Utilization of potential synergistic interactions among different bioactive agents is a promising approach to inhibit complex diseases such as cancer. Nobiletin (NBT) and tangeretin (TAN) are major polymethoxyflavones (PMFs) found in citrus fruits. Herein, we studied NBT and TAN in combination with atorvastatin (ATST, Lipitor, a cholesterol-lowering drug) in MDAMB231 and MCF-7 human breast cancer cells. Both NBT/ATST and TAN/ATST combinations at low doses produced much stronger inhibitory effect on cancer cell viability in comparison to those produced by NBT, TAN, or ATST alone at much higher doses. Isobologram analysis confirmed that both NBT/ATST and TAN/ATST combinations produced strong synergy in inhibiting the growth of two breast cancer cell lines. Flow cytometry analysis showed that both NBT/ATST and TAN/ATST combinations caused significant cell cycle arrest at G0/G1 phase in MDAMB231 cells (ER+). Consistent with these results, PMFs and ATST combinations decreased expression levels of phospho Rb, cyclin D1, and CDK4. Further experiments showed that the combination treatment induced autophagy and late apoptosis in MDA-MB-231 cells. Meanwhile, co-treatment of PMFs and ATST induce G2/M phase in MCF-7 (ER+) cells.. The combination of PMFs and ATST also caused autophagy in MCF-7 cells, which was evidenced by activation of LC3B and P62. In conclusion, our result demonstrated strong synergy between two major citrus PMFs (NBT and TAN) and ATST in inhibiting human breast cancer cell growth.

In Griechisch werden Transitivitätsänderungen durch non-aktives Genus Verbi (Voice) ausgedrückt, was zu einer morphologischen Unterspezifikation führt. Grundsätzlich können passive und reflexive Verben im Griechischen (neben anderen Strukturen) mit derselben Form ausgedrückt werden. Oft ist der Kontext das einzige Mittel zur Disambiguierung. Diese Studie untersucht den Erwerb von Transitivitätsalternationen (transitivity alternations) bei griechischen monolingualen Kindern im Vergleich zu zwei zweisprachigen Populationen, nämlich griechisch-deutschen und griechisch-englischen Kindern im Alter zwischen 4 und 8 Jahren. Es wird untersucht, ob beim Erwerb der jeweiligen morphologischen Systeme die dominante Sprache, in diesem Fall Deutsch bzw. Englisch, mit der Erbsprache (Heritage- sprache) Griechisch interagiert. Englisch und Deutsch unterscheiden sich vom Griechischen, indem sie Reflexivität und Passivierung anders ausdrücken, und stellen deswegen ein in- teressantes Forschungsgebiet dar. In der Studie wurden 80 zweisprachige Kinder sowie 40 einsprachige Kinder untersucht. Sie sollten die folgenden “Spiele” absolvieren: Ren- frews (1998) expressive Vokabelaufgabe, angepasst an das Griechische von Vogindroukas (2009), sowie zwei experimentelle Aufgaben: eine Wahrheitsbewertungsaufgabe (truth-value judgement task) und eine Handlungsaufgabe (act-out task). Die Ergebnisse zeigen, dass Passive Verben sind hingegen am schwierigsten zu verstehen und zu produzieren, sowohl für zweisprachige als auch für monolinguale griechische Kinder. / In Greek, transitivity alternations are expressed using the non-active voice resulting in a morphological underspecification. Essentially, passives and reflexives in Greek, among other structures, can be expressed using the same form; context is often the only means of disambiguation. This study investigates the acquisition of transitivity alternations in Greek comparing two bilingual populations namely, Greek-German and Greek-English bilinguals between the ages of 4 and 8. This study was motivated by the lack of research on the acquisition of transitivity alternations in bilingual populations. It examines whether the dominant language, in this case German and English respectively, interacts with the heritage language, Greek, as they both evolve morphologically. English and German differ from Greek in the way they express reflexivity and passivisation and posed an interesting area of research. 80 bilingual children as well as 40 monolingual children a baseline task: Renfrew’s (1998) Expressive Vocabulary Task adapted for Greek by Vogindroukas (2009) and two experimental tasks: a truth-value judgement task (TVJT) and an act-out task (AOT). The findings show that children across populations scored worst in passives thus replicating the results in previous literature.

Neuraminidase (NA) is one of two major antigens on the surface of influenza A viruses. It is comprised of a single N-terminal transmembrane domain (TMD), a stalk domain, and a C-terminal enzymatic head domain that cleaves sialic acid, most notably to release new particles from the host cell surface. NA is only enzymatically active as a homo-tetramer. However, it is not known which properties facilitate the oligomerization of NA during assembly. Our results show that, apart from anchoring the protein to the membrane, the NA TMD also contributes to the assembly process by keeping the stalk in a tetrameric conformation. The ability of the TMD to oligomerize is shown to be dependent on its amphiphilic characteristics that was largely conserved across the nine NA subtypes (N1-N9). Over time the NA TMDs in human H1N1 viruses were found to have become more amphiphilic, which correlated with stronger oligomerization. An old H1N1 virus with a more recent N1 TMD had impaired growth, but readily acquired compensatory mutations in the TMD to restore growth, by reverting the TMD oligomerization strength back to that of the old TMD, demonstrating a biological role of the TMD in folding and assembly. NA and the other viral proteins are spatially and temporally coordinated to achieve optimal viral production. By using a co-transfection analysis, the high AU-content in the NA and HA ER-targeting sequence coding regions (for NA TMD as well as the HA signal sequence) were found to inhibit their expression. The inhibition was alleviated by the early expressed influenza RNA-binding protein NS1, which promoted translation and showed enriched foci at the endoplasmic reticulum (ER). NS1, which expresses early during infection, is therefore likely the regulator of NA and HA to prevent premature expression. These results show that the NA TMD is under substantial selection pressure at both the nucleotide and amino acid level to accommodate its roles in ER-targeting, protein folding, and post-transcriptional regulation. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Accepted.</p>

Adipocytokines released by the adipose tissue play an important role in the regulation of immune and inflammatory responses. In obesity their production is dysregulated, which is one of the major factors contributing to the onset of a chronic low-grade systemic inflammation representing a risk factor for the progression of other diseases, such as atherosclerosis or type-2 diabetes. The main goal of this thesis was to analyze the secretion of selected adipocytokines (adiponectin, IL6 and MCP1) by in-vitro differentiated adipocytes, isolated from the adipose tissue prior to and after a dietary intervention, and this under basal conditions and during stimulated lipolysis. In case of adiponectin, the secretion of its isoforms was analyzed too. The concentration of adiponectin, IL6 and MCP1 was determined by the ELISA method, the Western Blot method was used to determine the distribution of the adiponectin isoforms. The thesis also concentrates on the gene expression of ATF3, ATF4 and HSPA5, factors engaged in the ER stress in the course of the differentiation of adipocytes. The changes in the gene expression were measured by the quantitative Real Time PCR method. At the same time the development of the endoplasmic reticulum (ER) in the course of adipogenesis was monitored by indirect...

O objetivo deste estudo foi o de avaliar os efeitos da irradiação laser de Er,Cr:YSGG em superfícies dentinárias no tratamento da hipersensibilidade dentinária cervical, em busca de um protocolo para ser aplicado clinicamente. Para o teste de permeabilidade, foram preparados 144 pré-molares, as coroas seccionadas e as raízes impermeabilizadas. Duas áreas de irradiação antagônicas foram delimitadas e livres da impermeabilização (controle e experimental). A seguir, os dentes foram subdivididos em dois sub-grupos, diferindo o condicionamento da superfície irradiada (EDTA 24% e ácido fosfórico 35%) e em grupos diferindo o protocolo:G1)Er:YAG, 60mJ/2Hz; G2)0,25W; G3)0,5W; G4)0,75W; G5)1W, G6)1,25W; G7)1,50W; G8)1,75W G9)2W. Após as irradiações, os espécimes foram imersos em azul de metileno 2% por 4 horas, e incluídos em resina epóxica para a realização dos cortes longitudinais. Estes tiveram suas imagens trabalhadas em computador para mensuração do nível de infiltração. Os dados foram submetidos à análise estatística. O Sub-grupo 1, condicionado com EDTA, apresentou diferenças entre os grupos sendo que as amostras irradiadas com Er:YAG demonstraram menor infiltração, diferindo estatisticamente dos grupos 3, 6 e 9. Os resultados do sub-grupo 2 mostraram que as médias das amostras irradiadas com Er:YAG tenderam a zero entretanto, não houve diferença entre os grupos. Na Fase B, fragmentos de dentina foram obtidos da região cervical de dentes humanos, preparados para análise em microscopia eletrônica de varredura. Morfologicamente, observou-se oclusão parcial dos túbulos dentinários após irradiação com Er:YAG e Er,Cr:YSGG nas condições de 0,25W e 0,50W. A partir de 0.75W, foi observado abertura dos túbulos e à medida que as energias aumentaram, foram observadas carbonização e fendas. De posse dos resultados dos trabalhos in vitro, foram selecionados os parâmetros para a aplicação e o acompanhamento clínico de 1 mês: G1)controle (placebo); G2)Er:YAG; G3)Er,Cr:YSGG 0,25W e G4)Er,Cr:YSGG 0,75W. Após a triagem, foram selecionados voluntários de acordo com critérios de exclusão e inclusão. O nível de sensibilidade de cada voluntário foi avaliado através da escala visual analógica de dor (VAS) com auxílio do ar da seringa tríplice 1 semana antes (Pré-1), 5 minutos anterior ao tratamento (Pré-2), após 5 minutos (Pós-1), 1 semana (Pós-2) e 1 mês após o tratamento (Pós-3). Os dados foram coletados e submetidos à análise estatística separadamente para os dois estímulos realizados: spray de ar e sonda exploradora. Tanto para o estímulo ar quanto para o estímulo sonda, não foram observadas diferenças estatisticamente significantes nas escalas Pré-1 e Pré-2, concluindo que os pacientes estavam padronizados e não houve efeito da profilaxia nos níveis de dor. Ao estímulo do spray de ar, nota-se uma redução nos níveis de dor na escala Pós-1, porém há uma estabilidade dos valores das escalas seguintes. O grupo 2 (Er:YAG) apresentou o menor nível de dor quando comparado aos outros grupos. Ao estímulo mecânico foi observado que o grupo 4 (Er,Cr:YSGG 0,50W) demonstrou o decréscimo de dor mais acentuado imediatamente após o tratamento, porém ao final do estudo, apresentou os maiores níveis de dor. Os grupos 1, 2 e 3 apresentam redução de dor, porém estatisticamente diferentes do grupo 4. Com base nos resultados apresentados e dentro dos limites e parâmetros estudados, pode-se concluir que nenhuma condição foi capaz de eliminar completamente a microinfiltração e diminuir a permeabilidade dentinária; porém o laser de Er:YAG e o laser de Er,Cr:YSGG 0,25W apresentaram resultados satisfatórios nos três estudos realizados. / The aim of the present study was to determine the correct parameters of the Er,Cr:YSGG laser in the treatment of cervical dentin hypersensitivity. This work was presented in 3 different stages. In the first one, it was evaluated the decrease in dentin permeability of dentinal tubules after Er,Cr:YSGG laser irradiation in radicular surfaces. Ninety premolars were prepared with its crows sectioned, and the roots completely impermeabilized. Two antagonist areas of irradiation were determined as control and experimental. Each surface was conditioned with EDTA 24% (sub-group1) and phosphoric acid 35% (sub-group2) and the selected parameters were: 1)Er:YAG, 60mJ, 2Hz, 4 irradiations of 20sec each, defocused mode; and groups 2 to 9 received irradiation with Er,Cr:YSGG laser, 20Hz, Z6 tip, 600µm diameter, defocus mode, 0% of air and water: 2)Er,Cr:YSGG 0,25W; 3) 0,5W; 4) 0,75W; 5) 1,0W; 6) 1,25W, 7) 1,50W, 8) 2W; 9) 2 W. After irradiation, samples were immersed in a solution of methylene blue for 4 hours, washed for 5 min and included in epoxy resin to allow longitudinal cuts. The images were digitalized and computed worked. Data was statistically analyzed. Although the samples irradiated with Er:YAG showed less microleakage, the sub-group 1 showed differences between the groups, statistically different from groups 3, 6 e 9. The results of the sub-group 2 showed that the mean values of the Er:YAG samples had a tendency to be negative, however, no differences were detected between the groups. In the second stage, cuts from the cervical area were obtained and prepared for scanning electron microscopy to evaluate the superficial morphology. It was observed the partial closure of dentinal tubules after the irradiation with Er:YAG and Er,Cr:YSGG laser in the 0.25 and 0.50W protocols. From the 0.75W until 2W, it was observed the dentinal tubules opened as the energy densities raised. With the results of the in vitro studies, the parameters were selected: G1)control; G2)Er:YAG; G3)Er,Cr:YSGG 0,25W e G4)Er,Cr:YSGG 0,75W. Thirty subjects, who met the entry criteria, were admitted into the study. The assessment method used to quantify sensitivity was the cold air syringe, recorded by the visual analogue scale (VAS), prior to treatment as baseline (Pre-1), immediately before (Pre-2) and immediately after (Post-1) the topical treatment, after 1 week (Post-2) and after 1 month (Post-3). Teeth were assigned to the 4 groups. After the follow-up, data was collected and submitted to statistical analysis for both kinds of stimulus: evaporative (air) and mechanical (probe). For both air and probe it was not observed differences among the Pre-1 and Pre-2 moments, for what it can be concluded that patients were standardized. Analyzing the evaporative stimulus, it can be observed the reduction in the pain level immediately after the treatment; however there was stability in the next values. Group 2 (Er:YAG) showed the least level of pain. Throughout the mechanical stimulus, it was observed that group 4 (Er,Cr:YSGG 0,50W) showed the most pronounced decrease of pain immediately after treatment, however, at the end of the study, the levels of pain increased. Groups 1, 2 e 3 showed reduction of pain, statistically different from the group 4. Based on the results and within the limits of this study, it can be concluded that none of the parameters and lasers studied were capable of eliminate the microleakage and decrease the dentin permeability, however, the Er:YAG and Er,Cr:YSGG 0,25W lasers showed satisfactory results in the three studies performed.

A erosão dental provoca alterações histológicas complexas na dentina e, apesar do aumento da ocorrência desta patologia na última década, a literatura apresenta poucos estudos sobre a adesão na dentina erodida. Com o aprimoramento da tecnologia e o conhecimento de que os lasers de érbio apresentam grande afinidade pela água e hidroxiapatita, sua utilização tem sido considerada um método alternativo para tratamento da superfície dentinária antes dos procedimentos adesivos. Assim, o objetivo deste trabalho in vitro foi avaliar o efeito de diferentes tratamentos de superfície (ponta diamantada, laser de Er:YAG 2,94 m, 60 mJ, 2 Hz, 0,2 W, 19,3 J/cm2 - e laser de Er,Cr:YSGG 2,78 m, 50 mJ, 30 Hz, 1,5 W, 4,5 J/cm2), prévios ao tratamento restaurador, tanto na dentina humana hígida quanto na dentina artificialmente erodida. Metade das amostras foi submetida à ciclagem erosiva, a qual foi realizada durante 5 dias por meio da imersão em solução de ácido cítrico 0,05 M (pH 2,3, 10 minutos, 6x/dia) e solução supersaturada (pH 7,0, 60 minutos entre os ataques ácidos). Na fase experimental 1, 32 fragmentos de dentina foram divididos aleatoriamente em 8 grupos (n = 4) e com o auxílio da Microscopia Eletrônica de Varredura (MEV), realizou-se a análise da morfologia da superfície da dentina hígida e da submetida à ciclagem erosiva, após os diferentes tratamentos de superfície. Na fase experimental 2, 192 terceiros molares humanos foram divididos aleatoriamente em 2 grupos (dentina hígida e dentina submetida à ciclagem erosiva) e 8 subgrupos (n = 12) para avaliação da resistência de união entre as superfícies tratadas e 2 sistemas adesivos (01 autocondicionante e 01 com condicionamento ácido prévio), por meio do ensaio de microtração e da análise do padrão de fratura. Para o ensaio de microtração, blocos de resina composta microhíbrida foram confeccionados na superfície de dentina e, após armazenados em água destilada-deionizada por 24h (37oC), palitos foram obtidos para serem tracionados na máquina de ensaio universal (0,5mm/min). A análise do padrão de fratura realizada em microscópio ótico (40x) e as avaliações das eletromicrografias foram feitas de forma descritiva. Os valores obtidos de resistência de união(MPa) foram submetidos aos testes de ANOVA (dois fatores) e de comparações múltiplas de Tukey (p<0,05). A análise morfológica de superfície mostrou alterações significativas da dentina hígida e da dentina artificialmente erodida, submetidas aos diferentes tratamentos de superfície. Quanto à resistência de união, na dentina hígida, o grupo controle, tanto do sistema autocondicionante (54,69 ± 7,80 MPa) quanto do condicionamento ácido total (47,36 ± 9,35 MPa), apresentou valores significativamente maiores que os outros grupos, sendo, porém, diferentes entre si. Na dentina erodida, a maior resistência de união foi alcançada com o grupo tratado com o laser de Er,Cr:YSGG associado ao adesivo autocondicionante (28,26 ± 9,22 MPa), o qual foi significativamente diferente de todos demais grupos. Com base nos parâmetros de irradiação utilizados neste estudo, pode-se concluir que em dentina hígida, os tratamentos de superfície com os lasers de Er:YAG e Er,Cr:YSGG apresentaram resultados de adesão inferiores, mas satisfatórios, em relação ao grupo controle. Já em substrato dentinário erodido, a irradiação com laser de Er,Cr:YSGG, associada ao uso de sistema adesivo autocondicionante, promoveu efeito positivo para a adesão à resina composta. / Dental erosion can cause complex histological changes in dentin and, despite the increase in its incidence during the last decade, limited information is available regarding adhesion to eroded dentin. With improvements in technology and due to the fact that the erbium lasers have high affinity to water and hydroxyapatite, the use of laser can consist on an alternative for the treatment of dentin surface before adhesive procedures. The aim of this in vitro study was to evaluate the effect of different surface treatments (diamond bur, Er:YAG laser 2.94 m, 60 mJ, 2 Hz, 0.2 W, 19.3 J/cm2 - and Er,Cr:YSGG laser 2.78 m, 50 mJ, 30 Hz, 1.5 W, 4.5 J/cm2) prior to restorative treatment, in sound and artificially eroded human dentin. Half of the samples was submitted to the erosive cycling, which was performed by immersion in 0.05 M citric acid (pH 2.3, 10 min, 6x/day) and in supersaturated solution (pH 7.0, 1h, between acid attacks), during 5 days. In phase 1, 32 dentin fragments were randomly divided into 8 groups (n = 4). The analysis of surface morphology of both sound and eroded dentin was performed by Scanning Electron Microscopy (SEM), after the different surface treatments. In phase 2, 192 human third molars were randomly divided into 2 groups (sound and eroded dentin) and 8 subgroups (n=12) to evaluate the bond strength between the treated surfaces and composite resin, with two adhesive systems (self-etching and total-etching) by means of the microtensile bond strength (TBS) test and fracture pattern analysis. For the microtensile bond strength test, blocks of composite resin were bonded to the samples and, after 24 h-storage in distilled/deionized water (37°C), stick-shapped samples were obtained and submitted to TBS test in a universal testing machine (0.5 mm/min). The fracture pattern was performed with an optical microscopy (40x) and the evaluations of the electromicrographs were made under a descriptive mode. The bond strength values (MPa) were analyzed by two-way ANOVA and Tukey tests ( = 0.05). In sound dentin, for the control group, both self-etching (54.69 ± 7.8 MPa) and total-etching adhesive system (47.36 ± 9.3 MPa) showed significantly higher values than all other groups (p<0.05), even though they differ from each other. However, in eroded dentin groups, the self-etching on the samples treated with the Er,Cr:YSGG laser presented the highest mean (28.3 ± 9.2 MPa), which was statistically significant higher than all the other treatments (p<0.05). Based on the irradiation parameters considered for this study, it can be concluded that in healthy dentin, the surface treatment with Er:YAG and Er,Cr:YSGG irradiation showed bonding values lower than the control group, but they presented satisfactory results. However, in eroded dentin, the use of Er,Cr:YSGG laser, associated with the self-etching adhesive system, promoted positive effect on adhesion to composite resin.

Scs2p ist ein integrales Membranprotein des Endoplasmatischen Retikulums (ER), dessen Deletion zu einer geringeren Expression der Inositol-1-P Synthase INO1 in Inositol-freiem Medium führt und dessen Überexpression die Inositol-Auxotrophie eines Stammes mit einer defekten "unfolded protein response" supprimiert. scs2 Mutanten weisen zudem eine erhöhte Sensitivität gegen Tunicamycin auf, eine Substanz, welche die N-Glykosylierung von Proteinen im ER inhibiert. Für die mutmaßlichen Orthologen von Scs2p in höheren Eukaryoten wurde eine Funktion dieser Proteine im vesikulären Transport postuliert. In dieser Arbeit wurde Scs2p als mit Cue1p, einer Komponente der ER-assoziierten Proteindegradation, quervernetzbares Protein identifiziert, und daraufhin begonnen, die Funktion von Scs2p näher zu charakterisieren. Eine Deletion von SCS2 hatte keinen Einfluß auf die bekannten Cue1p-abhängigen Degradationswege. Auch die Induktion der "unfolded protein response" (UPR) infolge einer Akkumulation von falsch gefalteten Proteinen wurde durch eine Deletion von SCS2 nicht beeinträchtigt. Eine Beeinträchtigung des Transportes durch den sekretorischen Weg konnte in scs2 Hefen ebenfalls nicht nachgewiesen werden. scs2 Mutanten zeigten eine generell verminderte Expression von Genen der Glycerolipid Biosynthese, deren Transkription über Ino2p/Ino4p reguliert wird. Dabei beeinträchtigte eine Deletion von SCS2 nicht die Induktion der UPR in Inositol-armem Medium und die darüber vermittelte Induktion der INO1 Expression. Ein analoger Phänotyp wurde auch in Mutanten beobachtet, in denen die Gene von Ubc7p, einer Komponente der ER-Degradation oder Lcb3p, einer Sphingosin-P Phosphatase, deletiert waren. Dabei bestand in bezug auf die Ino2p/Ino4p-abhängige Expressionsregulation keine epistatische Beziehung zwischen den drei Gendeletionen. Untersuchungen des Einflusses verschiedener Mutanten und Inhibitoren des Sphingolipid Stoffwechsels zeigten, daß Änderungen der Synthese von komplexen Sphingolipiden die Regulation der Inositol Synthese nur indirekt beeinflußten und gaben Hinweise auf eine Regulation des Glycerolipid Stoffwechsels durch Sphingosin. scs2 Mutanten erwiesen sich als sensitiv gegen Inhibitoren der Sphingolipid Biosynthese. Untersuchungen von Stämmen, in denen verschiedene Gene der Sphingolipid Synthese deletiert wurden, sowie Analysen der Lipidzusammensetzung zeigten, daß in scs2 Zellen eine Regulation beeinträchtigt ist, welche die IPC Synthase Aktivität bei einem verringerten Sphingolipid Gehalt der Zelle erhöht. Die geringere Expression von Genen der Glycerolipid Synthese und die niedrigere Kapazität zur Synthese von komplexen Sphingolipiden erwiesen sich als voneinander unabhängige Auswirkungen eines komplexen Phänotyps von scs2 Deletionsmutanten, wobei die primäre Funktion von Scs2p vermutlich nicht im Lipidstoffwechsel liegt. / Scs2p is an integral membrane protein of the endoplasmic reticulum (ER), whose deletion leads to a reduced expression of INO1 encoding inositol-1-phosphate synthase and whose over-expression suppresses the inositol auxotrophy of a strain with a defect unfolded protein response (UPR). scs2 mutants display an enhanced sensitivity to Tunicamycin, an inhibitor of protein N-glycosylation in the ER. It has been proposed that the putative orthologs of Scs2p in higher eukaryotes function in vesicular transport. In this work Scs2p, which was identified as a protein that can be crosslinked to Cue1p, a protein involved in ER-associated protein degradation, was further characterised. A deletion of SCS2 did not affect the known Cue1p-dependend degradation pathways. Cells lacking Scs2p normally induced the UPR upon an accumulation of misfolded proteins. An impairment of protein transport through the secretory pathway could also not be detected in scs2 cells. scs2 mutants displayed a generally reduced expression of genes involved in glycerolipid synthesis, whose transcription is regulated by Ino2p/Ino4p. The induction of the UPR in inositol-free medium and the subsequent upregulation of INO1 expression were not impaired in these cells. A similar effect was observed in strains lacking Ubc7p, a component of the ER -associated protein degradation system and in cells lacking the sphingoid base-1-phosphate phosphatase Lcb3p. Regarding the Ino2p/Ino4p-dependend gene expression no epistatic relationship was observed between Scs2p, Ubc7p and Lcb3p. An examination of the influence of various mutants and inhibitors of the sphingolipid pathway indicated that alterations of the synthesis of complex sphingolipids have only an indirect influence on the regulation of inositol synthesis and pointed towards a control of glycerolipid synthesis by sphingoid bases. scs2 cells were found to be sensitive to inhibitors of sphingolipid biosynthesis. The examination of strains lacking various enzymes involved in sphingolipid synthesis and direct analysis of lipid composition showed that a deletion of SCS2 impairs an upregulation of IPC synthase activity under conditions where the sphingolipid content of the cell is diminished. The reduced expression of genes involved in glycerolipid synthesis and the lower capacity for the synthesis of complex sphingolipids turned out to be mutually independent consequences of the complex phenotype of cells lacking Scs2p, whose primary function may not be in the lipid metabolism.

Din&ccedil / er-1 (1968) and South Din&ccedil / er-1 (1980) exploration wells are located at Sirnak Province of Southeast (SE) Anatolia. South Fields of SE Anatolia have received a significant attention after the completion of subjected wells and numerous studies have been implemented regarding this area. Many theories about the geological generation of these fields were put forward by people who studied this region.Both wells have penetrated the Arabian Plate autochthonous units. The Cudi Group, of this sequence, mainly consists of dolomites and anhydrites. The samples from the cores of this referred interval and the thin sections of these cores were examined in details by X-Ray Diffraction (XRD) Analyses and petrographic microscope. Thin sections taken from the core samples of the Cudi Group&rsquo / s Bak&uuml / k, &Ccedil / amurlu and Telhasan formations (from older to younger) stand out in the diagenetic manner. The analyses of these thin sections showed that dolomitization is the main diagenetic process along with some textural changes such as the increase in the deformation of algal structures, formation of stylolites and secondary porosity. Clay minerals, mainly illites, shows detritic behaviors rather than characters representing a diagenetic origin. Obtained results from this study showed that the dolomitization as diagenetic process plays an important role in oil and gas formations within Cudi Group. Dolomite stoichiometry studies indicated that Cudi Group formations have modern dolomites since they show poor ordering reflections. They are also younger formations which are subjected to longer periods of diagenetic effects in comparison with Uludere Formation&rsquo / s dolomites.

Die vorliegende Arbeit beschäftigt sich mit der genetischen Optimierung der Spalthefe S. pombe für die biotechnologische Produktion von Fremdproteinen. Hierbei werden vor allem zwei Aspekte näher untersucht, zum einen die Stabilität des zu produzierenden Proteins und zum anderen die Bildung von Disulfidbrücken. Von anderen Organismen ist bekannt, dass die N-terminale AS im Verbund mit einem Lysinrest ein Protein destabilisieren kann. Das Modellprotein vVEGF besitzt an Position 2 einen Lysinrest (K2) und damit ein Hauptmerkmal eines derartigen Destabilisierungselementes. Falls das Protein dem Ubiquitin-vermittelten Abbau unterliegt, ist es wahrscheinlich, dass K2 eine essenzielle Rolle für die Stabilität dieses Proteins spielt. Im Rahmen dieser Arbeit konnte gezeigt werden, dass K2 in S. cerevisiae destabilisierend wirkt, während es in S. pombe keinen destabilisierenden Effekt hat. Dieses Ergebnis spricht dafür, dass es Unterschiede im Ubiquitin-vermittelten Abbau von Proteinen in diesen beiden Hefen gibt. Der Schwerpunkt dieser Arbeit lag auf der Analyse und Optimierung der Bildung von Disulfidbrücken in S. pombe. Disulfidbrücken stellen eines der wichtigsten Elemente der korrekten Proteinfaltung dar und werden in Eukaryonten vorwiegend im oxidierenden Milieu des ER in das naszierende Protein eingeführt. Aus diesem Grunde wurden Proteindisulfid-isomerasen (PDIs) und ER-oxidoreduktin (Ero)-ähnliche Proteine, die die Schlüssel-komponenten der Bildung von Disulfidbrücken in Eukaryonten darstellen, näher untersucht. In S. pombe finden sich insgesamt drei PDI-Homologe (SpPdi1p, SpPdi2p und SpPdi3p) sowie zwei Ero-Homologe (SpEro1a p und SpEro1b p). Mit Ausnahme des nicht glycosylierten SpPdi2p, sind alle Proteine Membran-assoziierte glycosylierte Komponenten des ER. SpPdi2p und SpPdi3p sowie SpEro1a p und SpEro1b p liegen in vivo teilweise in oxidiertem Zustand vor. Des Weiteren konnte gezeigt werden, dass SpEro1b p, nicht jedoch SpEro1a p in der Lage ist, die temperatursensitive S. cerevisiae ero1-1-Mutante funktionell zu komplementieren. Interessanterweise ergab die Untersuchung konservierter Cysteine mittels gerichteter Mutagenese einerseits Unterschiede zwischen SpEro1a p und SpEro1b p sowie andererseits zwischen den S. pombe Ero-Proteinen und den Ero-Proteinen anderer Spezies. Im Gegensatz zu Ero1b p wird Ero1a p durch reduzierenden Stress und Hitzestress induziert. Dies deutet darauf hin, dass SpEro1b p für die Bildung von Disulfidbrücken unter normalen Wachstumsbedingungen nötig ist, während SpEro1a p vornehmlich bei der Adaption der Zellen an Stressbedingungen erforderlich ist. Abschließend konnte gezeigt werden, dass die gesteigerte Expression von SpEro1a p und SpEro1b p zu einer deutlich erhöhten Ausbeute des disulfidhaltigen heterologen Proteins Orf19p-HA führt. Dieser Befund impliziert, dass in S. pombe die Oxidation der Disulfidbrücken für die Faltung von Proteinen vermutlich limitierend ist.

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