Blastocystis is an important parasite that infects humans and a wide range of animals like rats, birds, reptiles, etc. infecting a sum of 60% of world population. It belongs to the Stramenopiles, a Heterologous group that includes for example the Phythophthora infestans the responsible for the Irish potato famine. Previous work had reported the presence of an unusual fusion protein that is composed of two of the main glycolytic enzymes; Triosephosphate isomerase-glyceraldehyde-3-phosphate dehydrogenase (TPI-GAPDH). Little is known about this protein. Blastocystis TPI-GAPDH and Blastocystis enolase were both characterized biochemically and biophysically in this project. The phylogenetic relationships of those two proteins among other members of either Stramenopiles, or other members of the kingdom of life were examined and found to be grouping within the chromalveolates. Our studies revealed that those two proteins, Blastocystis enolase and Blastocystis TPI-GAPDH, had a peptide signal targeting them to the mitochondria. This was an unusual finding knowing that text books always referred to the glycolytic pathway as a canonical cytoplasmic pathway. Structural studies had also been conducted to unravel the unknown structure of the fusion protein Blastocystis TPI-GAPDH. X-ray crystallography had been conducted to solve the protein structure and the protein was found to be a tetrameric protein composed of a central tetrameric GAPDH protein flanked with two dimmers of TPI protein. Solving its structure would be the starting point towards reviling the role that TPI-GAPDH might play in Blastocystis and other organisms that it was found in as well. Although a fusion protein, the individual components of the fusion were found to contain all features deemed essential for function for TPI and GAPDH and contain all expected protein motifs for these enzymes.

The role of CEA for stroke prevention in the presence of symptomatic carotid artery stenosis is well established. In order to maximize the benefit of surgery, several perioperative processes of care have been under scrutiny, of which one is the choice of anaesthetic method. The differing effects of GA vs. LA on the cerebral circulation after CEA may be of significance, since changes in the cerebral circulation post-CEA may give rise to cerebral hyperperfusion and intracerebral haemorrhage. This work assessed the effect of GA vs. LA on cerebral haemodynamic control after CEA using transcranial Doppler (TCD) techniques, and correlated these changes with serum markers of cerebral injury. Subjects undergoing CEA had perioperative TCD monitoring of middle cerebral artery blood flow velocity (MCAV). Pre- and postoperative (within 48 hours of surgery) testing of cerebral autoregulation [CA] (tilt-testing) and cerebral vasoreactivity to CO2 [CVR] (rebreathing expired air) was conducted. Cerebral haemodynamic parameters and clinical outcome were correlated with changes in jugular venous and peripheral levels of protein S100β and neurone-specific enolase (NSE).The change in CA and CVR was not different between GA (n=16) and LA (n=20). Overall, CA and CVR improved significantly within 48 hours of CEA for patients with preoperative impairment of these parameters, although some patients with normal baseline CA and CVR exhibited postoperative impairment. Increase of MCAV >100% from baseline after restoration of carotid blood flow was observed in patients with impaired CVR, but resolved by the first postoperative day. Transient elevation in jugular venous (but not peripheral) S100β during surgery was seen. Both jugular and peripheral NSE levels dropped during surgery. Neither anaesthetic method nor CA or CVR status had any effect on changes in serum S100β or NSE. Cerebral autoregulatory parameters thus improve rapidly after CEA, but appear unaffected by anaesthetic technique. This supports the concept that cerebral hyperperfusion is dependent on factors in addition to impaired CA or CVR. Changes in serum S100β or NSE do not reflect cerebral haemodynamic changes. However, the variability encountered between patients warrants further investigation. The implications for clinical practice and directions for further research are discussed.

The neuroendocrine (NE) cell is a minor cell population in normal human prostate glands. The number of NE cells is increased in advanced hormone-refractory prostate carcinomas (PCA). The mechanism of increased NE cell population in these advanced tumors is poorly understood. We examined molecular mechanisms which may be involved in the regulation of the transdifferentiation process of human PCA cells leading to a NE phenotype. We compared PCA cell lines LNCaP and PC-3 in the following medium conditions: steroid-reduced (SR), interleukin-6 (IL-6)-supplemented, or dibutyrate cAMP (db-cAMP)-supplemented. We found that androgen-responsive C-33 LNCaP cells responded to all treatments, having a neuronal-like morphology. In contrast, C-81 LNCaP cells, having a decreased androgen responsiveness, had a less pronounced effect although followed a similar trend. Androgen-unresponsive PC-3 cells showed little change in their morphology. Grown in the SR condition, the level of neuron-specific enolase (NSE), a marker of neuronal cells, was upregulated in C-33 LNCaP cells, while to a lesser degree in the presence of IL-6. In the presence of db-cAMP, the NSE level in C-33 cells was decreased, lower than that in control cells. An opposite effect was observed for C-81 LNCaP cells. Nevertheless, the NSE level was only elevated in db-cAMP-treated PC-3 cells, but no change was found in PC-3 cells grown in the SR- or IL-6-supplemented medium. Thus, a similar gross phenotypic change may correlate with differential molecular expressions. We also analyzed the expression of protein tyrosine phosphatase α (RPTPα) since it plays a critical role in normal neuronal differentiation and signaling. Our results showed that the expression of RPTPα correlates with the NE phenotypic change of LNCaP cells in the SR condition. In summary, our data clearly show that the molecular process by which cultured human prostate cancer cells undergo a transdifferentiation process to a NE cell-like phenotype is accompanied by differential expressions of different markers, and a gross NE cell-like phenotype can occur by exposing PCA cells to different pharmacological agents.

>Magister Scientiae - MSc / Knowledge of haematological 'normdata', of experimental animals, and the biological variables that affect it is essential in order to recognise variations from the normal. In addition, the haemopoietic system may be regarded in principle as good material for studies of the cellular events associated with ageing. These considerations, together with the well documented effects of age on various physiological processes, prompted an investigation into the effects of donor age on several blood parameters. Review of the literature revealed that age-related changes in blood parameters have been reported for several species, but the documentation thereof is incomplete, inconsistent and inconclusive in many respects. Blood samples from male Wistar rats of nine different biological ages, ranging from birth to 96 weeks of age, were analysed for haematological and biochemical parameters. These included the blood cell counts, erythrocytic indices, haemoglobin concentration, haematocrit, erythrocytic 2,3-diphosphoglycerate and adenosine triphosphate levels, and erythrocytic glucose 6-phosphate dehydrogenase and pyruvate kinase activities. Data was obtained which demonstrates that all blood parameters measured underwent significant, although not al~ays regular, age-related changes. These changes were found to be more marked during the first month of life than at any other period. Evidence is also presented to show that the depressed haemoglobin concentration during the early postnatal life may not imply a condition of 'physiologic anaemia' as was previously thought. Since the blood profile exhibits only slight changes from about 24 weeks of age, it does not seem that the haemopoietic system of the old rat deteriorates significantly as to constitute a limiting factor for the animal's life. However, the importance of taking an animal's age into account when blood parameters constitute experimental results is emphasised. The second phase of this study involved a detailed investigation of the effect of the animal's age on erythrocytes in particular. These cells have limited life-spans, and are often used as models in studies of cellular ageing. Special emphasis was therefore placed on comparing the relative effects of host and cellular ageing on the properties of these cells. Erythrocytes from rats between one and 48 weeks of age were separated into two populations by a modification of the conventional density gradient centrifugation technique. The two populations were assumed to differ in mean cell age and were analysed for erythrocytic indices, phosphate ester concentrations and the activities of glucose 6-phosphate dehydrogenase and pyruvate kinase. Evidence is presented to show that ageing rat erythrocytes exhibit a decrease in volume, phosphate ester content and enzyme activities while the cellular haemoglobin concentration increases. Differences in the mean cell age however, does not seem to account for the donor-age-related effects observed in the whole blood parameters. Rather, the significant differences found in the characteristics of similarly aged red cells, between variously aged donors, demonstrate that the biological age of the organism influences the red cells and probably the ageing thereof in vivo. The contribution of the changing status of the erythrocyte's environment of progressively older animals, to alterations which take place in the ageing red cell is discussed.

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Made available in DSpace on 2017-07-24T19:38:06Z (GMT). No. of bitstreams: 1 Cibeli May Villalba.pdf: 4939252 bytes, checksum: 5a6b5eaea0352844ad9868a7a9b79f0b (MD5) Previous issue date: 2012-03-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Proteins are the most abundant biomolecules in living organisms; they are present in all parts of a cell. They have different functions; their structural study is important because it brings greater insight into its functions and allows us to understand how they interact to each other and with the other molecules. Protein structures can be studied experimentally especially by the X-ray diffraction technique and computationally by homology modeling. Thus, in this work, structural studies were made with the alpha-amylase inhibitor from rye (Secale cereale), which inhibits the activity of amylase and then can be used in the treatment of Diabetes mellitus, obesity, pest control, amongst other applications. Through chromatographies, two different inhibitors could be separated, namely A2 and B2, which were crystallized, but did not show a minimum X-ray diffraction quality. Thus, a structural study was performed with data from a twinned crystal previously obtained, yet current refinement programs can now deal with such data. The structure was refined and compared with the alpha-amylase inhibitor 0.19 from wheat. Then, structural studies were also performed for the YchF GTPase and enolase from Trypanosoma cruzi; both have been studied with the possibility of being used as a target in the treatment of Chagas' disease. Initially, trials to express heterologously and to purify them for crystallization trials were performed; yet those were unsuccessful, a computational work was pursued, in which alignments and homology modelling for both proteins were made. The computational work was continued for Trypanosoma cruzi enolase,in which comparisons with the Homo sapiens enolase to seek and plan inhibitors for the former, through literature and data bank searches, were made; thus, docking of these was performed, which pointed more favorable binding energies for the substrates, phosphoenolpyruvate (PEP) and 2-phosphoglycerate (PG2), for the inhibitor phosphonacetohydroxamate (PAH) and for the compound coded ZINC25695689 from the ZINC (ZINC Is Not Commercial) data bank. Also, from the experimental position of the PAH inhibitor (deposited in the PDB, code 2PTZ), the interaction energies for these searched and planned molecules were estimated,through the AMBER molecular dynamics program, and, apparently, the presence of a chlorine atom conveniently bound to the inhibitor could promote an improvement of the interaction energy. / As proteínas são as biomoléculas mais abundantes nos seres vivos, estando presentes em todas as partes de uma célula. Elas possuem diferentes funções no organismo; seu estudo estrutural é importante, pois traz maior conhecimento sobre suas funções e possibilita entender como interagem entre elas e com outras moléculas. A estrutura de proteínas pode ser estudada experimentalmente principalmente por meio da técnica de difração de raios X e computacionalmente por meio da modelagem por homologia. Sendo assim, realizaram-se neste trabalho estudos estruturais com o inibidor de alfa-amilase do centeio (Secale cereale), que inibem a atividade amilásica e que podem ser utilizados em tratamento de Diabetes mellitus, obesidade, controle de pragas, entre outros usos. Por meio de cromatografias, puderam-se separar dois diferentes inibidores, denominados A2 e B2, que foram cristalizados, mas não apresentaram mínima qualidade de difração de raios X. Assim, realizou-se um estudo estrutural com dados de difração obtidos anteriormente para um cristal geminado, dada a possibilidade atual dos programas de refinamento de tratarem este problema. A estrutura foi refinada e comparada com o inibidor de alfa-amilase 0,19 do trigo. Em sequência, estudos estruturais também foram realizados para a proteína YchF da família das GTPases e para a enolase de Trypanosoma cruzi; ambas vêm sendo estudadas com a possibilidade de serem usadas como alvo no tratamento da doença de Chagas. Inicialmente tentou-se expressá-las de maneira heteróloga e purificá-las para a realização de ensaios de cristalização; com o insucesso disto, partiu-se para um trabalho computacional em que se fizeram alinhamentos e modelos por homologia para as duas proteínas. O trabalho computacional foi continuado para a enolase de Trypanosoma cruzi, comparando-a com a enolase de Homo sapiens para se buscar e planejar inibidores para a primeira, por meio de pesquisa na literatura e em bancos de dados; assim, fez-se a alocação ("docking") destes, obtendo-se energias de ligação mais favoráveis para os substratos, fosfoenolpiruvato (PEP) e 2-fosfoglicerato (PG2), para o inibidor fosfonacetohidroxamato (PAH) e para o composto codificado ZINC25695689 do banco de dados ZINC (ZINC Is Not Commercial). Também, a partir da posição experimental do inibidor PAH (depositada no PDB, código 2PTZ) estimaram-se as energias de interação para as moléculas pesquisadas e planejadas, através do programa de dinâmica molecular AMBER e, aparentemente, a presença de um átomo de cloro convenientemente ligado ao inibidor poderia promover melhoria da energia de interação.

Einleitung: Epidemiologische Studien zeigen den langfristigen Nutzen körperlicher Aktivität zur Prävention neurologischer Erkrankungen und kognitiver Defizite im Alter auf. Sportliche Betätigung kann jedoch, vor allem bei Ausübung von Kontaktsportarten, auch akute und chronische neuronale Schädigungen durch häufige mechanische Beeinflussungen des Gehirns hervorrufen. NSE und S-100B sind laborchemische Marker, die im Rahmen der Diagnostik von Hirnschädigungen zum Einsatz kommen. Einige Forschungsarbeiten fanden erhöhte Konzentrationen dieser Biomarker nach sportlichen Belastungen ohne offensichtliches Vorliegen von traumatischen Ereignissen oder neurologischer Beeinträchtigungen. Daher stellt sich die Frage, ob entsprechende Norm- bzw. Cut-Off-Werte für NSE und S-100B universell im klinischen Alltag geeignet sind oder durch belastungsinduzierte Einflüsse einer differenzierteren Beurteilung unterzogen werden sollten. In vorliegender Arbeit wird diese Fragestellung anhand der klassischen Ausdauersportarten Laufen und Radfahren untersucht. Ergebnisse und Diskussion: Zunächst erfolgte der Nachweis für die Reproduzierbarkeit von NSE bei Ausdauersportlern für Ruhe- und Belastungsbedingungen. Es wurden keine signifikanten Unterschiede für NSE im Belastungsvergleich von Radfahren und Laufen, wie beim Vergleich verschiedener Laufintensitäten im zeitlichen Verlauf festgestellt. Die denkbare Einflussnahme beim Laufen durch leichte Erschütterungen des Gehirns basierend auf dem Impact beim Bodenkontakt des Fußes, ist offensichtlich irrelevant. Physischer Stress, der durch einen Marathonlauf verursacht wird, führt offensichtlich in der Akutphase nach dem Wettkampf zu einem signifikanten Anstieg von NSE und S-100B, welche im späteren Verlauf wieder auf das Ausgangsniveau abfallen. Dies ist unabhängig von Alter und Geschlecht. Über dem Normwert für Gesunde liegende NSE-Konzentrationen nach einer Marathonbelastung, haben offensichtlich keine pathophysiologischen Auswirkungen oder klinische Korrelationen. In der klinischen Praxis sollten erhöhte NSE-Werte entsprechend vorsichtig interpretiert werden. Weitere marathonspezifische Faktoren wie Trainingshäufigkeit, Renneinteilung, Wetterbedingungen und Dysnatriämie, aber auch Alter und Geschlecht zeigten keine Zusammenhänge hinsichtlich der Serum-Konzentration von NSE und S-100B. Ebenfalls fanden sich keine signifikanten Veränderungen bei der Klassifizierung von Notfallpatienten bei Laufveranstaltungen bezüglich Diagnose und Streckenlängen. Fortführende Forschungsaktivitäten mit hohen Fallzahlen sind notwendig, um die Wertigkeit der beiden biochemischen Labormarker unter Einfluss körperlicher Aktivität herauszustellen.:1. Einleitung und allgemeine Problemstellung 1 1.1 Einleitung 1 1.2 Ziele der Arbeit 4 2. Theoretische Grundlagen 5 2.1 Theoretische Grundlagen zu NSE 5 2.2 Theoretische Grundlagen zu S-100B 8 2.3 Einfluss sportlicher Belastungen auf NSE 9 2.4 Einfluss sportlicher Belastungen auf S-100B 10 3. Grundlegende Aspekte zur Methodik dieser Arbeit 12 3.1 Blutanalytik 13 3.2 Messverfahren NSE und S-100B 13 3.3 Ausschluss Hämolyse 14 3.4 Plasmavolumenkorrektur 17 3.5 Leistungsdiagnostik 18 3.6 Auswertung und Statistik 19 4. Teilstudie 1 - Methodische Untersuchungen 21 4.1 Reproduzierbarkeit von NSE unter Ruhe- und Belastungsbedingungen 21 4.1.1 Einleitung 21 4.1.2 Methoden 21 4.1.2.1 Probanden 21 4.1.2.2 Studiendesign 23 4.1.3 Ergebnisse 24 4.1.4 Diskussion 30 4.2 Pilotstudie zur In-vivo-Bestimmung von zerebralen S-100B mittels 1H-Magnetresonanzspetroskopie 35 4.2.1 Einleitung 35 4.2.2 Methoden 35 4.2.2.1 Probanden 35 4.2.2.2 Messverfahren 36 4.2.2.3 Studiendesign 39 4.2.3 Ergebnisse 39 4.2.4 Diskussion 41 5. Teilstudie 2 - Untersuchungen zum Einfluss sportlicher Belastung und NSE 47 5.1 Belastungsart und NSE 47 5.1.1 Einleitung 47 5.1.2 Methoden 47 5.1.2.1 Probanden 47 5.1.2.2 Studiendesign 48 5.1.3 Ergebnisse 50 5.1.4 Diskussion 54 5.2 Belastungsintensität und NSE 56 5.2.1 Einleitung 56 5.2.2 Methoden 56 5.2.2.1 Probanden 56 5.2.2.2 Studiendesign 57 5.2.3 Ergebnisse 57 5.2.4 Diskussion 61 6. Teilstudie 3 - Untersuchungen zum Einfluss von Marathonbelastungen auf NSE und S-100B 62 6.1 Einleitung 62 6.2 Methoden 64 6.2.1 Probanden 64 6.2.2 Sonstige Blutparameter 69 6.2.3 Studiendesign 71 6.3 Ergebnisse 71 6.3.1 Zeitlicher Verlauf 71 6.3.2 Geschlecht und NSE 73 6.3.3 Einfluss des Alters 75 6.3.4 Sportanamnestische Daten 79 6.3.5 Renntaktik 81 6.3.6 Wetterbedingungen 82 6.3.7 Dysnatriämie 84 6.3.8 NSE und S-100B im Zusammenhang mit weiteren belastungsspezifischen Laborparametern 85 6.4 Diskussion 91 6.4.1 Zeitlicher Verlauf 91 6.4.2 Alter und Geschlecht 93 6.4.3 Trainingshäufigkeit und -umfang 94 6.4.4 Renneinteilung 95 6.4.5 Elektrolytstörungen 97 6.4.6 NSE und S-100B im Zusammenhang mit weiteren belastungsspezifischen Laborparametern 98 7. Teilstudie 4 - Untersuchungen bei laufassoziierten Notfällen 104 7.1 Laufassoziierte Notfälle und NSE 104 7.1.1 Einleitung 104 7.1.2 Methoden 105 7.1.2.1 Probanden 105 7.1.2.2 Studiendesign 105 7.1.3 Ergebnisse 106 7.1.4 Diskussion 108 7.2 Einzelfallstudie 111 7.2.1 Einleitung 111 7.2.2 Methoden 111 7.2.2.1 Proband 111 7.2.2.2 Studiendesign 112 7.2.3 Ergebnisse 112 7.2.4 Diskussion 114 8. Abschließende Diskussion 118 9. Zusammenfassung 123 10. Literaturverzeichnis 125 11. Anhang 140 12. Danksagung 145 13. Eidesstattliche Erklärung 146 / Introduction: Epidemiological studies show long term benefits of exercise and sports for neurological diseases and cognitive deficits in elderly populations. In contact sports however also acute and chronic neuronal impairment of the brain itself caused by repetitive mechanical impact have been found. NSE and S-100B are biochemical markers used in diagnostics of brain injuries. Through several studies found increased values of theses markers after exercise without evident existence of traumatological occasion or neurological impairment. Whether NSE and S-100B and its corresponding reference- and cut-off-values can be applied in clinical routine or if exercise-induced effects apply for a more sophisticated evaluation, should herein be examined by the effects of traditionally endurance sports running and cycling. Results and discussion: At first the proof for repeatability of NSE in endurance sportsmen at rest and during physical activity was given. When comparing the effects of cycling and running as well as different training intensities in these sports, no significant changes could be noticed. Possible causes of elevated NSE due to slight concussions of the brain based on repetitive impact forces during running are obviously irrelevant. Running a marathon tends to result in a significant raise of NSE and S-100B in the acute period after the competition. The further process shows a decline to the base level independent of age or gender. NSE values above norm values after completing a marathon have clearly no pathophysiological effects or clinical correlates. Thus high NSE- concentrations should be interpreted with caution in clinical praxis. Further marathon specific factors like training, pacing strategy, weather conditions, dysnatraemia show no significant correlations with NSE or S-100B. Clustering emergency cases by diagnosis and running distance revealed no relationships to NSE. Further research with larger populations is necessary, to emphasize the relevance for NSE and S-100B during exercise.:1. Einleitung und allgemeine Problemstellung 1 1.1 Einleitung 1 1.2 Ziele der Arbeit 4 2. Theoretische Grundlagen 5 2.1 Theoretische Grundlagen zu NSE 5 2.2 Theoretische Grundlagen zu S-100B 8 2.3 Einfluss sportlicher Belastungen auf NSE 9 2.4 Einfluss sportlicher Belastungen auf S-100B 10 3. Grundlegende Aspekte zur Methodik dieser Arbeit 12 3.1 Blutanalytik 13 3.2 Messverfahren NSE und S-100B 13 3.3 Ausschluss Hämolyse 14 3.4 Plasmavolumenkorrektur 17 3.5 Leistungsdiagnostik 18 3.6 Auswertung und Statistik 19 4. Teilstudie 1 - Methodische Untersuchungen 21 4.1 Reproduzierbarkeit von NSE unter Ruhe- und Belastungsbedingungen 21 4.1.1 Einleitung 21 4.1.2 Methoden 21 4.1.2.1 Probanden 21 4.1.2.2 Studiendesign 23 4.1.3 Ergebnisse 24 4.1.4 Diskussion 30 4.2 Pilotstudie zur In-vivo-Bestimmung von zerebralen S-100B mittels 1H-Magnetresonanzspetroskopie 35 4.2.1 Einleitung 35 4.2.2 Methoden 35 4.2.2.1 Probanden 35 4.2.2.2 Messverfahren 36 4.2.2.3 Studiendesign 39 4.2.3 Ergebnisse 39 4.2.4 Diskussion 41 5. Teilstudie 2 - Untersuchungen zum Einfluss sportlicher Belastung und NSE 47 5.1 Belastungsart und NSE 47 5.1.1 Einleitung 47 5.1.2 Methoden 47 5.1.2.1 Probanden 47 5.1.2.2 Studiendesign 48 5.1.3 Ergebnisse 50 5.1.4 Diskussion 54 5.2 Belastungsintensität und NSE 56 5.2.1 Einleitung 56 5.2.2 Methoden 56 5.2.2.1 Probanden 56 5.2.2.2 Studiendesign 57 5.2.3 Ergebnisse 57 5.2.4 Diskussion 61 6. Teilstudie 3 - Untersuchungen zum Einfluss von Marathonbelastungen auf NSE und S-100B 62 6.1 Einleitung 62 6.2 Methoden 64 6.2.1 Probanden 64 6.2.2 Sonstige Blutparameter 69 6.2.3 Studiendesign 71 6.3 Ergebnisse 71 6.3.1 Zeitlicher Verlauf 71 6.3.2 Geschlecht und NSE 73 6.3.3 Einfluss des Alters 75 6.3.4 Sportanamnestische Daten 79 6.3.5 Renntaktik 81 6.3.6 Wetterbedingungen 82 6.3.7 Dysnatriämie 84 6.3.8 NSE und S-100B im Zusammenhang mit weiteren belastungsspezifischen Laborparametern 85 6.4 Diskussion 91 6.4.1 Zeitlicher Verlauf 91 6.4.2 Alter und Geschlecht 93 6.4.3 Trainingshäufigkeit und -umfang 94 6.4.4 Renneinteilung 95 6.4.5 Elektrolytstörungen 97 6.4.6 NSE und S-100B im Zusammenhang mit weiteren belastungsspezifischen Laborparametern 98 7. Teilstudie 4 - Untersuchungen bei laufassoziierten Notfällen 104 7.1 Laufassoziierte Notfälle und NSE 104 7.1.1 Einleitung 104 7.1.2 Methoden 105 7.1.2.1 Probanden 105 7.1.2.2 Studiendesign 105 7.1.3 Ergebnisse 106 7.1.4 Diskussion 108 7.2 Einzelfallstudie 111 7.2.1 Einleitung 111 7.2.2 Methoden 111 7.2.2.1 Proband 111 7.2.2.2 Studiendesign 112 7.2.3 Ergebnisse 112 7.2.4 Diskussion 114 8. Abschließende Diskussion 118 9. Zusammenfassung 123 10. Literaturverzeichnis 125 11. Anhang 140 12. Danksagung 145 13. Eidesstattliche Erklärung 146

Le dégradosome d'E. coli est un complexe protéique impliqué dans la dégradation des ARNm constitué de quatre protéines majeures : RNase E, PNPase, RhlB et l'Enolase. Dans le but de mieux comprendre l'action du dégradosome, des études de l'interaction de RhlB et des autres hélicases DEAD-box de la cellule avec la RNase E ont été menées. Deux sites distincts d'interaction ont été mis en évidence dont l'un spécifique de RhlB. Ceci suggère l'existence de dégradosome alternatif différents selon les conditions de croissance. Des travaux menés sur l'Enolase afin de déterminer son rôle au sein du complexe semblent écarter un rôle structural du complexe ou global de la dégradation des ARNm. L'hypothèse d'un effet plus spécifique est privilégiée. Enfin, des travaux sur la régulation de la RNase E par phosphorylation ont conduit à plusieurs modèles de mécanismes son inhibition par ce processus. Une relation étroite entre le domaine NTH et CTH de la RNase E a ainsi été mise en évidence. De plus, il semble qu'une kinase endogène de E. coli puisse phosphoryler le CTH de la RNase E, uniquement en absence du NTH.

The accumulation of androstenone in the fat of mature boars results in boar taint; the conjugation of androstenone would decrease this important meat quality problem by decreasing the accumulation and increasing the excretion of androstenone. Leydig cells and testis microsomes from mature boars were incubated with radiolabeled pregnenolone, and the free and conjugated metabolites were examined by HPLC. Sulfated androstenone with a mass of 367 m/z was directly identified by MS, with a novel tentative structure of 3-keto-4- sulfoxy-androstenone. Addition of enolase to the microsomal incubations increased the formation of 3-keto-4-sulfoxy-androstenone. Overexpression of SULT2A1 in HEK cells resulted in the sulfoconjugation of dehydroepiandrosterone, but not androstenone, suggesting that SULT2A1 may not be involved in sulfoconjugation of androstenone. This thesis describes the novel direct characterization of androstenone sulfate and the importance of enolase in its formation. The relevance to boar taint metabolism is discussed.

Neuronspecifikt enolas (NSE) är en viktig biomarkör för att diagnostisera t.ex. neuroendokrina tumörer, särskilt småcellig lungcancer (SCLC) och neuroblastom. NSE används även som en del i utredning av hjärnskada vid hjärtstopp. Eftersom NSE finns i höga koncentrationer i erytrocyter, kan hemolys i blodprovet orsaka falskt förhöjda NSE-nivåer i serum utan hjärnskada. Syftet med studien var att utvärdera hur hemolys påverkar NSE-analysen på Cobas, ett helautomatiserat analysinstrument. Mätning av NSE-koncentration utfördes på Cobas 8000 från Roche Elecsys, baserad på immunokemisk sandwich-metod med ElectroChemi-LuminiscenceImmunoassay (ECLI) detektionsteknik. För att studera hemolysens inverkan, tillverkades hemolysat från 20 patientprover. Dessa hemolysat tillsattes till poolat serum, med NSE-nivåer inom referensintervallet (< 17 µg/L). Även graden av hemolys bestämdes på Cobas 8000. Resultatet visade ett linjärt samband mellan de uppmäta hemolysindex (HI) värden och S-NSE värden. Variationen i NSE-tillskott på individnivå undersöktes och resulterade i slutsatsen att varje hemolysenhet motsvarar ett NSE-tillskott på 0,33 ± 0,07 µg/L som frigörs från erytrocyter. Ett förslag för att lösa problemet med hemolys vid analys av S-NSE är att använda en kompenserande faktor för att korrigera NSE-koncentrationen. Kompensering kan utföras med hjälp av det erhållna sambandet i studien (1 HI = 0,33 ± 0,07 µg/L NSE-tillskott) genom att subtrahera tillskottet från den uppmätta NSE-koncentrationen. / Neuron Specific Enolase (NSE) is an important biomarker for diagnosing e.g. neuroendocrine tumors, especially small cell lung cancer (SCLC) and neuroblastoma. NSE is also used as a part of the investigation of brain damage in cardiac arrest. Because NSE is present in high concentrations in erythrocytes, hemolysis in the blood sample can cause falsely elevated NSE levels in serum without brain damage. The purpose of this study was to evaluate how hemolysis affects NSE analysis on Cobas, a fully automated analytical instrument. Measurement of NSE concentration was performed on Cobas 8000 from Roche Elecsys, based on immunochemical sandwich method with ElectroChemi-Luminescence Immunoassay (ECLI) detection technique. To study the effect of hemolysis, hemolysates were prepared from 20 patient samples. These hemolysates were added to pooled serum, with NSE levels within the reference range (<17 μg/L). The degree of hemolysis was also determined on Cobas 8000. The result showed a linear relationship between the measured hemolysis index (HI) values and S-NSE values. The variation in NSE contribution at the individual level was examined with the result that each hemolysis unit corresponds to an NSE contribution of 0.33 ± 0.07 µg/L, which is released from erythrocytes. A suggestion to solve the problem of hemolysis relating to NSE analysis is to use a compensatory factor to correct the NSE concentration. Compensation can be performed by using the relationship obtained in the study (1 HI = 0.33 ± 0.07 µg/L NSE contribution) and subtracting the contribution from the measured NSE concentration.