Les tiques sont des arthropodes ectoparasites obligatoires qui se nourrissent sur une grande variété de vertébrés sur une large partie du globe. Au cours de leur repas, les tiques sécrètent dans leur salive de nombreux facteurs leur permettant de contourner bon nombre des défenses de l’hôte. Bien que la littérature rapporte beaucoup d’informations au sujet des effets du repas de la tique sur l’hôte, la nature des facteurs actifs exprimés par les glandes salivaires de la tique est peu connue. Au cours d’anciens travaux au sein du laboratoire, le crible de deux banques d’ADN complémentaires - issues de la rétro-transcription des ARN messagers synthétisés par les glandes salivaires de la tique Ixodes ricinus – a permis l’identification de 27 protéines dont l'expression est spécifiquement induite ou régulée positivement pendant le repas sanguin de la tique I. ricinus. Parmi ces protéines, la protéine Seq24, induite au cours du repas sanguin, présente la capacité de moduler les immunités innée et acquise de l’hôte. En conséquence, la protéine Seq24 a été nommée Iris pour « Ixodes ricinus Immunosuppressor ». Au cours de la présente étude, notre but fût de caractériser le rôle d’Iris et de déterminer son importance dans le repas sanguin de la tique I. ricinus.<p>La protéine Iris appartient à la famille des inhibiteurs de sérine protéases et présente une homologie significative avec l’inhibiteur d’élastase de leucocytes. Une analyse in silico a confirmé qu’Iris présentait la structure des serpines, et notamment le RCL (Reactive Center Loop), boucle responsable de l’activité anti-protéasique. Comme attendu (sur base de l’analyse in silico), Iris inhibe de manière spécifique l’activité de plusieurs sérine protéases, et en particulier l’élastase de leucocyte. Ces tests effectués, nous avons essayé de comprendre quel(s) pouvai(en)t être le(s) rôle(s) d’Iris dans l’accomplissement du repas sanguin de la tique, c’est à dire dans la lutte contre les différents systèmes de défenses de l’hôte.<p>Tout d’abord, des tests ont démontré la capacité d’Iris à inhiber les mécanismes de l’hémostase. Des tests sur du plasma et du sang complet ont montré qu’Iris allonge le temps de fibrinolyse, la voie intrinsèque de la coagulation et l’adhésion plaquettaire. L’utilisation de mutants a également démontré que si les deux premières activités sont dépendantes du RCL, et donc d’un mode de fonctionnement anti-protéolytique, l’adhésion plaquettaire est indépendante de ce système. Ce résultat met en évidence l’existence d’autres sites actifs, isolés par analyse in silico, nommés Receptor Binding Domain (RBD).<p>Un travail antérieur du laboratoire avait permis d’indiquer la capacité de la protéine recombinante Iris semi-purifiée à inhiber la production de TNF-a, d’IL-6, et d’IL-8 (cytokines pro-inflammatoires) ainsi que l’IFN-g par des PBMCs (Peripherical Blood Mononuclear Cells) humaines. Ces résultats ont été confirmés avec de la protéine purifiée. Des analyses complémentaires ont démontré qu’un mutant d’Iris - dépourvu d’activité anti-protéasique - conserve l’activité pro-inflammatoire. Là encore, ce mécanisme semble impliquer un ou plusieurs RBD. L’utilisation d’anticorps dirigés contre ces zones a permis de déterminer le domaine d’interaction (aa :105-120) impliqué dans cette fonction. D’autre part, une analyse par FACS a permis de démontrer qu’Iris interagit uniquement avec les cellules d’origine monocytaire.<p>Enfin, nous avons également analysé l’importance d’Iris au cours du repas sanguin de la tique par une approche vaccinale. Les résultats observés indiquent que 30 % des tiques nourries sur des lapins immunisés par la protéine rIris ne survivent pas au repas. / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

Einleitung: Coxiella burnetii ist ein gramnegatives und obligat intrazelluläres Bakterium und Erreger der meldepflichtigen Zoonose Q-Fieber. Den Hauptübertragungsweg stellt die Inhalation infizierter Stäube dar, aber auch Zecken werden seit der Isolation des Pathogens aus der Spezies Dermacentor andersonii als Vektoren diskutiert. Es liegen jedoch keine gesicherten Berichte über Infektionen des Menschen durch Zecken oder eine Beteiligung von Vektoren bei Q-Fieber-Ausbrüchen vor. Zudem steht durch die genetische Nähe zu überwiegend apathogenen Endosymbionten die Spezifität molekularer Untersuchungsmethoden in Frage, wodurch die Relevanz von Zecken in der Übertragung von Q-Fieber in der Literatur kontrovers diskutiert wird. Ziele der Untersuchung: Es sollten mithilfe einer systematischen Literaturanalyse die Daten zur Prävalenz von C. burnetii in Zecken in Europa dargelegt werden. Zudem sollte mittels eines in-vitro-Fütterungssystems untersucht werden, inwieweit die heimischen Spezies Ixodes ricinus und Dermacentor marginatus den Erreger aus dem Blut aufnehmen und ausscheiden, sowie ob das Bakterium transstadial in I. ricinus übertragen wird, wodurch Aussagen über die Vektorkompetenz dieser Arten möglich sind. Tiere, Material und Methoden: Im experimentellen Ansatz wurde die Infektion der Zecken in einem Silikonmembran-basierten Fütterungssystem mit heparinisiertem Rinderblut vorgenommen. Die Gruppengrößen betrugen 7-9 Weibchen mit fünf Männchen oder 50-60 Nymphen. Es wurden verschiedene Konzentrationen C. burnetii Nine Mile RSA439 Phase II eingesetzt: 10^4 (zwei Gruppen adulte I. ricinus), 10^5 (zwei Gruppen adulte I. ricinus) und 10^6 (fünf Gruppen adulte I. ricinus, vier Gruppen adulte D. marginatus, drei Gruppen I. ricinus Nymphen) Genomäquivalente (GE)/ml Blut sowie insgesamt sieben Negativkontrollgruppen. Zudem wurden vier Gruppen adulte I. ricinus für 36 Stunden zu Beginn der Fütterung mit 10^6 GE C. burnetii/ml infiziert und anschließend mit sterilem Blut gefüttert. Täglich wurde Zeckenkot entfernt, zudem wurden adulte I. ricinus zu unterschiedlichen Zeitpunkten während oder nach der Fütterung entnommen. Die Proben wurden nach DNA-Extraktion mittels quantitativer Echtzeit-PCR (qPCR) auf das C. burnetii-Gen icd untersucht. Ein Teil (n = 46) der infizierten Nymphen wurde vor bzw. nach der Häutung mittels qPCR untersucht. Ein anderer Teil (acht Weibchen in zwei Gruppen) wurde nach der Häutung erneut auf sterilem Blut gefüttert. Dabei wurden Kot sowie Blut getestet. Weiterhin fand in L929-Zellen und axenischem Medium eine Anzucht von C. burnetii aus einem Filtrat des Zeckenkots sowie aus dem Filtrat eines qPCR-positiven Weibchens statt, welches sich von einer zuvor infizierten Nymphe gehäutet hatte. Die statistische Auswertung erfolgte mittels SPSS V 22.0, wobei je nach Voraussetzung der t-Test, der Mann-Whitney-U-Test sowie der Chi²-Test nach Pearson verwendet wurden. Das Signifikanzniveau wurde jeweils auf p < 0,05 festgelegt. Ergebnisse: Im Fütterungssystem haben 49 % der adulten I. ricinus und 29 % der D. marginatus vollständig gesaugt. Die Häutungsrate der I. ricinus Nymphen betrug 92 %. I. ricinus-Weibchen nahmen den Erreger auf, welcher innerhalb von sieben Wochen in einer Konzentration von ca. 10^3 GE/mg in den Zecken nachweisbar war. Eine Ausscheidung von infektiösen C. burnetii mit dem Kot wurde bei adulten Zecken ab einer Konzentration von 10^5 GE/ml im verfütterten Blut festgestellt. Dabei erfolgte in allen Versuchen mit beiden Zeckenspezies eine signifikant höhere Ausscheidung von C. burnetii an den Tagen 10-13, welche bis zu 10^5 GE/mg erreichte. Es wurde eine transstadiale Übertragung bei I. ricinus mit einer Rate von 25 % nach-gewiesen. Infizierte Nymphen, die nach der Häutung steriles Blut bekamen, schieden ebenfalls C. burnetii im Kot aus; im Blut wurde hingegen keine DNA von C. burnetii nachgewiesen. Schlussfolgerungen: Das genutzte Fütterungssystem eignet sich für die Untersuchung der Vektorkompetenz von Zecken. Beide Zeckenspezies sind in der Lage, infektiösen Zeckenkot auszuscheiden, wodurch eine Gefahr einer möglichen aerogenen Übertragung entsteht. Dabei muss jedoch eine hohe Erregerkonzentration im Blut des Wirts erreicht werden. Es wurde eine transstadiale Übertragung von C. burnetii in I. ricinus von Nymphen zu Adulten und damit die Möglichkeit der Bildung eines Reservoirs nachgewiesen. Die Ergebnisse legen eine Vermehrung von C. burnetii im Mitteldarm der Zecken nahe. Dadurch konnten historische Aussagen aus der Literatur über andere Zeckenspezies zur Ausscheidung von C. burnetii mittels Zeckenkot bestätigt werden. In der Studie konnte weiterhin gezeigt werden, dass heimische Zecken unter Laborbedingungen zur Verbreitung von Q-Fieber beitragen können. Weitere Untersuchungen sind jedoch notwendig, um die tatsächliche Bedeutung der Vektorkompetenz von Zecken unter natürlichen Bedingungen abschätzen zu können.:1 Einleitung 2 Literaturübersicht 2.1 Coxiella burnetii 2.1.1 Historischer Ursprung und taxonomische Einordnung 2.1.2 Morphologie und Replikation 2.1.3 Coxiella burnetii als Krankheitserreger 2.1.3.1 Epidemiologie 2.1.3.2 Coxiellosen der Tiere 2.1.3.3 Q-Fieber des Menschen 2.1.3.4 Nachweis und Therapie 2.2 Zecken 2.2.1 Taxonomie 2.2.2 Verbreitung und Habitat 2.2.3 Morphologie 2.2.4 Lebenszyklus 2.2.5 Wirtssuche und sensorische Fähigkeiten 2.2.6 Blutmahlzeit und Verdauung 2.2.7 Zeckenmikrobiom und Coxiella-like Endosymbionten 2.2.8 Zecken als Vektoren 2.2.8.1 Infektionserreger 2.2.8.2 Coxiella burnetii in Zecken 2.3 Fütterung von Zecken 2.3.1 Fütterungssysteme 2.3.1.1 Membranbasierte Fütterungssysteme 2.3.1.2 Fixierungsstimuli im Zeckenfütterungssystem 2.3.1.3 Experimentelle Infektionen im membranbasierten in-vitro-System 3 Veröffentlichungen 3.1 Stellungnahme zum Eigenanteil an den Arbeiten zur Publikation 3.1.1 Publikation 1 3.2 Stellungnahme zum Eigenanteil an den Arbeiten zur Publikation 3.2.2 Publikation 2 4 Diskussion 5 Zusammenfassung 6 Summary 7 Literaturverzeichnis 8 Anhang 8.1 Bilder der Zeckenfütterung 9 Danksagung

Orientador: Carlos Alexandre Costa Crusciol / Banca: Rogério Peres Soratto / Banca: Heitor Cantarella / Resumo: O objetivo deste trabalho foi avaliar a influência de épocas de aplicação, fontes e doses de nitrogênio em híbridos de mamona de porte baixo em sistema plantio direto, possibilitando a obtenção de informações para o manejo adequado da adubação nitrogenada tanto em safra como em safrinha. O projeto de pesquisa foi constituído de três experimentos conduzidos em safra e safrinha por dois anos agrícolas (2005/2006 e 2006/2007) na Fazenda Experimental Lageado pertencente a Faculdade de Ciências Agronômicas da UNESP, em Botucatu-SP. No Experimento 1 o delineamento experimental foi em blocos casualizados em esquema parcela subdividida, com quatro repetições. As parcelas foram constituídas por dois híbridos de mamona (híbrido Lyra e híbrido Savana) e as subparcelas, por cinco doses de N (0, 50, 100, 150 e 200 kg ha-1). A fonte de N utilizada foi o nitrato de amônio (32% de N), sendo que a aplicação foi realizada aos 20 dias após a emergência. No Experimento 2 o delineamento experimental foi em blocos casualizados em esquema parcela subdividida, com quatro repetições. As parcelas foram constituídas por dois híbridos de mamona (híbrido Lyra e híbrido Savana) e as subparcelas, por seis formas de parcelamento da adubação nitrogenada (0-0, 0-100, 100-0, 30-70, 70-30, 50-50 kg ha-1 de N) aplicados em duas épocas (20 DAE e aos 40 DAE). A fonte de nitrogênio utilizada também foi o nitrato de amônio. Por fim, no Experimento 3 o delineamento experimental foi em blocos casualizados em esquema fatorial 2 x 4, com quatro repetições. Os tratamentos foram constituídos pela combinação de duas fontes de nitrogênio (sulfato de amônio e uréia), com quatro doses de nitrogênio (0, 30, 60 e 120 kg ha-1 de N) aplicadas em cobertura aos 20 dias após a emergência, sendo utilizado o híbridoLyra. Vale ressaltar que na safrinha de 2007 o híbrido Savana foi substituído pelo... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to evaluate the effect's time, as well as sources and nitrogen doses in castor bean hybrids in no-till system, enabling the appropriate nitrogen fertilization management both in summer crop and fall crop for two crop years (2005/2006 and 2006/2007) in an experimental area located in Botucatu, São Paulo State, Brazil. In the experiment 1, the experimental design was a randomized complete block design, in splitplot scheme with four replications, was used. The plots were constituted by two castor bean hybrids (Lyra and Savana). Five N levels (0, 50, 100, 150, and 200 kg ha-1) side dressing fertilization constituted the subplots. Ammonium nitrate was the nitrogen source utilized (32% of N) applied 20 days after emergence. In the experiment 2, the experimental design was a randomized complete block design, in splitplot scheme with four replications, was used. The plots were constituted by two castor bean hybrids (Lyra and Savana). Six combination of times out rates (0-0, 0-100, 100-0, 30-70, 70-30, 50-50 kg ha-1 of N) side dressing nitrogen fertilization constituted the subplots. The N source used was also ammonium nitrate, applied 20 days after emergence and 40 days after emergence. Finally, in experiment 3, the experimental design was a randomized blocks, in 2 x 4 factorial design, with four replications. The treatments comprised the combination of two nitrogen sources (ammonium sulfate and urea), with four doses of nitrogen (0, 30, 60 and 120 kg ha-1 of N) surface applied 20 days after emergence, plus a control. In the latter experiment, hybrid Lyra was utilized. It's important to highlight that in the 2007 fall crop, Savana hybrid was replaced by Sara. In the summer crop, the conclusions were: a) Lyra hybrid was more productive than Savana; b) Lyra was more efficient in N utilization as to the application of 150 kg ha-1; e) the components ...(Complete abstract click electronic access below) / Mestre

<p>Ricin je prirodni protein, toksin koji spada među najpristupačnije i najsmrtonosnije otrove. Nalazi se u biljci Ricinus (Ricinus communis), sa najvećim sadržajem u semenu (1-5 %). Ricin se smatra potencijalnim bioterorističkim oružjem i prema riziku za ljudsko zdravlje svrstan je u B kategoriju biolo&scaron;kog oružja. U novije vreme kori&scaron;ćen je za konstruisanje imunotoksina protiv tumorskih ćelija u terapiji maligniteta. Dokumentovana su mnoga trovanja ricinom, kako zadesna, tako i samoubilačka i ubilačka. U tu svrhu koristilo se intaktno seme ricinusa ili ekstrahovani ricin. Osim ricina, u semenu ricinusa je prisutan toksični alkaloid ricinin u količini 0.3-0.8 %. Ricinus je jedini poznati prirodni izvor ricinina, koji se ko-ekstrahuje sa ricinom iz semena biljke. Ricinin se jednostavno detektuje u kliničkim uzoracima metodom tečne hromatografije i masene spektrometrije i, s obzirom na komplikovanu identifikaciju ricina u biolo&scaron;kim uzorcima, smatra se biomarkerom za intoksikaciju ricinusom, odnosno ricinom. Osnovni ciljevi ovog istraživanja su da se uz pomoć HS-GC metode i patohistolo&scaron;kom&nbsp; analizom dokaže prisustvo ricinina u krvi laboratorijskih pacova u odnosu na vremenski interval koji je protekao od oralne aplikacije suspenzije do vremena žtvovanja, da se odredi distribucija i koncentracija ricinina u organima laboratorijskih pacova u različitim vremenima žrtvovanja, kao i da se utvrdi da li postoji značajna razlika u razvoju patomorfolo&scaron;kih promena na organima laboratorijskih pacova u različitim vremenima žrtvovanja. Istraživanje je bilo otvoreno, randomizirano i prospektivnog tipa. Laboratorijski pacovi su u istom vremenu oralno tretirani suspenzijom koja je sadržaja subletalnu koncentraciju ricina. Nakon žrtvovanja u precizno definisanim vremenskim intervalima uzeti su uzorci krvi i unutra&scaron;njih organa radi daljih analiza. Odgovarajući uzorci su analizirani metodom HC-GS u cilju određivanja koncentracije i distribucije ricinina, kao pouzdanog markera trovanja ricinom, u krvi i unutra&scaron;njim organima. Takođe je izvr&scaron;ena patohistolo&scaron;ka analiza uzoraka tkiva unutra&scaron;njih organa u cilju utvrđivanja promena izazvanim delovanjem ricina u odnosu na vreme proteklo od aplikacije suspenzije. Dobijeni rezultati su obrađeni odgovarajućim statističkim metodama. Rezultati istraživanja omogućavaju standardizaciju postupaka odabira reprezentativnih uzoraka prilikom sumnje na trovanje ricinusom i metode dokazivanja akutnog trovanja. Na taj način može se pouzdano i efikasno dokazati trovanje ricinusom.</p> / <p>Ricin is a naturally occurring protein, a toxin which belongs to the category of the most accessible and the most lethal poisons. It is obtained from the castor oil plant ( Ricinus communis), whose seeds contain its highest content (1-5%). Ricin is also thought to be a potential weapon of bioterrorism and taking into account the risk for human health, it is classified as a biological weapon category B. Lately it has been used for the construction of the immunotoxins against tumor cells in the therapy of malignant diseases. Numerous poisonings using ricin have been documented, not only accidental poisoning, but also in case of suicides and homicides. In those cases, intact ricin seeds or extracted ricin were used. Apart from ricin, castor oil plants also contain a toxic alkaloid ricinine (0.3-0.8%). Castor oil plants are the only known natural source of ricinine, which is co-extracted with ricin from the seeds of this plant. Ricinine is simply detected in clinical samples by using the method of liquid chromatography and mass spectrometry. Taking into account a complicated identification of ricin in biological samples, it is considered to be a biomarker for the intoxication by castor oil plant, or ricin itself. The main aim of this research is to use the HS-GC method and pathohistological analysis in proving the existence of ricinine in the blood of experimental rats in relation to the time interval between the oral application of solution of castor seeds in water and the time of sacrificing, to determine the distribution and concentration of ricinine in the organs of experimental rats, as well as to establish whether there was a significant difference in the development of pathomorphological changes on the organs of experimental rats at various points of sacrificing. The research was open, randomised and prospective. Experimental rats were simultaneously orally tested by the solution which contained sublethal concentration of ricin. After sacrificing, blood samples were taken from inner organs in specifically defined intervals of time and used for further analysis. The appropriate samples were analysed by HC-GS method in order to determine the concentration and distribution of ricinine as a reliable marker of ricin poisoning in blood and inner organs. Also, pathohistological analysis of the samples of inner organ tissues was made with the purpose of establishing the changes caused by the effects of ricinine in relation to time which passed from the application of the solution. The obtained results were processed by appropriate statistical methods. The results of this research allow for the standardisation of the actions in selecting the representative samples in case there is a possibility of ricin poisoning and the method of proving the acute poisoning. Following these steps, ricin poisoning can be proved in a reliable and an efficient way.</p>

The spotted fever rickettsia, Rickettsia helvetica, is an endemic tick-borne bacteria in Sweden. It causes infections in humans, manifested as aneruptive fever, headache, arthralgia and myalgia, and sometimes an inoculation eschar or a rash. There have also been two known cases of human infections with R. felis in Sweden. The present thesis starts by investigating dispersal of ticks and Rickettsia spp. by migrating birds flying from Africa to Europe. Almost 15,000 birds were searched and 734 ticks collected, mainly of the species Hyalomma marginatum complex. Almost half (48%) of the ticks were infected with Rickettsia spp., 96% of which was R. aeschlimannii, the remaining R. africae and undefined species. The next study focused on questing ticks over a large area in Sweden and determining the prevalence of Rickettsia spp., Anaplasma spp. and Coxiella burnetii. Rickettsia spp. was found in 9.5-9.6% of the ticks and A. phagocytophilum in 0.7%; no C. burnetii was found. The last three papers in the thesis focused on the clinical presentation of rickettsiosis, the symptoms associated with the infection in general and particularly in patients with neurological complications. A tick-exposed population in Sweden was investigated to gain a better understanding of symptoms due to rickettsioses, also in relation to co-infections with other tick-borne bacteria. Based on symptoms, it was not possible to distinguish what pathogen caused the infections. Most patients had erythema migrans, some had serological reactions to Rickettsia spp., Borrelia spp. or co-infections by Rickettsia spp., Borrelia spp. and/or Anaplasma spp. In the fourth and fifth papers, we found associations between antibodies against Rickettsia spp. and sudden deafness (in 10-24% of patients) and facial nerve paralysis (in 8.3-25% of patients). In three patients R. felis was detected in the cerebrospinal fluids.    Briefly, the thesis helps to clarify our knowledge about tick dispersal, shows a narrower prevalence estimate of Rickettsia spp. in Swedish ticks, and illuminates symptoms of rickettsioses and co-infections with other tick-borne infections. It also shows that presence of erythema migrans may be explained by more than Lyme disease and indicates a possible association between rickettsiosis and sudden deafness and facial nerve paralysis.

This thesis examines the parasite dynamics and the mechanisms affecting parasite load and transmission focalising on the role played by host and habitat heterogeneities. This study is based on the gastrointestinal nematode Heligmosomoides polygyrus and the small mammal yellow necked mouse and uses data gathered from experimental field manipulations of parasites intensities and data gathered from trapping monitoring. Initially the parasite community of yellow-necked mouse (Apodemus flavicollis) was explored in North-Eastern Italian Alps with the aim to describe the major patterns and identify the factors affecting parasite community structure. Despite the observed spatial variability it has been found that differences within the host population such age and secondly sex and breeding conditions, were the major factors acting on parasite occurrence and intensity. Habitat differences had a less apparent effect on parasite community structure. The consequences of H. polygyrus infection on other parasite species infections have been analysed, in specific the infestation of the tick Ixodes ricinus in populations of A. flavicollis. H. polygyrus load and tick infestation were monitored as well as were carried out field manipulations of H. polygyrus intensity and were monitored changes in tick infestation. It has been found that H. polygyrus load was negatively related to I. ricinus infestations. Host factors mediated the H. polygyrus-I. ricinus interaction such that young and non-breeding mice exhibited higher I. ricinus to H. polygyrus intensity respect breeding adults. The role of host sex on parasite abundance was then investigated carrying out a field experiment where the H. polygyrus intensity were manipulated in relation to mice gender. In specific, H. polygyrus was removed alternately from either sexes and the parasite load was analysed in the untreated sex. It was found that males mice were responsible to drive parasite transmission in the host population and this was observed in absence of sex-bias in parasite infection, suggesting that this pattern was not a mere consequence of quantitative differences in parasite loads between sexes. To disentangle the possible mechanism causing this sex bias in parasite transmission mathematical simulations based on parameters obtained for the field experiment were used. Two non mutually exclusive hypotheses causing sex bias in parasite transmission were tested: a- males immune response is less efficient and this causes the development of more successful parasite infective stages or b-males behaviours allow them to be more efficient is spreading in more exposed areas parasite infective stages. Multi-host models were developed and simulations were compared with field results. While it was not disentangled the most dominant mechanism causing sex bias in parasite transmission this study underlined the importance of host sexes in affecting parasite dynamics and host-parasite interaction. In conclusion this thesis highlighted the importance of considering host and environmental differences when investigating host parasite interactions. This finding could be extremely important when planning measured of disease control or to avoid disease outbreak. Controlling target group of individuals host could avoid economical losses and a more effective measure of intervention.

O objetivo deste estudo foi analisar a modulação da expressão de metaloproteinases da matriz-2 e -9, no tecido ósseo neoformado na interface do implante derivado do polímero da mamona (Ricinus communis) com o canal medular da tíbia de coelhos, por meio de análise histológica por microscopia óptica, tomografia computadorizada e imunoistoquímica. Foram selecionados 44 coelhos machos, Oryctolagus cuniculus, da linhagem Nova Zelândia, albinos, divididos em dois grupos, sendo o Grupo 1, composto por 12 animais controle, cujas fresagens do canal medular foram produzidas bilateralmente nas tíbias e não preenchidas e, o Grupo 2, com 30 animais, cujos canais medulares da tíbia, após fresagem, foram preenchidos bilateralmente com os cilindros derivados da poliuretana da mamona. Os animais do Grupo 1 e Grupo 2 foram divididos aleatoriamente em subgrupos experimentais, conforme as datas de eutanásia pré-determinadas em 90, 120, 150 dias após o ato operatório. Um animal não foi submetido ao procedimento de fresagem, sendo utilizado para controle histológico e outro submeteu-se à eutanásia após o implante do polímero, sendo utilizado para controle de imagem do estudo com tomografia computadorizada. Decorridos os períodos experimentais, os animais foram submetidos à eutanásia, as peças removidas e encaminhadas para o exame tomográfico; posteriormente ao processamento histológico, as lâminas foram analisadas em microscópio óptico. As Metaloproteinases da Matriz (MMPs) são um importante grupo de enzimas proteolíticas zinco-dependentes responsáveis pela degradação de matriz extracelular e membranas basais. As enzimas são secretadas em uma forma latente e se tornam ativadas no ambiente pericelular, sendo relacionadas a processos fisiológicos e patológicos. No presente estudo, foram revisados alguns aspectos importantes das MMPs, discutindo-se o papel dessas enzimas em processos fisiológicos como a neoformação e maturação óssea (MMP-2). Dentre os processos patológicos que envolvem a participação das MMPs, destacam-se a reabsorção óssea e processos inflamatórios (MMP-9). A expressão de metaloproteinase da matriz-2 e -9 nos tecidos foi avaliada por meio de imunoistoquímica. Os dados obtidos foram submetidos à análise estatística por meio do teste ANOVA seguido pelo pós-teste de Tukey (&alpha; = 0,05). No grupo experimental, aos 90 dias, a interface com o polímero apresentava uma camada espessa de tecido ósseo neoformado rico em osteócitos, o qual apresentou uma maturação com o passar do tempo, aos 120 e 150 dias pós-implantação. No grupo controle, a superfície interna junto ao canal medular apresentava-se revestida por osteoblastos, seguida de faixa de tecido ósseo, com poucas lacunas preenchidas por osteócitos. O amadurecimento do tecido da superfície interna medular acontece na região interior, sendo o osso alamelar, constituído por fibras colágenas menos amadurecidas que o osso lamelar. As imagens tomográficas demonstraram não haver espaço entre a superfície do material e do osso na interface implante/medula óssea, sendo a densidade dos tecidos nesta interface semelhante à densidade das demais porções da medula óssea. O processo de remodelação óssea observado histologicamente foi acompanhado pela modulação positiva de metaloproteinase da matriz-2 durante todo o período de avaliação com baixa expressão de metaloproteinase da matriz-9. / The aim of this study was to evaluate the modulation of matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9) expression in newly formed bone tissue at the interface between implant derived from castor oil (Ricinus communis) polymer and the medullary canal of rabbit tibia, by histological examination under optical microscopy, computed tomography (CT) and immunohistochemical analysis. For such purpose, 44 rabbits (Oryctolagus cuniculus, New Zealand, albinus) were selected and assigned to two groups. In Group 1, composed of 12 animals (control), reamings of the medullary canal were produced bilaterally in the tibiae of the rabbits and were not filled. In Group 2, composed of 30 animals, the tibial medullary canals, after reaming, were filled bilaterally with cylinders derived from castor oil polyurethane. The animals of Groups 1 and 2 were randomly divided in experimental subgroups, according to the periods of predetermined euthanasia, which were 90, 120, 150 days postoperatively. One animal was not subjected to the reaming procedure, and served as a histological control; another animal was killed after placement of the polymer implant, and served as a control for CT imaging. Euthanasia was undertaken at the established experimental periods, and the anatomic specimens were removed and subjected to CT analysis. Then, after histological processing, the slides were examined under optical microscopy. MMPs are an important group of zinc-dependent proteolytic enzymes responsible for the degradation of extracellular matrix and basal membranes. The enzymes are synthesized in a latent form and are activated in the pericellular environment, being involved in physiological and pathological processes. In the present study, some important aspects of MMPs were reviewed, and the role of these enzymes in physiological processes, such as new bone formation and bone maturation (MMP-2), was discussed. Among the pathological processes that have the participation of MMPs, the most relevant are bone resorption and inflammatory processes (MMP-9). MMP-2 and MMP-9 expression in the tissues was evaluated by immunohistochemistry. Data were subjected to statistical analysis by ANOVA and Tukey post-test (&alpha; = 0.05). In the group experimental, at 90 days, the interface with the polymer presented a thick layer of newly formed bone tissue rich in osteocytes. This tissue exhibited an ongoing maturation at 120 and 150 days post-implantation. In the control group, the internal surface close to the medullary canal was lined by osteoblasts, followed by a bone tissue zone with few lacunae filled with osteocytes. Maturation of the tissue of the medullary internal surface occurred in the inner region, with the bone being alamellar, that is, constituted of collagen fibers less maturated than the lamellar bone. The CT scans showed no space between the material surface and the bone at the implant/bone marrow interface, and the density of the tissues at this interface was similar to the density measured in the other regions of the bone marrow. The bone remodeling process observed histologically was accompanied by positive modulation of MMP-2 during the entire evaluation period and low MMP-9 expression.

De nombreuses études suggèrent que linhibition de la voie alterne du complément de lhôte est nécessaire aux parasites hématophages pour leur permettre daccomplir leur repas sanguin. Une revue décrivant ces études a été publiée dans Developmental and Comparative Immunology (Schroeder et al., (2009) Dev. Comp. Immunol. 33, 5-13). Plusieurs études suggèrent que linhibition de la voie alterne de lhôte par les protéines salivaires de tiques est importante pour lacquisition du repas sanguin et la transmission subséquente de pathogènes à lhôte infesté. Confortant cette hypothèse, une protéine salivaire capable dinhiber la voie alterne du complément a été clonée chez la tique américaine Ixodes scapularis (Valenzuela et al., (2000) J. Biol. Chem. 275, 18717-18723). Cette protéine, appelée Isac, ne présente aucune homologie avec les autres molécules anti-complément connues à ce jour, suggérant que cette protéine a été acquise au cours de lévolution par un mécanisme dévolution convergente. En plus de cet aspect fondamental, Isac représente un candidat antigénique prometteur pour le développement dun vaccin anti-tique potentiellement capable dinduire le rejet de la tique et/ou de prévenir la transmission des pathogènes. Le but initial de cette thèse était didentifier le ou les homologue(s) de la protéine Isac de la tique américaine Ixodes scapularis chez la tique européenne Ixodes ricinus. De façon intéressante, deux séquences différentes ont été isolées du transcriptome des glandes salivaires de la tique I. ricinus (Daix et al., (2007) Insect Mol. Biol. 16 (2), 155-166). Lexpression de ces séquences a révélé quelles codent pour des protéines secrétées capables dinhiber la voie alterne du complément. Ces protéines ont été appelées IRAC I et II pour « Ixodes ricinus anti-complement protein I and II ». La caractérisation des IRACs à laide danticorps monoclonaux a permis de révéler que ces deux protéines sont exprimées de façon constitutive au sein des glandes salivaires de la tique Ixodes ricinus et sont sur-exprimées au cours du repas sanguin. Létude de lexpression des protéines IRAC I et IRAC II au sein de la population dIxodes ricinus a révélé que ces deux protéines sont des paralogues codés par des gènes différents et non par des allèles dun même locus. Enfin, des analyses phylogéniques portant sur les différentes séquences codant pour les protéines homologues à Isac isolées chez les tiques Ixodes scapularis, Ixodes ricinus et Ixodes pacificus ont révélé que les tiques appartenant au complexe Ixodes ricinus codent pour une famille encore non décrite de molécules anti complément qui se sont diversifiées au cours de lévolution par un processus de sélection darwinienne positive. Les analyses phylogéniques des IRACs suggèrent que ces séquences ont subi une diversification par un processus de pression de sélection darwinienne positive, menant probablement à des molécules possédant des propriétés biologiques différentes. Dans la seconde étude, nous avons testé lhypothèse de travail selon laquelle chaque paralogue pourrait posséder des activités inhibitrices différentes à lencontre du complément de différentes espèces dhôtes naturels, contribuant ainsi à élargir le spectre dhôte des tiques I. ricinus. Les résultats obtenus démontrent que cette hypothèse est correcte (Schroeder et al., (2007) Microbes Infect. 9 (2), 247-250). Dans la troisième et dernière étude de ce manuscrit, nous avons testé le potentiel des protéines IRACs comme candidat antigénique pour le développement dun vaccin anti tique. Des recombinants de lherpèsvirus bovin 4 (BoHV-4) exprimant IRAC I ou IRAC II ont été produits. De façon intéressante, nous avons observé que bien que les recombinants BoHV-4 expriment de hauts taux de protéines IRACs fonctionnelles in vitro, les lapins immunisés à laide des recombinants BoHV-4 exprimant les IRACs ne développent pas de réponse humorale détectable à lencontre des transgènes. Dans le but daugmenter limmunogénicité des IRACs exprimés comme transgène, une seconde génération de recombinants BoHV-4 a été produite. Ceux-ci induisent lexpression des IRACs sous la forme de protéines de fusion transmembranaires à la surface des cellules infectées. Linoculation de ces recombinants à des lapins a engendré le développement dune forte réponse humorale à lencontre des IRACs. Néanmoins, cette réponse immune na pas engendré deffet majeur sur le repas sanguin de femelles Ixodes ricinus placées sur les lapins immunisés. / An increasing number of studies demonstrate that inhibition of host complement activation is crucial for completion of the blood feeding process of hematophagous parasites. A review of these studies has been published in Developmental and Comparative Immunology (Schroeder et al., (2009) Dev. Comp. Immunol. 33, 5-13). Several observations suggest that inhibition of the host complement alternative pathway by tick salivary proteins is crucial for the achievement of blood feeding and efficient transmission of the pathogens transmitted by the parasite. Strongly supporting this conclusion, a salivary protein able to inhibit the alternative pathway was cloned from the American tick Ixodes scapularis (Valenzuela et al., (2000) J. Biol. Chem. 275, 18717-18723). Interestingly, this molecule, termed Isac, has no similarity to any previously reported anti-complement molecules suggesting that it has been acquired through a mechanism of convergent evolution. In addition to this fundamental aspect, Isac is also a promising candidate antigen for the development of an anti-tick vaccine potentially able to induce the reject of the tick and/or to prevent the transmission of the pathogens. The initial goal of the present work was to clone the orthologue of Isac from the European tick Ixodes ricinus. Interestingly, two different sequences were isolated from the transcriptome of I. ricinus salivary glands (Daix et al., (2007) Insect Mol. Biol. 16 (2), 155-166). Expression of these sequences revealed that they both encode secreted proteins able to inhibit the complement alternative pathway. These proteins were called I. ricinus anticomplement (IRAC) protein I and II. Further characterization of IRACs using monoclonal antibodies revealed that both proteins are expressed constitutively in I. ricinus salivary glands and are up-regulated during blood feeding. Analysis of a series of individual ticks revealed that all ticks tested express both IRAC I and IRAC II, demonstrating that they are the products of different genes and not of alleles of the same locus. Finally, phylogenetic analyses of the I. ricinus IRAC I and II sequences together with homologues from I. scapularis and I. pacificus demonstrates that ticks belonging to the Ixodes ricinus complex encode a new family of relatively small anti-complement molecules undergoing diversification by positive Darwinian selection. Phylogenetic analyses of IRACs suggested that these sequences were diversifying by a process of positive Darwinian selection, possibly leading to molecules with different biological properties. In the second study, we tested the hypothesis that each paralogue may have different inhibitory activities against the complement of different natural host species, thereby contributing to broaden the host range of I. ricinus ticks. The data obtained demonstrated that this working hypothesis is correct (Schroeder et al., (2007) Microbes Infect. 9 (2), 247-250). In the third and last chapter of the present manuscript, we addressed the potential of IRAC I and II as candidate antigens for the development of an anti-tick vaccine. Bovine herpesvirus 4 (BoHV-4) recombinants expressing IRAC I or II were produced. Interestingly, we observed that although both recombinants expressed high levels of functional IRAC proteins in vitro, our attempts to immunize rabbits against IRACs via infection with these viruses invariably failed. In order to improve the immunogenicity of IRACs expressed as transgene, a second generation of BoHV-4 recombinants was produced. The latter expressed IRACs as transmembrane fusion proteins on cell surface. Comparison of the vaccine potential of BoHV-4 recombinant viruses expressing either secreted or transmembrane IRAC proteins revealed that while the former did not induce a detectable immune response against IRACs, the latter led to high titers of anti-IRAC antibodies. However, the immune response induced against IRACs did not lead to the reject of the tick but only slightly increased the duration of the blood feeding process.

L'esca est un syndrome cryptogamique vasculaire de la vigne (Vitis vinifera). Une phase pionnière, sous la dépendance de Phaeoacremonium aleophilum (PA), Phaeomoniella chlamydospora (PC) et éventuellement de Eutypa lata (EL) permet la mise en place d'autres espèces. Depuis l'interdiction de l'arsénite de sodium, cette maladie n'est plus contrôlée.<br /> Nous avons étudié les propriétés de PA, PC et EL, en particulier l'impact sur leur croissance de fongicides commerciaux et de fongicides systémiques synthétisés au laboratoire, ainsi que de molécules naturelles. Ces pathogènes ne présentent pas la même sensibilité à ces molécules et l'un d'eux (PA) est peu affecté par divers traitements. En parallèle, nous avons étudié les propriétés d'ambimobilité de l'acide salicylique (AS) et de quelques-uns de ses dérivés halogénés.<br /> Nous avons montré que F 30, un dérivé acide du fenpiclonil, est mobile dans les boutures de vigne après application foliaire. Il est en partie retenu dans le bois et libère la molécule parent dans les racines. L'acide 5-chlorosalicylique (5-ClAS), connu pour être plus actif que AS pour stimuler les défenses naturelles, présente une mobilité voisine de celle de AS. Sur ces bases, F 30 et 5-ClAS ont été retenus pour des tests préliminaires de traitement par voie foliaire de boutures de vigne infectées.<br /> Cette recherche exploratoire souligne la complexité de la problématique, une lutte chimique (fongicide), génératrice de contraintes, devant s'intégrer dans une stratégie globale de contrôle.

Ticks are of global interest as the pathogens they spread can cause diseases that are of importance to both human health and economies. In Scotland, the most populous tick species is the sheep tick Ixodes ricinus, which is the vector of pathogens causing diseases such as Lyme borreliosis and Louping-ill. Recently, both the density and spread of I. ricinus ticks have grown across much of Europe, including Scotland, increasing disease risk. Due to the nature of the tick lifecycle they are particularly dependent on environmental factors, including temperature and habitat type. Because of this, the recent increase in tick-borne disease risk is believed to be linked to climate change. Many mathematical models have been used to explore the interactions between ticks and factors within their environments; this thesis begins by presenting a thorough review of previous modelling of tick and tick-borne pathogen dynamics, identifying current knowledge gaps. The main body of this thesis introduces an original mathematical modelling framework with the aim to further our understanding of the impact of climate change on tick-borne disease risk. This modelling framework takes into account how key environmental factors influence the I. ricinus lifecycle, and is used to create predictions of how I. ricinus density and disease risk will change across Scotland under future climate warming scenarios. These predictions are mapped using Geographical Information System software to give a clear spatial representation of the model predictions. It was found that as temperatures increase, so to do I. ricinus densities, as well as Louping-ill and Lyme borreliosis risk. These results give a strong indication of the disease risk implications of any changes to the Scottish environment, and so have the potential to inform policy-making. Additionally, the models identify areas of possible future research.