O conhecimento a respeito de mecanismos de reprodução e geração de diversidade genética de organismos do gênero Leishmania vem sendo alterado com o advento de novas tecnologias. Estudos de laboratório demonstraram ocorrência de reprodução sexuada experimentalmente em organismos do gênero Leishmania. Relatos na literatura mostram a ocorrência de híbridos no ambiente natural, associada a casos de leishmaniose tegumentar americana (LTA). A partir da caracterização fenotípica realizada utilizando anticorpos monoclonais e eletroforese de isoenzimas em linhagens isoladas de pacientes com LTA na região Amazônica do Brasil, sete isolados (M15983, M15984, M15987, M15988, M19672, M19676 e M19697) apresentaram um padrão intermediário entre as espécies L. (V.) shawi shawi e L. (V.) guyanensis. O presente estudo visou realizar a confirmação da identidade híbrida desses parasitas através da clonagem dos isolados, seguida da análise do polimorfismo pela clivagem do produto da reação em cadeia da polimerase (PCR RFLP) tendo como alvo a enzima de choque térmico de 70 quilodaltons (hsp70). O hsp70 PCR RFLP também foi utilizado para análise comparativa da intensidade dos produtos de digestão, para estimar a ploidia dos híbridos e a proporção da herança dos alelos de hsp70. Foi realizada ainda, avaliação do comportamento biológico in vitro e experimental in vivo desses parasitas. A curva de crescimento foi realizada na 4° e 12° passagem após isolamento do parasita. Infecções experimentais de BALB/c foram realizadas para avaliação da evolução da infecção causada pelo isolado híbrido selvagem M19672 e sua comparação com as cepas parentais L. (V.) guyanensis e L. (V.) s. shawi. Foi determinada a carga parasitaria em pele, linfonodo e baço dos animais infectados e análise histopatológica das lesões. Os resultados obtidos a partir da clonagem dos isolados demonstraram que se tratavam de populações homogêneas de parasitas. Três isolados apresentaram padrão heterozigoto de L. (V.) guyanensis/L. (V.) s. shawi e quatro isolados apresentaram padrão homozigoto, apresentando somente os alelos de L. (V.) s. shawi. O ensaio de clivagem de sequências polimórficas amplificadas (SNP-CAPS) mostrou que os homozigotos apresentaram o mesmo número de cópias de hsp70 de L. (V.) s. shawi, enquanto que nos heterozigotos os alelos de L. (V.) guyanensis são mais representativos. Os parasitas híbridos apresentaram uma maior plasticidade fenotípica no crescimento in vitro. A infeção de BALB/c pelos parasitas híbridos apresentou uma evolução rápida tendo seu maior pico entre 14 e 28 dias pós-infecção, com regressão posterior da lesão. Esse período foi acompanhado da maior carga parasitária na pele e linfonodo. A infecção causada pelas cepas parentais mostrou aumento progressivo da lesão a partir do 21° dia de infecção para L. (V.) guyanensis e 35° dia para L. (V.) s.shawi. As alterações histopatológicas da lesão causada pelo híbrido apresentaram um padrão intermediário entre as de L. (V.) s. shawi e L. (V.) guyanensis, guardando, no entanto, mais características desta última. Nossos achados confirmam que as cepas analisadas são híbridas entre L. (V.) guyanensis e L. (V.) s. shawi da região do Baixo Amazonas, estado do Pará, área com alta ocorrência de casos de LTA na Brasil e grande diversidade de espécies do parasita, vetores e reservatórios / Knowledge about the mechanisms of reproduction and generation of genetic diversity of the genus Leishmania organisms has been changed with the advent of new technologies for it studies. Laboratory researches have shown the occurrence of experimentally sexual reproduction in the genus Leishmania. Reports in the literature shows the occurrence of hybrids in the natural environment, associated with cases of american cutaneous leishmaniasis (ACL). From the phenotypic characterization performed using monoclonal antibodies and isozymes in strains isolated from patients with ACL in the Amazon region of Brazil, seven isolates (M15983, M15984, M15987, M15988, M19672, M19676 and M19697) showed an intermediate pattern between species L. (V.) shawi shawi and L. (V.) guyanensis. This study aimed to perform the confirmation of the hybrid identity of these parasites by performing cloning of the isolated, followed by analysis of polymorphism by cleavage of the polymerase chain reaction product (PCR RFLP) targeting heat shock enzyme 70 kilodaltons (hsp70). The hsp70 PCR RFLP was also used for comparative analysis of the intensity of the digestion products to estimate the ploidy of the hybrids and the proportion of the inheritance of hsp70 alleles. It was also observed in this study the biological in vitro behavior of these parasites. The growth curve in vitro was held at 4° and 12° passage after the isolation of mammalian host. Experimental infections of BALB /c mice were carried out to evaluate the evolution of the infection caused by the hybrid wild strain M19672 compared to the parental strains L. (V.) guyanensis and L. (V.) s. shawi. The parasitic load was determined on the skin, lymph nodes and spleen of infected animals and histopathology of the lesions. The results from the cloning of the isolates showed that these are homogeneous populations of parasites. Three isolates presented heterozygous L.(V.) guyanensis/ L.(V.) s. shawi pattern and four presented homozygous pattern with only the L.(V.) s. shawi alleles. The cleavage amplified polymorphic sequences assay (SNP-CAPS) showed that the homozygous had the same number of copies of hsp70 L. (V.) s. shawi, whereas in the heterozygous alleles L. (V.) guyanensis are more representative. Hybrid parasites demonstrated a higher phenotypic plasticity growth in vitro. BALB /c infection by M19762 hybrids showed a rapid evolution with a peak between 14 and 28 days post-infection, with subsequent lesion regression. The period of greatest development of the lesion was accompanied by higher parasite burden in the skin and lymph nodes. The infection caused by parental strains showed a progressive increase in the lesion from the 21th day of infection for L. (V.) guyanensis, and day 35 PI for L. (V.) s. shawi The alteration in the histopathological lesions caused by the hybrid strain showed an intermediate pattern between those of L. (V.) s. shawi and L. (V.) guyanensis, keeping, however, more characteristics of the latter. Our findings confirm that the strains analyzed are true hybrids between L. (V.) guyanensis and L. (V.) s. shawi from Amazon region, Pará state, an area with high occurrence of ACL in Brazil and diversity of parasite species, vectors and reservoirs

A diversidade de tripanossomas é atribuída a um arsenal gene vasto. HSP compreendem várias famílias que atuam como uma chaperona moleculares em condições de estresse e fisiológicas. A HSP70 em tripanossomas consiste em 9 genes: Canonical, HSP70.4, HSP70.c, GRP78, Lc2.2, HSP70.b, Grp170, HSP110 e HSP70.a. Análises filogenéticas indicam que a evolução de HSP70 segue um padrão de ramificação que coincide com a compartimentação celular. As inferências filogenéticas de Trypanosoma obtidas a partir de genes que codificam HSP70s foram compatíveis com os obtidos por gGAPDH indicando que as HSP70s são uteis como marcadores moleculares. Os genes que codificam proteínas possuindo domínio P3/alfa-cristalino (ACD) na sua estrutura atuam como chaperonas envolvidas na proliferação e migração celular, citoesqueleto, na resposta a agentes patogénicos e desenvolvimento. Nove chaperonas putativas com o ACD foram recuperadas em Trypanosoma: TryDYX1C1, TrySGT1, Tryp23A, Tryp23B, TryNudC1, TryNudC2, HSP20, TryACDP, TryACD-TPR. / The diversity of trypanosomes is attributed to a vast gene arsenal. HSP comprehends several families which acts as a molecular chaperone in stress and physiological conditions. The HSP70 in trypanosomes consists of nine genes: Canonical, HSP70.4, HSP70.c, Grp78, Lc2.2, HSP70.b, Grp170, HSP110 and HSP70.a. Phylogenetic analysis shows that the evolution of HSP70 from Trypanosoma follows branching pattern that coincides with the cellular compartmentation. Phylogenetic Inferences of Trypanosoma obtained from genes encoding HSP70s were compatible with those obtained by gGAPDH indicating that HSP70 gene is a useful molecular maker. Genes encoding proteins having p3/alpha-crystalline domain (ACD) in its structure act as chaperones and co-chaperones involved in cell proliferation and migration, cytoskeleton and in response to pathogens and development. Nine putative chaperones containing the ACD were recovered in Trypanosoma: TryDYX1C1, TrySGT1, Tryp23A, Tryp23B, TryNudC1, TryNudC2, HSP20, TryACDP, TryACD-TPR.

A leishmaniose tegumentar é uma zoonose que ocorre endemicamente em 88 países, caracterizando-se como um grave problema de saúde pública nos países tropicais e subtropicais. Estima-se que o número de pessoas infectadas por Leishmania seja de cerca de 12 milhões e que ocorram entre 700 mil a 1,2 milhões de novos casos anualmente. O Brasil apresenta a maior prevalência de leishmaniose tegumentar na América, sendo que já foram identificadas seis espécies como causadoras de leishmanise tegumentar: Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis, Leishmania (Viannia) guyanensis, Leishmania (Viannia) lainsoni, Leishmania (Viannia) naiffi, Leishmania (Viannia) shawi. Apesar de termos diferentes espécies causadoras de leishmaniose tegumentar no Brasil e com diferentes formas clínicas, a discriminação das espécies de Leishmania responsáveis por determinada lesão ainda é um desafio. Utilizando as técnicas qPCR, HRM e PCR multiplex, com pares de oligonucleotídeos contruídos e direcionados para o gene alvo hsp70, propomos uma alternativa para os ensaios utilizados atualmente para a identificação de Leishmania e diferenciação dos subgêneros Leishmania e Viannia em cultura de promastigota. A identificação do subgênero Leishmania obtida nos nossos resultados em amostras de cepas referência e em amostras de isolados de pacientes com suspeita de leishmaniose tegumentar atendidos no Instituto de Infectologia Emílio Ribas, correspondem ao obtido por PCR-RFLP direcionada para o alvo kDNA com a enzima de restrição HaeIII realizada num estudo anterior. No entanto, este estudo permitiu a identificação do subgênero em apenas um passo, o que contribui para a redução do tempo, custo e manuseio de amostras. Diante desses resultados, sugere-se a validação da técnica em DNA de amostras de amastigotas de lesões tegumentares de pacientes diagnosticados com esta doença / The cutaneous leishmaniasis is a zoonotic disease that is endemic in 88 countries, characterized as a serious public health problem in tropical and subtropical countries. It is estimated that the number of people infected by Leishmania to be about 12 million, occurring between 700 thousand to 1.2 million new cases annually. Brazil has the highest prevalence of cutaneous leishmaniasis in America and six species have been identified as causes of cutaneous leishmaniasis: Leishmania (Leishmania) amazonensis, Leishmania (V.) braziliensis, Leishmania (V.) guyanensis, Leishmania (V.) lainsoni, Leishmania (V.) naiffi, Leishmania (V.) shawi. Although we have different species that cause cutaneous leishmaniasis in Brazil and with different clinical forms, discrimination against Leishmania species responsible for certain injury is still a challenge. Using the techniques qPCR, HRM and multiplex PCR with oligonucleotide engineer to the target gene hsp70, propose an alternative to the currently used assays for Leishmania identification and Leishmania and Viannia subgenus differentiation in parasite promastigote culture. The identification of Leishmania subgenus obtained in our results with parasite promastigote culture of reference strains samples and isolates in patients with suspected tegumentary leishmaniasis attended at the Institute of Infectious Diseases Emilio Ribas, corresponded to that obtained by PCR-RFLP directed to the target kDNA with HaeIII restriction enzyme carried out in a previous study. However, this review allowed the identification of the subgenus in just one step, contributing to the reduction of time, cost and handling of samples. Given these results, we suggest the validation of this technique in DNA of amastigote samples from tegumentary lesions of patients diagnosed with this disease

A proteína Cpn60 faz parte de um grupo de proteínas altamente conservadas que estão envolvidas em funções celulares essenciais. camundongos BALB\\c foram imunizados com 5 ou 10 µg da proteína recombinante (Cpn60r) sozinha ou adicionada à vacina DTP sem hidróxido de alumínio (NADTP). A vacina DTP do Instituto Butantan (DTP) foi usada como controle. Foi avaliada a produção de citocinas por células esplênicas após reestímulo in vitro com a Cpn60r. Os animais foram desafiados após o protocolo de imunização. A Cpn60r sozinha ou misturada à vacina NADTP foi capaz de induzir níveis de anticorpos contra pertussis mais altos do que os induzidos pela DTP. Os níveis de IgG1 e IgG2a foram similares para todos os grupos. Pôde-se observar a produção de de IL-6 e IFN-&#947 nos grupos imunizados com Cpn60r. Os grupos imunizados com Cpn60r+NADTP apresentaram um índice de proteção entre 60 e 80% contra o desafio pela bactéria virulenta, semelhante ao grupo imunizado com DTP. A proteína Cpn60r é bastante promissora não somente como imunógeno, mas também como adjuvante. / The Cpn60 protein is a member of a group of higly conserved proteins linked to essencial cell functions. The Cpn60 was cloned, expressed and its immune response has been evaluated. BALB\\c mice were immunized with 5 or 10 µg of the recombinant protein (Cpn60r) alone or mixed with DPT vaccine without aluminum hidroxyde (NADPT). The DPT vaccine from Instituto Butantan was used as control. We evaluated the cytokines production by spleen cells after they have been reestimulated in vitro with Cpn60r. The animals were challenged after the immunization protocol. The Cpn60r alone or mixed with NADPT vaccine was able to induce higher antibodies levels than DPT. IgG1 and IgG2a levels were similar in all groups. We could detect levels of IL-6 and IFN-&#947 on groups immunized with Cpn60r. The groups immunized with Cpn60r+NADTP showed a 60 and 80% protection rate against the challenge with the live bacteria, similar to the group immunized with DPT. These results show the immune response of the recombinant protein that could be included in immunization protocols for pertussis.

O dermatófito Trichophyton rubrum é um fungo filamentoso, queratinofílico e antropofílico, sendo o principal agente etiológico de micoses cutâneas em humanos. Sua distribuição cosmopolita e seu acometimento grave em pacientes imunocomprometidos fazem dele um dos desafios a ser enfrentado pelos serviços de saúde pública mundial. As interações patógeno-hospedeiro envolvem diferentes processos relacionados com a degradação de queratina e com modificações metabólicas que permitem sua adesão e posterior penetração nos tecidos infectados. Essas alterações são importantes para o sucesso do processo infeccioso e envolvem mecanismos que modulam a expressão gênica, a secreção de proteínas específicas, a adaptação metabólica e as alterações no pH cutâneo, fundamentais para o estabelecimento da infecção. Dentre as proteínas que participam do processo de interação patógeno-hospedeiro estão as proteínas de choque térmico HSPs (Heat Shock Proteins), relacionadas aos mais diversificados processos celulares. Nesse sentido, a hipótese desse trabalho foi avaliar se os genes hsps de T. rubrum, bem como seu principal fator de transcrição (Hsf1), estão envolvidos nos processos de resposta a situações adversas e na interação com o microambiente hospedeiro, e se estes genes são modulados pelo fator de transcrição pacC, um regulador envolvido na sinalização do pH ambiente. Para tanto, a expressão dos genes hsps foi analisada em resposta ao cultivo de T. rubrum em diferentes meios de cultura, durante a exposição a antifúngicos, estresse térmico e interação com fragmentos de unha e pele humanas. O envolvimento da Hsp90 na modulação da expressão gênica, na suscetibilidade a antifúngicos e na interação de T. rubrum com fragmentos de unha humana foi avaliado utilizando-se um inibidor químico específico para esta proteína. Os resultados indicam uma expressão variável dentre os genes hsps e até mesmo dentro de cada família HSP, em resposta a cada condição ambiental ou interação a que o fungo foi exposto. Além disto, temos indício de que a expressão dos genes hsps seja modulada pelo fator de transcrição PacC, através da modulação do fator de transcrição Hsf1. Constatamos ainda, a influência de Hsp90 na susceptibilidade de T. rubrum às drogas Itraconazol e Micafungina, e no desenvolvimento de T. rubrum em fragmentos de unha humana. Esses resultados revelam a participação das proteínas HSPs em diversos aspectos do metabolismo de T. rubrum, e sugerem a participação de Hsp90 na patogenicicade e na suscetibilidade a drogas deste dermatófito / The dermatophyte Trichophyton rubrum is a filamentous, keratinophilic, and anthrophophilic fungi, being the major etiologic agent of cutaneous mycoses in humans. Its cosmopolitan distribution and the severe infection in immunocompromised patients make it one of the challenges to be faced by public health agencies worldwide. Hostpathogen interactions involve different processes related to keratin degradation and metabolic changes that allow adhesion and subsequent penetration of the infected tissue. These changes are important to the success of the infectious process and involve mechanisms that modulate gene expression, secretion of specific proteins, and metabolic adaptation, and cutaneous pH changes, essential to the establishment of the infection. Among the proteins that participate in the host-pathogen interaction are the heat shoch proteins (HSPs), related to diverse cellular processes. Thus, the hypothesis of this work was to evaluate whether T. rubrum hsp genes, as well as their major transcription factor (Hsf1), are involved in the response to adverse situations and in the interaction with the host microenvironment, and if these genes are regulated by the transcription fator PacC, a regulator of the pH signaling pathway. The expression of the hsp genes was evaluated in response to the cultivation of T. rubrum in different culture medium, during exposure to antifungal drugs, heat stress, and interaction with human nail and skin. The involvement of T. rubrum Hsp90 in the modulation of gene expression, susceptibility to antifungal drugs, and interaction with human nails was evaluated by using a chemical inhibitor, specific to this protein. Our results indicate a variable expression of the hsp genes, even among members of the same HSP family, in response to each environmental condition or interaction, to which the fungus was exposed. Furthermore, we have evidence that the hsp gene expression is modulated by the PacC transcription factor, by modulating the expression of the Hsf1 coding gene. We also found that Hsp90 is involved in T. rubrum susceptibility to the drugs Itraconazole and Micafungin, and in the development of this dermatophyte in human nails. These results reveal the involvement of HSPs in several aspects of T. rubrum metabolism, suggesting a role for Hsp90 in the pathogenicity and drug susceptibility in this dermatophyte

Die einzellige Grünalge C. reinhardtii dient als Modellorganismus und ist aufgrund des GRAS-Status, des schnellen, kostengünstigen Wachstums und der Möglichkeit posttranslationaler Modifikationen im Kerngenom, als Expressionssystem für beispielsweise orale Impfstoffe sehr interessant. Herausforderungen sind die im Vergleich zu konventionell verwendeten Expressionssystemen sehr geringen Expressionsraten im Kerngenom. Daher sollten in dieser Arbeit neuartige, teils induzierbare, Promotorkonstrukte verwendet und mittels Luciferase-Reporter auf ihre Expressionssteigerung hin getestet werden. Zunächst wurden ausgewählte Promotoren des Chlorella-Virus-1 (PBCV-1) gewählt, diese führten allerdings zu keiner Expression. Außerdem wurden synthetische, aneinandergereihte Hitzeschockelemente mit dem endogenen RBCS2-Promotor fusioniert und die Expressionsraten analysiert. Dabei ergab sich bei der Kombination aus dem synthetischen Hitzeschockelement in achtfacher Wiederholung (HSE8x) mit RBCS2 nach der Hitzeinduktion eine Steigerung der Expressionsrate um das bis zu dreifache. Die Basalexpression war hierbei bei HSE1x-RBCS2 am höchsten und erreichte Expressionslevels, welche um das fünffache höher lagen als die Positivkontrolle HSP70A-RBCS2. Mittels Chromatinimmunopräzipitation mit dem Antikörper gegen HSF1 konnte gezeigt werden, dass eine Bindung an das synthetische Hitzeschockelement vorliegt und deshalb die Expression über die konventionelle Hitzeschockantwort in Chlamydomonas reinhardtii funktioniert. Im Gegensatz zur Luciferase benötigen Fluoreszenzproteine als Reporter kein Substrat. Infolge dieses Vorteils und der Möglichkeiten der FACS-Analyse und Fluoreszenzmikroskopie wurde tagRFP als neuer Reporter in C. reinhardtii etabliert. Die Erhöhung der Expressionsrate durch neue Promotorkombinationen und die Anwendung von tagRFP als neuen Fluoreszenzreporter bedeuten wichtige Schritte in der Etablierung von C.reinhardtii als Expressionssystem für Produkte in der Biotechnologie. / The unicellular green alga C.reinhardtii which is used as a model organism could be an interesting expression system for oral vaccines. This is because the alga is generally regarded as safe, it shows fast growth rates and culturing is cheap. Furthermore it offers the possibility of posttranslational modifications. Challenges lie in the low expression rates in the nuclear genome when compared to other expression systems. Therefore the first step in this work was to test whether selected Chlorella virus PBCV-1 promoters, do lead to enhanced expression rates, but there was no detectable expression. Furthermore synthetic repeats of heat shock elements were used in combination with the endogenous RBCS2-promoter and analysed for expression rates via reporter measurements. The combination of synthetic heat shock elements in eightfold repeats in combination with RBCS2 enhanced expression rates of luciferase after heat shock up to threefold in comparison with the up to now strongest known promoter combination HSP70A-RBCS2. Basalexpression turned out to be best for the HSE1x-RBCS2 promoter and reached expression levels fivefold higher compared to HSP70A-RBCS2. To examine if the artificial heat shock elements (HSEs) are bound by the heat shock factor 1 in C. reinhardtii a ChIP assay with HSE8x-RBCS2 and the antibody of HSF1 of C. reinhardtii was done. It could be shown that HSF1 binds HSEs and therefore one can explain the heat shock inducibility of HSEs is regulated via the conserved heat shock response. In contrast to luciferase fluorescence reporters do not need substrate. Because of this advantage and the possibility of FACS analyses and fluorescence microscopy tagRFP was established as new reporter in C.reinhardtii. Enhancement of expression rates through new constitutive and inducible promoter combinations and the possible use of tagRFP as new fluorescence reporter are significant steps in establishing C.reinhardtii as expression system for products in biotechnology.

HSP25 ist ein Vertreter der ubiquitär verbreiteten kleinen Hitzeschockproteine, einer Familie innerhalb der großen Klasse der Streßproteine. Es ist an der Vermittlung von zellulärer Streßtoleranz beteiligt, besitzt Chaperoneigenschaften, hemmt die Actinpolymerisation in vitro und ist in der Lage, supramolekulare Komplexe auszubilden. Die hier vorgelegte Arbeit befaßte sich mit der Isolierung, der strukturellen und funktionellen Charakterisierung des Proteins und seinem Vorkommen in verschiedenen Geweben von Ratten mit pathologisch erhöhtem Blutdruck: · Es wurde eine Methode zur schonenden Isolierung von HSP25 aus Ehrlich-Ascites-Tumor (EAT) etabliert. Daraus konnte niedrig- und hochmolekulares Material gewonnen werden. · Unter Anwendung elektronenmikroskopischer und hydrodynamischer Methoden konnte für die hochmolekularen Komplexe des nativen HSP25 ein Strukturmodell abgeleitet wer-den. Es ist durch eine zylinderförmige Struktur aus vier gestapelten Ringen mit je acht HSP25-Monomeren charakterisiert. · Hochmolekulare Komplexe des rekombinanten HSP25 liegen demgegenüber als kom-pakte globuläre Strukturen vor. Elektronenmikroskopische Analysen verschiedener Mutanten und von phosphoryliertem HSP25 zeigen, daß die HSP25-Komplexe mit zu-nehmender Phosphorylierung kleiner werden. Dies belegt einen Zusammenhang zwischen dem Phosphorylierungszustand des Proteins und seiner supramolekularen Organisation. · Mittels Elektronenmikroskopie und Fluoreszenzspektroskopie konnte gezeigt werden, daß nur natives HSP25 aus EAT, aber nicht rekombinantes HSP25 oder HSP25-Mutanten die Actinpolymerisation hemmen. Dies bestätigt den Befund, daß nur unphosphorylierte nati-ve HSP25-Monomere für die Inhibierung der Actinpolymerisation verantwortlich sind. · Es konnten zwei HSP25-Peptide identifiziert werden, die eine Hemmung der Actinpoly-merisation auslösen. Damit konnte erstmals experimentell nachgewiesen werden, daß be-stimmte Sequenzabschnitte von HSP25 eine spezifische Wechselwirkung mit Actin ein-gehen. Mit Hilfe phosphorylierter HSP25-Peptide konnte die Abhängigkeit dieser Reaktion vom Phosphorylierungszustand bestätigt werden. · Vorläufige Ergebnisse mit Zellulose-gebundenen Peptid-Bibliotheken deuten auf eine Wechselwirkung von HSP25 mit einem exponierten Loop in Actin-Domäne IV hin, einem Bereich, der an Actin-Actin-Wechselwirkungen beteiligt ist. · HSP25 hat aufgrund seiner verstärkten Synthese bei vielen pathologischen Prozessen eine medizinische Relevanz. Untersuchungen an verschiedenen Tiermodellen zeigen, daß es bei Hypertonie-belasteten Herzen verstärkt im rechten Ventrikel akkumuliert wird. · Es wird ein Modell vorgestellt, in dem die Struktur-Funktions-Beziehungen für HSP25 zusammengefaßt sind. / HSP25 is a member of the ubiquitous family of small heat shock proteins belonging to the big class of stress proteins. It is related to acquiring of cellular thermotolerance, can act as molecular chaperone, is able to inhibit polymerization of actin in vitro and can form high molecular weight complexes. In this thesis the isolation, structural and functional characteri-zation of this protein as well as its abundance in different tissues of rats suffering on patho-logical forms of hypertension is analyzed: · A method for rapid isolation of HSP25 out of Ehrlich-ascites-tumor (EAT) was estab-lished. From isolated HSP25 low and high molecular weight material could be obtained. · Analysis of high molecular weight complexes by means of electron microscopy and ana-lytical ultracentrifugation results in a structural model characterized by a cylindrical structure composed of four stacked rings each containing eight HSP25 monomers. · High-molecular weight complexes of recombinant HSP25 are organized as compact globular structures. Electron microscopic investigations of different mutants and of in vi-tro phosphorylated HSP25 show a connection between phosphorylational status and su-pramolecular organization of the protein: the higher the degree of phosphorylation the smaller are the HSP35-complexes. · By means of electron microscopy and fluorescence spectroscopy it could be shown that only native HSP25 from EAT but not recombinant HSP25 nor HSP25 mutants inhibit polymerization of actin. This is in agreement with recent results showing that only un-phosphorylated native HSP25 monomers are active inhibitors of actin polymerization. · Two HSP25 derived peptides could be identified as competent inhibitors of actin polym-erization. This is the first experimental evidence for a specific interaction of HSP25 se-quences with actin. This interaction is dependent on the phosphorylational status as con-firmed by phosphorylated HSP25 peptides. · Preliminary results with cellulose bound peptide libraries indicate an interaction of HSP25 with an exposed loop in actin domain IV, an area involved in actin-actin interactions. · HSP25 is of medical relevance because of its increased synthesis in a bright variety of pathological processes. Investigations on different animal models of hypertension show an enhanced accumulation of HSP25 in the right ventricle. · A model is presented summing up the structure-function relationships of HSP25.

Die Zytokine Interleukin-2 und Interferon-a2a nehmen eine zentrale Stellung bei der Behandlung von Patienten mit metastasierten Nierenzellkarzinomen ein. Im klinischen Teil der Arbeit wurde die Effektivität der Chemoimmuntherapie monozentrisch an 107 Patienten untersucht. Es zeigte sich eine Rate objektiver Remissionen von 22 % (95 % CI 14,6 - 31,3 %) mit einer zusätzlichen Rate an vorübergehenden Stabilisierungen von 46 %. Das mediane Überleben lag bei 19 Monaten mit einer kalkulierten 5-Jahres-Überlebensrate von 17 %. In der multivariaten Analyse zeigte sich eine hochsignifikante Abhängigkeit vom Karnofsky-Performance-Index der Patienten. Bei den 83 Patienten mit einem Karnofsky-Performance-Index von über 80 % lag das mediane Überleben bei 23 Monaten mit einer kalkulierten 5-Jahres-Überlebensrate von 21 %. Diese Untersuchungsergebnisse rechtfertigen eine weitere Prüfung der Kombination von Interleukin-2, Interferon-a2a und 5-Fluorourazil im Rahmen prospektiver klinischer Studien. In Weiterentwicklung der klinischen Aspekte zur Zytokin-basierten Immuntherapie war es die Aufgabenstellung des experimentellen Teils dieser Arbeit, die Bedeutung von 70 kDa Hitzeschockproteinen (HSP70) beim Nierenzellkarzinom zu untersuchen. Neben ihrer Funktion im Rahmen der zellulären Stressverarbeitung haben Mitglieder der HSP70-Familie (HSP72 und HSP73) eine außerordentliche Bedeutung für tumorimmunologische Fragestellungen erlangt, die sich aus der immunologischen Signalfunktion, der HSP72-vermittelten Interaktion von immunologischen Effektorzellen und Tumorzellen und der Beteiligung von HSP70 an der Peptidprozessierung in malignen Zellen ergeben. An einer Nierenkarzinomzelllinie und an Primärzellen von Nierenkarzinomen konnte die HSP72-Oberfächenexpression durchflusszytometrisch auf der Tumorzelloberfläche nachgewiesen und an der Zelllinie mit einer erhöhten Lyse der Tumorzellen durch Interleukin-2 stimulierte NK-Zellen korreliert werden. Im Gegensatz zu bisherigen Literaturbefunden zeigte sich in diesem Modell jedoch, dass die Membranexpression von HSP72 nicht nur auf maligne Zellen beschränkt, sondern auch bei renalen Zellen aus Normalgewebe nachzuweisen war. In einer immunhistochemischen Studie zur basalen, zytoplasmatischen HSP72-Expression zeigte sich univariat eine signifikante Korrelation einer hohen HSP72-Expression und dem Überleben der Patienten. In einer multivariaten Analyse konnte das Ausmaß der HSP72-Expression als unabhängiger prognostischer Parameter identifiziert werden, der auch mit dem Ansprechen auf eine Chemoimmuntherapie korreliert. Die klinische Etablierung spezifisch wirksamer Vakzinen nimmt eine zentrale Rolle bei der Entwicklung neuer Behandlungsmethoden des metastasierten Nierenzellkarzinoms ein. Dabei spielen Vakzinen auf der Basis von HSP-Peptid-Vakzinen eine besondere Rolle. Ein weiterer Bestandteil der experimentellen Arbeiten war daher in der Entwicklung einer Methode zur Aufreinigung von HSP70-Peptid-Komplexen. Mit der ADP-Affinitätschromatographie konnte eine Methode zur Anwendung gebracht und patentiert werden, durch die sich HSP70-Peptid-Komplexe aus Tumorzellen anreichern lassen. Die HSP70-Peptid-Komplexe sind einerseits für HSP-basierte Vakzinekonzepte verfügbar oder können andererseits für die Identifikation und Charakterisierung HSP70-gebundenener biologisch-aktiver (immunogener) Peptide genutzt werden. Die vorliegenden Untersuchungsergebnisse demonstrieren, dass HSP72 per se eine prognostische Bedeutung beim Nierenzellkarzinom besitzt und weisen auf die potentielle Bedeutung von HSP72 als immunologische Zielstruktur hin. Die Resultate stellen neue Ansätze zum tieferen Verständnis der immunogenen Eigenschaften des Nierenzellkarzinoms dar und liefern Ausgangspunkte für die Etablierung innovativer Vakzinationskonzepte bei der Behandlung des fortgeschrittenen Nierenzellkarzinoms. / The cytokines interleukin-2 and interferon-a2a are of central importance in the treatment of patients with metastatic renal cell cancer. In the clinical part of the thesis the efficacy of chemoimmunotherapy was investigated monocentrically on 107 patients. It appeared a rate of objective remissions of 22% (95% CI 14.6 - 31.3%) and an additional rate of temporary disease stabilisations of 46%. The median survival was 19 months, with a calculated 5-year survival rate of 17%. Multivariate analysis revealed a highly significant dependence on the Karnofsky-Performance-Index of the patients. In 83 patients with Karnofsky-Performance-Index over 80% the median survival was 23 months, with a calculated 5-year survival of 21%. These results justify the further investigation of the combination of interleukin-2, interferon-a2a, and 5-fluorouracil in prospective clinical studies. In the context of the clinical aspects of cytokine-based immunotherapy it was the task of the the experimental part of the study to investigate the importance of 70 kDa heat shock proteins (HSP70) in renal cell cancer. Besides their function in cellular stress processing, members of the HSP70 family (HSP72 and HSP73) are especially important for answering questions of tumor immunology resulting from their immunological signal function, the HSP72-mediated interaction of immunological effector cells with tumor cells, and from the involvement of HSP70 in peptide processing in malignant cells. On the basis of a renal cancer cell line and of primary cells of renal carcinomas the HSP72 expression on the surface of tumor cells could be demonstrated by flow cytometry and correlated with an increased lysis of tumor cells on the cell line by interleukin-2 stimulated natural killer cells. In contrast to the findings so far reported in the literature, the present model revealed that the membrane expression of HSP72 was not limited to malignant cells but was detectable in renal cells of normal tissue too. In an immunohistochemical study on basal cytoplasmatical HSP72 expression, a univariate analysis showed a significant correlation of high HSP72 expression and patient survival. In a multivariate analysis the extent of the HSP72 expression could be identified as an independent prognostic parameter that was also correlated with the response of patients to chemoimmunotherapy. The clinical establishment of specifically effective vaccines plays a central part in the development of new treatment strategies for metastatic renal cell cancer. In this context, vaccines on the basis of HSP-peptides are of special interest. Therefore, the experimental part of the work was also aimed at developing a method for enriching HSP70-peptide complexes. With ADP affinity chromatography a method for concentrating HSP70-peptide complexes from tumor cells was created, which proved to be usable and was patented. On the one hand, HSP70-peptide complexes can be used for HSP-based vaccine programmes, on the other hand, they can serve for identifying and characterizing HSP70-bound biologically active (immunogenic) peptides. The present findings demonstrate that HSP72 per se has a prognostic value in renal cell cancer and point to the potential importance of HSP72 as an immunological target structure. The results represent new approaches to a deeper understanding of the immunological properties of renal cell carcinoma and give a basis from which to establish innovative vaccination programmes for the treatment of advanced renal cell carcinoma.

Gesteigerter oxidativer Stress ist ein typisches Zeichen in der Pathogenese der Herzinsuffi-zienz. Anhand von 2 Rattenmodellen sollen charakteristische Veränderungen des enzymatischen antioxidativen Systems, repräsentiert durch die Glutathionperoxidase (GSH-Px) und Superoxiddismutase (SOD) sowie Änderungen im Hitzeschockproteinsystem (Hsp) untersucht werden, für das stellvertretend Hsp25 und Hsp72 bestimmt wurden. Daneben wurde die Lipidperoxidation durch die Konzentration von Thiobarbitursäure-reaktiven Substanzen (TBARS) quantifiziert. Im ersten Rattenmodell, der Hypertonie-induzierten Herzinsuffizienz durch permanente Renin-Angiotensin-System-Aktivierung in doppelt transgenen Ratten konnte eine linksventrikuläre Hypertrophie mit CK-Isoenzym-Shift mit vermehrter Expression von CK-MB und -BB beobachtet werden. Es kam in beiden Ventrikeln der transgenen Tiere zu einer Zunahme der Lipidperoxidation begleitet von einer Erniedrigung der Serum-Vitamin E-Konzentration durch Verbrauch. SOD und Hsp72 blieben unverändert. Durch die biventrikuläre Zunahme der GSH-Px-Aktivität bei linksventrikulär erhöhtem Hsp25-Gehalt konnte die Toleranz gegenüber Hypoxie/Reoxygenierungsstress erhöht werden. In der 2. Teilstudie - der Myokardinfarkt-induzierten Herzinsuffizienz - wurden die Parameter oxidativer Schädigung und antioxidativen Schutzes im Akutstadium (14-16h) und 3, 6 und 9 Wochen nach experimentellem Infarkt (MI) bestimmt. In der Akutphase zeigten sich in beiden Ventri-keln und im Papillarmuskel gesteigerte GSH-Px- und SOD-Aktivitäten, ohne dass kardiale Hypertrophie vorlag. Hsp72 und Hsp25 waren während der Akutphase nach MI im Papillar-muskel und im linken Ventrikel erhöht. In der Folge resultiert eine verbesserte kontraktile Funktion bei experimentellem Hypoxie/Reoxygenierungsstress. Damit gelang es erstmals eine Selbstprotektion des Myokards während eines akuten MI nachzuweisen. Erst ab der 6. Woche trat kardiale Hypertrophie auf, begleitet vom charakteristischen CK-Isoenzym-Shift. Während die SOD und die Hitzeschockproteine nach der akuten Phase auf das Niveau der Kontrollen absanken, blieb die hohe GSH-Px-Aktivität im linken Ventrikel über den gesamten Untersuchungszeitraum bestehen, bei zunehmender Toleranz des Herzens gegenüber Hypoxie/Reoxygenierungsstress. Die vorgelegten tierexperimentellen Untersuchungen zeigen, dass das Herz sowohl unter akut als auch unter chronisch gesteigertem oxidativen Stress Mechanismen zur Selbstprotektion aktivieren kann, die eine Prävention bzw. Minimierung von Schädigungsreaktionen durch Sauerstoffradikale ermöglichen. / Elevated oxidative stress is typical in the pathogenesis of heart failure. Characteristical changes in antioxidant enzyme status, represented by glutathionperoxidase (GSH-Px) and superoxide dismutase (SOD), and changes in heat shock protein status (Hsp), denoted by Hsp25 and Hsp72, have been revealed in two different rat-models. Lipidperoxidation was quantified by the concentration of thiobarbituric acid reactive substances (TBARS). In the first model of heart failure caused by permanent activation of renin-angiotensin system in double transgenic rat, leftventricular hypertrophy accompanied by a shift of creatine kinase (CK) isoenzyme pattern to higher concentration of fetale CK-MB an -BB-isoforms was found. Higher TBARS concentrations and lower alpha-tocopherol levels caused by consumption have been measured. SOD and Hsp72 remained unchanged. The tolerance against experimental hypoxia/reoxygenation was improved by higher levels of GSH-Px and Hsp25 in both right and left ventricular tissue. In the second study of heart failure caused by experimental myocardial infarction (MI) the parameters of oxidant demolishing and antioxidant defence were evaluated under acute condi-tions (14-16h) and 3, 6 and 9 weeks after infarction. In the acute period higher activities of GSH-Px and SOD in non-hypertrophied left and right ventricular tissue and papillary muscle have been reported. Leftventricular and papillary muscle Hsp25 and Hsp72 content showed higher levels 14-16 hours after MI compared with controls, improving the contractile function in hypoxia/reoxygenation experiments. These findings suggest for the first time a myocardial self-protection after acute myocardial infarction. 6 and 9 weeks after MI leftventricular hyper-trophy occurred attended by the characteristic CK-isoenzymeshift. While SOD-activity and Hsp-content decreased to the levels of the controls, GSH-Px remained on higher altitude in leftventricular tissue in all examined periods after MI. This was accompanied by better toler-ance against hypoxia/reoxygenation stress. These findings of experimental-induced heart failure show the activation of myocardial self protecting mechanisms under acute and chronic oxidative stress conditions, minimizing or even preventing demolishing reactions of oxygen radicals.

A lesão pulmonar é determinante da morbi-mortalidade na pancreatite aguda (PA). Neutrófilos (PMN) e mediadores inflamatórios são responsáveis pelo desenvolvimento da lesão. A solução hipertônica modula a reposta inflamatória tendo como resultante um efeito imunomodulatório capaz de prevenir a lesão tecidual. Neste trabalho, investigamos os efeitos da solução hipertônica sobre os níveis de HSPs, MMPs e citocinas no tecido pulmonar, bem como, o mecanismo envolvido em sua regulação. Ratos machos Wistar foram submetidos a pancreatite pela injeção retrógrada de 1 ml/Kg de taurocolato de sódio 2,5%. Os animais foram randomizados em 4 grupos: 1) controle: não foi submetido a qualquer procedimento; 2) pancreatite sem tratamento (ST); 3) pancreatite e tratamento com solução fisiológica (SF); 4) pancreatite e tratamento com solução hipertônica (SH). Os animais dos grupos 3 e 4 tiveram a veia jugular cateterizada e receberam infusão de solução hipertônica ou fisiológica 1 hora após a indução de pancreatite. Os animais com PA foram sacrificados após 4, 12 e 24 horas. Os pulmões foram processados e submetidos a histologia com HE e dosagem de mieloperoxidase (MPO) para quantificação do infiltrado de neutrófilos. A produção e atividade das MMPs 2 e 9 foi analisada por zimografia. Western Blotting foi utilizado na detecção das HSPs 60, 70 e 90. As alterações na expressão gênica foram analisadas por RTPCR. Os níveis de peroxidação lipidica dosados por TBARs. A concentração de citocinas foi medida por ELISA. A reposição volêmica com SHT diminui o infiltrado inflamatório (PMN) 12 horas após a indução da PA. Concomitante a redução dos PMNs vimos a queda na atividade e expressão da MMP-9 e aumento da expressão protéica de HSP70 no tecido pulmonar. Tardiamente, 24 horas, o grupo tratado com SHT mostrou aumento na produção de IL-10, uma citocina anti-inflamatória. A SHT modula a resposta inflamatória, promovendo proteção ao tecido pulmonar contra os efeitos deletérios decorrentes da PA. Seus efeitos benéficos envolvem alterações celulares e moleculares que levam à redução da lesão tecidual. / Acute Pancreatitis (AP) is an inflammatory process of the pancreas with variable involvement of other organs and systems. The lungs are the most common distant organs affected by severe acute pancreatitis. The immunomodulatory effects of hypertonic solution (HS) provide potential strategies to attenuate inappropriate inflammatory reactions. This study tested the hypothesis that administration of HS modulates the development of lung injury in pancreatitis model. HS resuscitation results in a significant attenuation of lung injury following AP by modulate MMP 2 and 9 activity and protected tissue by increased HSPs 70 and 90 expression.