Fehlgefaltete Proteine des sekretorischen Weges werden aus dem Endoplasmatischen Retikulum (ER) in das Zytosol transportiert und dort durch das Ubiquitin-Proteasom-System abgebaut. Dieser Qualitätskontrollmechanismus wird als Endoplasmatisches Retikulum-assoziierte Proteindegradation bezeichnet (ERAD). In der Bäckerhefe Saccharomyces cerevisiae stellt die HRD-Ubiquitinligase eine zentrale Komponente dieses Abbausystems dar. Eine Untereinheit dieses Multienzymkomplexes ist das ER-ständige Membranprotein Der1, das über den Faktor Usa1 an die Ubiquitinligase Hrd1 rekrutiert wird und ausschließlich für den Abbau löslicher luminaler ERAD-Substrate notwendig ist. Im Rahmen dieser Arbeit konnte gezeigt werden, dass der C-Terminus von Der1 die Interaktion zu Usa1 und damit die Rekrutierung des Proteins zur HRD-Ligase vermittelt. Usa1 wirkt nicht nur als Rekrutierungsfaktor, sondern induziert auch die Der1-Oligomerisierung. Punktmutationen in den Transmembrandomänen von Der1 beeinträchtigen die Dislokation luminaler Substratproteine aus dem ER. Um weitere Hinweise für eine Beteiligung von Der1 beim Substrattransport zu erhalten, wurde die Methode des zielgerichtetem in vivo photocrosslinking für Der1 angewendet. Hierbei wurden bestimmte Positionen von Der1 mit dem photoreaktiven Aminosäureanalogon p-Benzoylphenylalanin markiert, was die Ausbildung von Quervernetzungen von Der1 zu Interaktionspartnern nach einer UV-Bestrahlung ermöglichte. Schließlich konnte auf diese Weise eine räumliche Nähe der luminal exponierten Bereiche von Der1 zum Substratrezeptor Hrd3 gezeigt werden, während die Transmembransegmente Quervernetzungen zu Hrd1 ausbildeten. Beide Bereiche von Der1 konnten zudem mit einem luminalen ERAD-Substrat quervernetzt werden. Anhand dieser Ergebnisse wurde somit erstmals eine direkte Beteiligung von Der1 insbesondere in den ersten Schritten der Substratdislokation gezeigt, was eine Funktion von Der1 als zentrale Komponente des Exportkomplexes nahelegt. / Newly synthesized proteins of the secretory pathway are subjected to an efficient quality control system in the endoplasmic reticulum. In order to prevent a harmful aggregation misfolded proteins are exported via a largely unknown mechanism into the cytosol and degraded by the ubiquitin-proteasome system in a process termed ER associated degradation (ERAD). In the yeast Saccharomyces cerevisiae the HRD-ligase constitutes a central component of ERAD. A subunit of this multi-enzyme complex is the small multispanning membrane protein Der1, which is exclusively required for the degradation of misfolded ER luminal proteins but dispensable for the turnover of membrane-bound substrates. In this study a short conserved motif in the cytosolic carboxyterminus of Der1 was identified that mediates the binding to the HRD-ligase. Moreover, co-immunoprecipitation experiments show that Der1 forms oligomers, which relies on its assembly into the degradation complex. Mutations in the transmembrane domains of Der1 block the export of soluble proteins across the ER-membrane. To further investigate the function of Der1 in substrate dislocation an in vivo site-specific photocrosslinking approach was applied. Various positions of Der1 were labelled with the photoreactive amino acid analogue p-benzoyl-phenylalanine followed by UV irradiation of living cells expressing these Der1 constructs. The crosslinking experiments reveal a spatial proximity of ER luminal exposed parts of Der1 to the substrate receptor Hrd3. By contrast, the membrane-embedded domains of Der1 reside adjacent to the ubiquitin ligase Hrd1. Intriguingly, both regions also form crosslinks to a client protein. In summary the data of this work imply that multimeric Der1 initiates the export of aberrant polypeptides from the ER-lumen by threading such molecules into the ER-membrane and routing them to Hrd1 for ubiquitylation.

INTRODUÇÃO: O câncer de ovário é o mais letal de todos os cânceres ginecológicos e requer ser tratado por ginecologistas especializados em centros terciários para se obter melhor prognóstico. Este trabalho tem como objetivo analisar e comparar quatro estratégias diferentes para predizer a benignidade ou malignidade de tumores pélvicos supostamente de origem ovariana utilizando para este fim, marcadores tumorais CA 125 e HE4, índice de risco de malignidade (IRM) e algoritmo ROMA. MÉTODOS: Neste estudo prospectivo foram avaliadas 128 pacientes com diagnóstico de tumores pélvicos supostamente de origem ovariana atendidas na Divisão de Clínica Ginecológica do Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo e Instituto do Câncer do Estado de São Paulo entre julho de 2008 e janeiro 2011. Foram calculadas a sensibilidade e a especificidade e construídas curvas ROC para comparar os quatro parâmetros (CA 125, HE4, ROMA e IRM) na eficácia de diferenciar tumores ovarianos. RESULTADOS: A sensibilidade obtida para CA 125, HE4, ROMA e IRM foi de, respectivamente, 70,4%, 79,7%, 74,1% e 63,0%. A especificidade para CA 125, HE4, ROMA e IRM foi de, respectivamente, 74,2%, 66,7%, 75,8% e 92,4%. Não houve diferença na comparação das áreas abaixo da curva ROC entre os quatro parâmetros. CONCLUSÕES: Nenhum dos quatro métodos estudados é o ideal na diferenciação de tumores ovarianos. Entre os quatro parâmetros analisados o HE4 foi o parâmetro com melhor sensibilidade na diferenciação de tumores ovarianos. A acurácia dos quatro métodos é equivalente e podem ser utilizados indistintamente para referenciar pacientes para serviços especializados no tratamento de câncer de ovário / BACKGROUND: Ovarian cancer is the most lethal of all gynecological cancers and requires to be treated by gynecologic oncologists in tertiary centers accustomed to treating this disease to achieve the best prognosis. This study aims to compare four different strategies to predict the benignity or malignancy of pelvic tumors presumably of ovarian origin using, for this purpose, tumor markers CA 125 and HE4, risk malignancy index (RMI) and algorithm ROMA. METHODS: This prospective study evaluated 128 patients supposedly with ovarian tumors treated at the Divisão de Clínica Ginecológica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo and at Instituto do Câncer do Estado de São Paulo between July 2008 and January 2011. We calculated sensitivity, specificity and ROC curves to compare the four parameters (CA 125, HE4, ROMA and RMI) ability to differentiate the ovarian tumors. RESULTS: The sensitivity obtained for CA 125, HE4, ROMA and RMI was, respectively, 70.4%, 79.7%, 74.1% and 63.0%. The specificity obtained for CA 125, HE4, ROMA and RMI was, respectively, 74.2%, 66.7%, 75.8% and 92.4%. There was no difference the areas under the ROC curve among the four parameters. CONCLUSIONS: None of the four studied methods is best in the differentiation of ovarian tumors. Among the four parameters analyzed, HE4 was the parameter with highest sensitivity in the differentiation of ovarian tumors. The accuracy of the four methods is equivalent and can be used interchangeably to refer patients for specialized services in the treatment of ovarian cancer

INTRODUÇÃO: O câncer de ovário é o mais letal de todos os cânceres ginecológicos e requer ser tratado por ginecologistas especializados em centros terciários para se obter melhor prognóstico. Este trabalho tem como objetivo analisar e comparar quatro estratégias diferentes para predizer a benignidade ou malignidade de tumores pélvicos supostamente de origem ovariana utilizando para este fim, marcadores tumorais CA 125 e HE4, índice de risco de malignidade (IRM) e algoritmo ROMA. MÉTODOS: Neste estudo prospectivo foram avaliadas 128 pacientes com diagnóstico de tumores pélvicos supostamente de origem ovariana atendidas na Divisão de Clínica Ginecológica do Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo e Instituto do Câncer do Estado de São Paulo entre julho de 2008 e janeiro 2011. Foram calculadas a sensibilidade e a especificidade e construídas curvas ROC para comparar os quatro parâmetros (CA 125, HE4, ROMA e IRM) na eficácia de diferenciar tumores ovarianos. RESULTADOS: A sensibilidade obtida para CA 125, HE4, ROMA e IRM foi de, respectivamente, 70,4%, 79,7%, 74,1% e 63,0%. A especificidade para CA 125, HE4, ROMA e IRM foi de, respectivamente, 74,2%, 66,7%, 75,8% e 92,4%. Não houve diferença na comparação das áreas abaixo da curva ROC entre os quatro parâmetros. CONCLUSÕES: Nenhum dos quatro métodos estudados é o ideal na diferenciação de tumores ovarianos. Entre os quatro parâmetros analisados o HE4 foi o parâmetro com melhor sensibilidade na diferenciação de tumores ovarianos. A acurácia dos quatro métodos é equivalente e podem ser utilizados indistintamente para referenciar pacientes para serviços especializados no tratamento de câncer de ovário / BACKGROUND: Ovarian cancer is the most lethal of all gynecological cancers and requires to be treated by gynecologic oncologists in tertiary centers accustomed to treating this disease to achieve the best prognosis. This study aims to compare four different strategies to predict the benignity or malignancy of pelvic tumors presumably of ovarian origin using, for this purpose, tumor markers CA 125 and HE4, risk malignancy index (RMI) and algorithm ROMA. METHODS: This prospective study evaluated 128 patients supposedly with ovarian tumors treated at the Divisão de Clínica Ginecológica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo and at Instituto do Câncer do Estado de São Paulo between July 2008 and January 2011. We calculated sensitivity, specificity and ROC curves to compare the four parameters (CA 125, HE4, ROMA and RMI) ability to differentiate the ovarian tumors. RESULTS: The sensitivity obtained for CA 125, HE4, ROMA and RMI was, respectively, 70.4%, 79.7%, 74.1% and 63.0%. The specificity obtained for CA 125, HE4, ROMA and RMI was, respectively, 74.2%, 66.7%, 75.8% and 92.4%. There was no difference the areas under the ROC curve among the four parameters. CONCLUSIONS: None of the four studied methods is best in the differentiation of ovarian tumors. Among the four parameters analyzed, HE4 was the parameter with highest sensitivity in the differentiation of ovarian tumors. The accuracy of the four methods is equivalent and can be used interchangeably to refer patients for specialized services in the treatment of ovarian cancer

Fascioloides magna (the giant liver fluke) originated from North America, is known in the Czech Republic since 1930s. This pathogenic fluke invades mostly cervids, but livestock too. Excretory-secretory products (ES products) contain number of esential biomolecules which are produced by excretory and secretory system of the fluke. These molecules play key role in many biological process during the life cycle not only of fascioloid flukes (e.g. migration in the host tissues, immune evasion and digestion). Due to their antigenic properties they could be also used in immunodiagnostics. Excretory-secretory proteins from adult Fascioloides magna and comparative related species Fasciola hepatica were purified and separated by the basic biochemical methods (1D, 2D electrophoresis, ion-exchange chromatography) and their activity was confirmed by specific (fluorogenic peptide) and nonspecific (gelatine) substrates. By using the mass spectrometry methods (MALDI TOF/TOF), the most abundant peptidolytically active proteins from ES products of F. magna were identified as cathepsin L (FmCL). Recombinant analog of FmCL was expressed in Pichia pastoris expression system. The peptidolytic activity was again confirmed using the synthetic fluorogenic substrates; the specifity of recombinant FmCL active site was...

Text in English / Corridor disease is an economically important tick-borne disease of cattle in southern Africa. The disease is caused by Theileria parva and transmitted by the vectors, Rhipicephalus appendiculatus and R. zambeziensis. There is currently no vaccine to protect cattle against T. parva that is permitted in South Africa. To develop recombinant anti-tick vaccines against Corridor disease, comprehensive databases of genes expressed in the tick’s salivary glands are required. Therefore, in Chapters 2 and 3, mRNA from the salivary glands of R. appendiculatus and R. zambeziensis was sequenced and assembled using next generation sequencing technologies. Respectively, 12 761 and 13 584 non-redundant protein sequences were predicted from the sialotranscriptomes of R. appendiculatus and R. zambeziensis and uploaded to public sequence domains. This greatly expanded the number of sequences available for the two vectors, which will be invaluable resources for the selection of vaccine candidates in future. Further, in Chapter 3, differential gene expression analysis in R. zambeziensis revealed dynamic expression of secretory protein transcripts during feeding, suggestive of stringent transcriptional regulation of these proteins. Knowledge of these intricate expression profiles will further assist vaccine development in future. In Chapter 4, comparative sialotranscriptomic analyses were performed between R. appendiculatus and R. zambeziensis. The ticks have previously shown varying vector competence for T. parva and this chapter presents the search for correlates of this variance. Phylogenetic analyses were performed using these and other publically available tick transcriptomes, which indicated that R. appendiculatus and R. zambeziensis are closely related but distinct species. However, significant expression differences were observed between the two ticks, specifically of genes involved in tick immunity or pathogen transmission, signifying potential bioinformatic signatures of vector competence. Furthermore, nearly four thousand putative long non-coding RNAs (lncRNAs) were predicted in each of the two ticks. A large number of these showed differential expression and suggested a potential transcriptional regulatory function of lncRNA in tick blood feeding. LncRNAs are completely unexplored in ticks. Finally, in Chapter 5, concluding remarks are given on the potential impact the R. appendiculatus and R. zambeziensis sialotranscriptomes may have on future vaccine developments and some future research endeavours are discussed. / Life and Consumer Sciences / Ph. D. (Life Sciences)