Die Verfütterung von Fetten mit mehrfach ungesättigter Fettsäuren (PUFA) stellt seit Jahren eine nebenwirkungsfreie Alternative in der Behandlung atopischer Erkrankungen wie der Caninen Atopischen Dermatitis (CAD) dar. Zahlreiche Studien belegen die antiinflammatorische Wirkung der PUFA und die damit verbundene Besserung der klinischen Symptomatik. Diese antiinflammatorische Wirkung kommt auf molekularer Ebene zustande, indem die supplementierten Fettsäuren die Eigenschaften zellulärer Membranen verändern, intrazelluläre Signalwege und Enzyme beeinflussen sowie die Expression verschiedener Gene modulieren. Die cytosolische Phospholipase A2 (cPLA2) stellt eines der Schlüsselenzyme im Rahmen der Synthese proinflammatorischer Eikosanoide dar, die allergische Symptome wie Juckreiz und Hautrötungen bedingen. Sie spaltet bevorzugt Arachidonsäure (AA) aus der sn2-Position membranständiger Phospholipide ab und stellt somit das Substrat für die Synthese von Entzündungsmediatoren wie Prostaglandinen, Leukotrienen und Thromboxanen zur Verfügung. Peroxisomen-Proliferator-aktivierte Rezeptoren (PPAR) gehören zur Großfamilie der Kernrezeptoren. Besonders die Isoform PPARγ ist an der Regulation der Expression verschiedener proinflammatorischer Proteine beteiligt, wie z.B. der Cyclooxygenase (COX). PUFA gehören zu den natürlichen Liganden dieser Rezeptoren, was die Vermutung nahe legt, dass zumindest ein Teil der Wirkungen über die Beeinflussung dieser Rezeptoren vermittelt wird. Über die Beeinflussung der Expression des cPLA2-Gens durch PPAR existieren zurzeit noch keine Erkenntnisse. Mastzellen spielen eine zentrale Rolle im Rahmen der Pathogenese allergischer Erkrankungen. Auf einen allergenen Stimulus hin sind sie in der Lage, präformierte (z.B. Histamin) und de novo synthetisierte Entzündungsmediatoren (z.B. Eikosanoide) freizusetzen und damit die für allergische Erkrankungen typischen Symptome hervorzurufen. Das Ziel der vorliegenden Arbeit war es, an einer caninen Mastozytomzelllinie (C2) die zeitabhängigen Effekte der Supplementierung des Zellkulturmediums mit jeweils 20 μM n3- PUFA (α-Linolensäure LE, C18:3n3; Eikosapentaensäure EPA, C20:5n3) und n6-PUFA (Linolsäure LA, C18:2n6; Arachidonsäure AA, C20:4n6) auf das Wachstum, das zelluläre Fettsäurenmuster, die Histaminfreisetzung, die Aktivität und Expression der cPLA2 sowie die Expression von PPARγ zu untersuchen. Vergleichend wurden die C2 auch im Grundmedium kultiviert. Zur Untersuchung des Einflusses der PUFA auf das Wachstum der Zellen erfolgte die Kultivierung über einen Zeitraum von 8 Tagen. Um die zeitabhängigen Effekte feststellen zu können, wurden die Zellen 6 h, 48 h bzw. 6 Tage in den entsprechenden Medien bzw. dem Grundmedium kultiviert und danach die folgenden Verfahren angewendet. – Bestimmung des zellulären Fettsäurenmusters mittels Gaschromatographie (GC) – Bestimmung der Histaminfreisetzung mit Hochleistungsflüssigkeitschromatographie (HPLC) – Bestimmung der Aktivität der cPLA2 über Messung der freigesetzten [3H]Arachidonsäure – Bestimmung der Expression der cPLA2 und des PPARγ mittels Western Blot. Die Ergebnisse zeigen, dass die Fettsäuren-Supplementierung keinerlei Auswirkung auf das Zellwachstum hat. Sie führt aber zur Anreicherung der jeweils angebotenen Fettsäure sowie ihrer Elongations- und Desaturierungsprodukte. Die Effekte auf die spontane und auch auf die Mastoparan-stimulierte Histaminfreisetzung waren zeitabhängig sehr unterschiedlich. Die Zugabe der Fettsäuren hatte auf die spontane cPLA2-Aktivität der Zellen nur bei längerer Kultivierungsdauer (6 d) einen Einfluss, wobei es zu einer Minderung der Aktivität nach PUFA- Gabe kam. Im Gegensatz dazu zeigten die Mastoparan-stimulierten C2 nur nach 6- stündiger Kultivierung eine signifikante Erhöhung der cPLA2-Aktivität in den mit den n6-FS supplementierten Medien. Die cPLA2-Expression war in ihrer Höhe von der Kultivierungsdauer abhängig. Nach 6 h zeigten die PUFA-supplementierten C2 eine im Vergleich zum Grundmedium gesteigerte Expression, unabhängig von der Art der zugesetzten Fettsäure. Nach 48 h war die Expression in den C20-PUFA-supplementierten C2 deutlich erhöht, während sie nach 6-tägiger Kultivierung in den mit C20-PUFA angereicherten Medien signifikant reduziert war. Der fehlende Zusammenhang zwischen der Aktivität des Enzyms und der Höhe der cPLA2-Expression lassen auf eine unterschiedliche Regulation dieser beiden Parameter schließen. Die PPARγ-Expression verhielt sich, besonders nach 48-stündiger und 6- tägiger Kultivierung, umgekehrt zur cPLA2-Expression. Dabei deutet das annähernd gegensätzliche Verhalten von cPLA2 und PPARγ nach 48 h und 6 d auf eine mögliche Beteiligung des Kernrezeptors an der Regulation der cPLA2-Expression hin. Aus den vorliegenden Untersuchungen geht somit deutlich hervor, dass die C2 mit der cPLA2 und dem PPARγ zwei bedeutende Regulatoren von Entzündungsprozessen exprimieren und somit als Modell zur Erforschung der Pathogenese der CAD geeignet sind.

Francisella tularensis is a gram-negative, highly virulent, intracellular bacterium which causes the zoonotic disease tularemia. The subspecies tularensis and holarctica are clinically important, and the former is the more virulent. The intracellular lifestyle of F. tularensis is not completely understood, but after uptake in monocytes, the bacterium escapes from the phagosome within hours and replicates massively in the cytosol. The escape is dependent on factors encoded by the Intracellular Growth Locus (igl) operon, located in the Francisella Pathogenicity Island, FPI. The thesis was aimed to clarify and understand the interaction of F. tularensis strains with the endosomal pathway of monocytic cells in general and the roles of the Igl proteins and the global regulator MglA for this interaction in particular. A focus has also been to elucidate the roles of reactive oxygen and nitrogen species for the intracellular host-parasite interaction. We show that mutants in the IglB, IglC, or IglD proteins or their regulator MglA of the live vaccine strain, LVS (subspecies holarctica), all demonstrated reduced replication rates and lowered cytopathogenicity compared to the wild type in a J774 mouse macrophage cell model. Colocalization with LAMP-1 was significantly increased for the IglC, IglD and MglA mutants compared to LVS. This indicated an impaired ability to escape into the cytoplasm, while at the same time they, like LVS, partly prevented fusion with lysosomes. IFN-γ activation of the J774 host cells prior to infection had a bactericidal effect on LVS and all of the mutants, though the cidal effect was significantly more pronounced for the mutants. Following IFN-γ activation, a majority of the mutant-containing phagosomesfused with lysosomeswhile LVS remained localized in the cytosol without significantly increased interactions with the endosomal pathway. Previous studies have revealed that IFN-γ activation of F. tularensis-infected macrophages leads to control of infection but conclusions about the importance of reactive nitrogen and oxygen species on bacterial killing are inconsistent. We found that the growth inhibition resulting from IFN-γ activation could not be attributed to an increased oxidative burst since PMA-induced superoxide production was still inhibited by LVS to the same extent as in non-activated macrophages. On the other hand, reactive nitrogen species may in part have contributed to the cidal effect. To further assess the role of reactive nitrogen species to the killing of F. tularensis, nitric oxide was administrated exogenously to J774 cells infected with LVS. This led to significant killing of intracellular LVS with a concomitant increased phagosomal localization and downregulation of the virulence gene regulator mglA. These effects were reversed by addition of a peroxynitrite decomposition catalyst. A spontaneous avirulent mutant of subspecies tularensis, strain FSC043, was previously demonstrated to provide protective immunity in mice. Here, microscopic analyses of the strain revealed an unusual intracellular localization with a delayed phagosomal escape. This may account for the low virulence, while at the same time FSC043 remains immunogenic and thereby confers protection. The igl operon is intact in strain FCS043 and we hypothesize that a defect in the FPI gene pdpC contributed to the observed phenotype. Altogether, this thesis work demonstrates the importance of the mglA and igl genes for the virulence of F. tularensis and specifically their important roles for a functional phagosomal escape and inhibition of the host cell oxidative burst. Also, addition of exogenous nitric oxide likely leads to formation of peroxynitrite intracellularly, a reactive molecule which confines the bacterium to the phagosome and confers a significant bactericidal effect on intracellular F. tularensis.

L'interferon-γ (IFNγ) est une cytokine immunomodulatrice puissante, également dotée d'une activité antivirale. Il possède deux ligands de haute affinité : un récepteur par lequel il transmet ses signaux et des polysaccharides complexes de la famille des héparanes sulfates (HS), tous deux situés à la surface cellulaire. In vivo, la liaison aux HS permet de concentrer localement la cytokine et de réguler son activité biologique par le biais d'une protection partielle du domaine C-terminal de la protéine. Ce domaine C-terminal, caractérisé par deux domaines basiques D1 et D2, est impliqué dans la reconnaissance du récepteur et des HS. Dans ce contexte, nos travaux se sont attachés à définir les aspects structuraux de l'interaction de l'IFNg, et plus précisément de son extrémité C-terminale, avec ses deux ligands. Pour cela, de divers mutants ponctuels, multiples et de délétion de l'IFNg ont été produites, purifiées et étudiées. Leur capacité à lier les HS et le récepteur de l'IFNg est déterminée par SPR puis leur influence sur l'activité antivirale de l'IFNg est déterminée. Les paramètres thermodynamiques de l'interaction IFNg:HS-oligosaccharides sont investigués. Par ailleurs, nous avons préparé une banque oligosaccharidique dérivée d'HS. Le criblage de cette banque, pour sa capacité à lier l'IFNγ, a permis de démontrer que l'IFNg reconnaissait des motifs de sulfatation particuliers. Finalement, nous avons tenté de cristallisé le complexe IFNg:HS-oligosaccharide, jusqu'à présent sans obtention de cristaux qui diffractent. Ces différentes approches visent à élucider le mécanisme de reconnaissance d'IFNg par des HS. Ceci afin de concevoir un mime de ce site d'interaction inhibant la signalisation de l'IFNg. Enfin, une compréhension plus détaillé de l'interaction de l'IFNg avec les HS et son récepteur reste à établir afin d'entièrement comprendre comment l'IFNg migre des HS vers l'IFNgR. / Interferon-γ (IFNγ) is a strong immunomodulating cytokine with some antiviral activity. It has two ligands for which it has high affinities: a receptor through which it transmits its signals, and complex polysaccharides of the heparan sulphate (HS) family. Both are situated on the cellular surface. In vivo, binding on the HS permits local concentration of the cytokine, and regulates its biological activity via a partial protection of the C-terminal region. This C-terminal region, characterised by two basic domains, D1 and D2, is implicated in the recognition of the receptor and the HS. In this context, we investigated the structural features for the interaction of IFNγ, and more specifically the importance of his C-terminus, with both of his cellular ligands. Therefore, we produced, purified and examined various mutants of IFNγ, including points, multiples and deletions mutants. There ability to bind to HS and the IFNγ receptor is examined by SPR and there influence on IFNγ's antiviral activity is determined. The thermodynamics complexation of IFNγ with the HS-oligosaccharides is examined. Moreover, we have prepared an oligosaccharidic library derived from HS. By screening this library for its capacity to bind IFNγ, we have demonstrated that the cytokine recognizes a particular sulphated pattern. Finely, we tried to crystallize the IFNγ:HS-oligosaccharide complex, without obtaining diffracting crystals yet. These studies contribute to clarify the mechanism of recognition of IFNγ by the HS. This would enable us to design a mimic of the interaction site for IFNγ on the HS, who inhibits inappropriate signaling of the cytokine. Finely, a detailed comprehension of the interaction of IFNγ with his receptor and with the HS needs to be established to fully understand how IFNγ migrates for the HS to its receptor.

Influenza is a serious illness worldwide, causing high morbidity and mortality. 10-20% of world population fall ill with influenza each year and 250 000 - 500 000 people die annually. The most efficacious protection to date is vaccination. Current vaccines are efficient only one season because of fast mutation rate of influenza virus. The effort to create an effective vaccine faces lack of potent adjuvant, which can adequately stimulate and modulate immune system to protect organism from virus infection. Moreover, todays vaccines administered parenterally do not induce immune response on mucosal surfaces. Bacillus firmus, a Gram-positive non-pathogenic bacterium, has strong immmune-modulating properties and is able to induce cross-protection when administered with influenza virus antigens. Immunization with Bacillus firmus stimulates production of neutralizing antibodies, but other mechanisms of its action remain to be elucidated. To better understand the mechanisms how is antiviral immunity enhanced by Bacillus firmus (delipidated fraction, DBF), the effect of immunization with DBF only was studied on mouse model. In last decade it has become obvious that intranasal immunization can induce both systemic and mucosal immune response and in case of influenza it can induce cross-protection. Therefore...

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First section of chapter 1 deals with gold catalyzed synthesis of ring fused furo[3,2,b]pyrans and furo[3,2,b]furans. Furo[3,2,b]pyrans and furo[3,2,b]furans are ubiquitous structural segments found in a number of natural products including polyether containing marine toxins. Synthesis of furo[3,2,b]pyrans 2a was accomplished from the bis-propargyl ethers 1a, while the synthesis of furo[3,2,b]furans 2b was accomplished from the prenyl propargyl ethers 1b. Scheme 1: Synthesis of furo[3,2,b]pyrans and furo[3,2,b]furans Second section of chapter 1 describes an unusual ring-contraction rearrangement route to functionalized 2,8-oxymethano-bridged di and triquinane. During the course of investigations concerning the total synthesis of 6-oxabicyclo[3.2.1]octane framework containing natural products, an unusual ring-contraction rearrangement sequence was observed in the reaction of 5-substituted 1-methyl-4-isopropenyl-6-oxabicyclo[3.2.1]octan-8-ones 4 to the 2,8-oxymethano-bridged diquinanes 5. The reaction was further demonstrated in the synthesis of triquinane 7 framework. Scheme 2: Synthesis of functionalized di and triquinane In third section of chapter 1 gold catalyzed synthesis of isochromanones and isoquinolones from suitable substituted allyl propargyl ethers was discussed. Synthesis of isochromanones and isoquinolones comprising a quaternary center with high diastereoselectivity was realized via AuCl3 catalyzed tandem intramolecular exo-dig heterocyclization/enol isomerization/Claisen rearrangement sequence in excellent yields. The reaction was general and amenable for the synthesis of structurally diverse analogues. Scheme 3: Synthesis of isochromanones and isoquinolones Forth section of chapter 1 consists of gold catalyzed intramolecular hydroalkoxylation assisted ring opening of furans to the corresponding saturated -keto esters. During the course investigations concerning gold catalyzed intramolecular enyne cyclization reactions, an interesting ring opening of furans in furyl propargyl alcohols to the corresponding tetrahydrofuran appended saturated -keto esters exclusively driven by intramolecular hydroalkoxylation of the alkyne was observed. Reaction of furyl propargyl alcohols without free hydroxyl group, under similar conditions afforded the conjugated enynes involving dehydration/ketalization. Scheme 4: Synthesis of saturated -keto esters and enynes Chapter 2 delineates the enantiospecific synthesis of bicyclo[4.2.2]decadienes 15 via gold catalyzed tandem enyne cyclization, semipinacol rearrangement reaction. Bicyclodecadienes are key structural units of natural products nakafuran-8 and pallescensin B. Scheme 5: Synthesis of bicyclo[4.2.2]decadienes

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The aim of this study was to measure the levels of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin 1 (IL-1) and interleukin 6 (IL-6) in serum of rats experimentally infected with Trypanosoma evansi and to correlate with the hematological parameters. Seventy-six rats (Wistar) were divided into two groups. Group C (control) composed of twenty-eight non-inoculated rats distributed in four subgroups with seven animals each (C3, C5, C10 and C20), which received 0.2 mL saline by intraperitoneally. The group T (infected) formed of forty-eight rats was inoculated intraperitoneally with cryopreserved blood containing 1x106 trypomastigotes per animal. These, eight animals died between 5th -7th days post-infection. The remaining animals were divided into four subgroups with ten animals (T3, T5, T10 and T20) according to parasitemia degree. The blood samples were collected by cardiac puncture at the day 3 (C3, T3), 5 (C5, T5), 10 (C10, T10) and 20 (C20, T20) post infection (pi) to perform the complete blood count and determination of IFN-γ, TNF-α, IL-1 and IL-6 levels using an ELISA quantitative sandwich. Immediately after collection the animals were euthanized. The levels of all measured cytokines increased significantly (P < 0.01) in infected animals compared to the controls. T. evansi infection in rats caused an increase in serum IFN-γ, TNF-α, IL-1 and IL-6 and this increase was observed during the whole experimental infection. In addition, the increase in the cytokine levels was concomitant and directly correlated with parasitemia and anemia development at the parasitemia peak. These results suggest a synergism between these cytokines contributing to the development of anemia and the regulation of the immune response against the parasite. / O objetivo deste estudo foi avaliar os níveis séricos das citocinas pró-inflamatórias interferon-gama (INF-γ), fator de necrose tumoral-alfa (TNF-α), interleucina 1 (IL-1) e interleucina 6 (IL-6) em ratos experimentalmente infectados por Trypanosoma evansi e estabelecer uma correlação com os parâmetros hematológicos. Setenta e seis ratos (Wistar) machos foram divididos em dois grupos experimentais. O Grupo C (controle) foi composto por vinte e oito ratos não inoculados distribuídos em quatro subgrupos com sete animais cada (C3, C5, C10 e C20), que receberam 0,2 mL de solução fisiológica pela via intraperitoneal. O grupo T (infectados) formado por quarenta e oito ratos inoculados intraperitonealmente com sangue criopreservado, contendo 1x106 tripomastigotas de T. evansi por animal. Destes, oito morreram entre o 5º e 7º dia pós-infecção. Os animais restantes foram divididos em quatro subgrupos de dez animais cada (T3, T5, T10 e T20) de acordo com o grau de parasitemia. As amostras de sangue foram coletadas por punção cardíaca, nos dias 3 (C3, T3), 5 (C5, T5), 10 (C10, T10) e 20 (C20,T20) pós-infecção (pi) para a realização do hemograma e determinação dos níveis séricos de INF-γ, TNF-α, IL-1 e IL-6 pela técnica de ELISA tipo sanduíche. Imediatamente após as coletas os animais eram submetidos à eutanásia. Os níveis de citocinas pró-inflamatórias aumentaram significativamente (P<0,01) nos animais infectados em relação ao grupo controle. A infecção por T. evansi em ratos provocou um aumento nos níveis séricos de INF-γ, TNF-α, IL-1, IL-6 e esse aumento foi observado durante toda a infecção experimental. Além disso, o aumento nos níveis de citocinas foi diretamente correlacionado com a parasitemia e o desenvolvimento da anemia. Estes resultados sugerem um sinergismo entre essas citocinas contribuindo para o desenvolvimento da anemia e regulação da resposta imune contra o parasito.

There has been increasing concern regarding the emergence of multi-drug resistant pathogens. The resistance develops when pathogens, especially bacteria, are frequently exposed to conventional antibiotics, as they are heavily used in both human and livestock. This is due to the high target specificity of conventional antibiotics, which places pathogens in high selective pressures and eventually results in drug resistant by mutations. To address this issue, global actions and cooperation are needed. At the same time, new technologies and strategies need to be developed. Host defense peptides (HDPs) are widely found in the innate immune system. They show both direct antimicrobial properties and immunomodulatory activities. The multifaceted functions of HPDs make them less likely to promote antimicrobial resistance. Thus, they are promising as new therapeutics to treat multi-drug resistant infections. In fact, several drug candidates derived from HDPs have entered the clinical trial, but none of them got into the clinic. This is due to several challenges associated with HDPs, such as low in vivo stability, high cost of manufacturing, and toxicity to mammalian cells. In this dissertation, we explored the ability of a new type of unnatural scaffolds (γ-AApeptides) to mimic the functions of HDPs, including both broad spectrum antimicrobial properties and immunomodulatory activities. Furthermore, the efforts to identify simpler and more drug like γ-AApeptide based antimicrobial agents were also discussed. The findings in this dissertation may lead to the development of potential drug candidates to treat multi-drug resistant infections.

The redox cycling of iron is intimately linked to the cycling of C, S, N, P as well as the speciation, mobility, and bioavailability of various toxic contaminants in soils and sediments. Within these environments, the cycling of iron is catalytically driven by iron-oxidizing (FeOB) and iron reducing bacteria (FeRB) which mediate the formation, transformation, and dissolution of various iron-bearing minerals. Under oxic conditions, FeOB promote the formation of iron oxides on or in close proximity of their cell walls and extracellular polymeric substances, and such composite, termed biogenic iron oxides (BIOS), offers highly reactive heterogenous sites that efficiently immobilize trace metals and contaminants alike. However, under reducing conditions, FeRB mediate the reductive dissolution of BIOS and in turn lead to the remobilization of associated contaminants. Conversely, contaminants may become immobilized by secondary iron minerals that form from the metabolic activity of FeRB. Therefore, determining the factors that influence the reactivity of BIOS, as well as the formation of secondary iron minerals is of critical importance to develop a better understanding of the geochemical cycling of iron and in turn the transport of contaminants in the environment. This thesis investigated (1) the impact of simulated diagenesis (ageing for ~5 years at 4ºC) on the mineral stability and reactivity of BIOS towards reduction by Shewanella putrefaciens CN32, (2) the effects of phosphate at an environmentally relevant (10µM) and excess (3.9mM) concentration on the rates and extent of microbial reduction of synthetic 2-line ferrihydrite and BIOS, as well as the formation of secondary iron minerals, and (3) the impact of sterilization by γ-irradiation on the mineral stability and reactivity of BIOS. It was found that simulated diagenesis did not affect the mineralogical composition of BIOS but significantly lowered the reactivity of BIOS towards microbial reduction. The concentration of phosphate was found to have contrasting effects on the rates of reduction of ferrihydrite and BIOS, but in general, excess concentration of phosphate enhanced the extent of Fe(III) reduction. The formation of a specific secondary iron mineral was also found to depend on the concentration of phosphate, as well as, in the case for BIOS, the presence of intermixed cell derived organic matter. γ-irradiation did not alter the mineralogy and reactivity of BIOS towards microbial reduction, and it was concluded to be a suitable technique to sterilize BIOS.

After kidney transplantation the recipient's immune system responds to the donor's antigens and the graft rejection occurs. Borderline changes are a frequent diagnosis after kidney transplantation, representing only mild rejection signs. Some patients with borderline changes undergo progression to rejection. The identification of these at- risk patients by biomarkers will allow enhanced treatment and help to prevent the development of rejection. The aim of my work was to verify biomarkers of rejection in patients with borderline changes. Chemokines CXCL9, CXCL10 and CCL17 in urine/serum of 40 patients with subclinical borderline changes at 3 months and in 25 patients with early borderline changes were determined by ELISA. At 3 months, the higher CXCL10 level predicted rejection with AUC=0.749, p=0.024. High levels of CXL10 had also been found in patients with BKV infection. We did not confirm the relationship between rejection and the CXCL9 and CCL17. In the early posttransplant period the levels of CXCL10 and CXCL9 were elevated in all patients and therefore couldn't be used to predict rejection. The alloreactivity was examined using IFN-γ ELISPOT (n=38). No association between the frequency of IFN-γ producing cells after stimulation with donor cells or CMV peptides and the development of...