene genus which belong to the Dalbergoid legume clade use a different class of cysteine rich peptides (NCR-like) to govern bacteroid differentiation. This mechanism is similar to the one previously described in Medicago which was up to now assumed to be restricted to the advanced IRLC legume clade, to which it belongs. I have also shown that the Bradyrhizobium symbionts of Aeschynomene legumes possess a multidrug transporter, named BclA, which mediates the import of a diversity of peptides including NCR peptides. In the absence of this transporter, the rhizobia do not differentiate and do not fix nitrogen. BclA has a transmembrane domain of the same family as the transmembrane domain of the BacA transporter of Rhizobium and Sinorhizobium species which is known to be required in these rhizobia to respond to the NCR peptides of IRLC legumes. Again this is a mechanism which is analogous to the one described in S. meliloti the symbiont of Medicago.This study broaden our knowledge on the evolution of symbiosis by showing that the modus operandi involving peptides derived from innate immunity used by some legumes to keep their intracellular bacterial population under control is more widespread and ancient than previously thought and has been invented by evolution several times.