No description available.

Plasma volume (PV) rises by up to 50% in normal pregnancy, a phenomenon associated with a favourable pregnancy outcome. A previous study of pregnant women with sickle cell (haemoglobin SS) disorder found that PV paradoxically contracts in late pregnancy. A cross-sectional study was performed to determine PV (Evans blue method) and volume regulatory hormones and electrolytes in pregnant women with haemoglobin (Hb) SS and in non-pregnant and Hb AA controls. PV rose in pregnant HbAA and was significantly correlated with plasma angiotensinogen. Non-pregnant Hb SS women had supranormal PV measurements and reduced glomerular filtration rate (GFR). Their PV did not rise in pregnancy and was not correlated with angiotensinogen. Their plasma renin concentration also failed to rise significantly by 36 weeks gestation and was significantly less than in Hb AA pregnancy although aldosterone concentration was raised as expected. A general vasoconstriction in pregnancy can cause inactivation of the renin-angiotensin system and could explain this, with aldosterone being elevated by non Angiotensin II dependent stimulation such as plasma potassium, which was significantly higher in the pregnant Hb SS women. Further studies demonstrating a deficiency of vasodilator substances in pregnant Hb SS women will strengthen this hypothesis.

The prevalence of cardiovascular disease is lower in premenopausal women compared to age-matched males and postmenopausal females. Differences in risk may be due to sex differences in endothelial function. Therefore, this thesis examined the effects of gender on endothelium-dependent vasorelaxation in porcine isolated coronary arteries (PCAs). Distal PCAs were studied under myographic conditions and pre-contracted with U46619. Concentration-response curves to bradykinin, an endothelium-dependent vasorelaxant, were constructed in the presence of various inhibitors. Inhibition of NO and prostanoid synthesis (EDH-type response) produced greater inhibition in males compared to females. Eliminating H2O2 using PEG-catalase significantly reduced the bradykinin-induced vasorelaxation in the absence, but not in the presence of L-NAME and indomethacin in females, and had no effect in males. Inhibition of gap junctions with carbenoxolone and 18α-GA inhibited the EDH-type response in females but not in males. Inhibition of SKCa channels reduced the EDH-type response in PCAs from both sexes but inhibition of IKCa had an effect only in females but not males. Western blot did not detect any differences in the expression of Cx40, 43 or IKCa between sexes. H2O2 caused concentration-dependent vasorelaxations which were significantly inhibited by PEG-catalase, TEA, 60 mM K+ and 500 nM ouabain. Inhibition of NOS, cyclo-oxygenase, gap junctions, SKCa, IKCa, BKCa, Kir, KV, KATP, cGMP, Na+-Ca2+ exchanger or removal of endothelium had no effect on the H2O2-induced vasorelaxation. 1 mM H2O2 inhibited both KCl-induced vasorelaxation and rubidium-uptake consistent with inhibition of the Na+/K+-pump activity. The effects of the antioxidant Tiron® under different gassing conditions (95% O2/5% CO2 or 95% air/5% CO2) were investigated. The bradykinin-induced vasorelaxations in PCAs were unaffected by different levels of oxygenation. Tiron® increased the potency of bradykinin only when gassed with 95% O2/5% CO2 and the enhancement in vasorelaxation was prevented by catalase. Similarly, Tiron® enhanced the EDH-type response when gassed with 95% O2/5% CO2 in PCAs from both sexes. Biochemical analysis using Amplex Red demonstrated that H2O2 was generated in Krebs’-Henseleit solution when gassed with 95% O2/5% CO2, but not with 95% air/5% CO2. Inhibition of Nox had no effect in PCAs from females but DPI, a non-selective Nox inhibitor reduced the potency of the bradykinin-induced vasorelaxation in males. In the EDH-type responses, inhibition of Nox had no effect in females, but in males, ML-171 (a selective Nox inhibitor) and DPI enhances while VAS2870 (a selective Nox inhibitor) reduces the bradykinin-induced vasorelaxation. ML-171 had no effect on the forskolin-induced vasorelaxation but decreased the potency of U46619-induced tone in both sexes in the absence or presence of endothelium. Nox activity was reduced by DPI and ML-171, but not VAS2870 in PCAs from both sexes. Sex differences in the functional study of Nox could be attributed to the differential expression of Nox proteins where expression of Nox1 and Nox2 were greater in males but Nox4 was greater in females. This may underlie the greater oxidative stress observed in males. Bradykinin-induced EDH-type responses in PCAs from both sexes were essentially abolished by 2-APB (TRPC&TRPM antagonist). SKF96365 (TRPC antagonist) inhibited the bradykinin-induced vasorelaxation in males, and EDH-type response in both sexes. Pyr3 (TRPC3 antagonist) inhibited both the NO and EDH components of the bradykinin-induced vasorelaxation in males, but not females. RN1734 (TRPV4 antagonist) reduced the potency of the NO component of the bradykinin-induced vasorelaxation in females only, but inhibited the EDH-type response in both sexes. 2-APB, SKF96365 and RN1734 all reduced the H2O2-induced vasorelaxation, whereas Pyr3 had no effect. No differences in expression level of TRPC3 and TRPV4 between sexes were detected using Western blot. In conclusion, present study demonstrated clear sex differences in endothelial function in PCAs where H2O2, MEGJs, IKCa and TRPV4 channels play a role in the bradykinin-induced vasorelaxation only in female pigs while Nox-generated reactive oxygen species and TRPC3 channels play a role in the bradykinin-induced vasorelaxation only in male pigs. Therefore, gender-specific drug treatment for cardiovascular disease may be a novel therapeutic strategy.

En la present tesi doctoral es presenta una aportació als estudis relacionats amb la construcció i reivindicació de la memòria i dels processos de representació implicats en l'escriptura de la història i en particular en la història dels vençuts. L'actual treball de recerca exposa i estudia, en un primer moment, la concepció d'història presentada per Benjamin. Aquesta se centra en particular en la seva última obra, les "Tesis de filosofia de la història", però així mateix ofereix les respostes que el filòsof alemany presenta al llarg de tot el seu recorregut intel·lectual fins arribar a les seves últimes formulacions, que s'han d'entendre com una resposta de caràcter ètic i profundament polític. La tesi, doncs, pren com a punt de partida metodològic i conceptual les aportacions realitzades per Walter Benjamin al debat contemporani al voltant de la filosofia de la història. La seva interpretació de la història com discontinuïtat i redempció serveix per oferir una suggerent forma de narrar i recuperar de l'oblit la veu dels vençuts que trenca amb la historiografia clàssica, basada en una interpretació lineal i causal dels esdeveniments històrics. Conceptes com "instant", "temps-ara", "discontinuïtat", "rememoració" estan al servei d'una construcció de la història que posa l'accent en les llacunes i en els intersticis de tota narració històrica amb pretensió de totalitat. Aquesta estratègia permet rescatar el valor del testimoni i trencar així amb l'homogeneïtat de la cronologia clàssica. Al llarg d'aquest estudi, es presenta com s'articula aquesta nova concepció de la història, a partir de la idea central de rememoració, que pretén presentar un relat històric que, tal i com proposa Benjamin, se centri no ja en l'èpica de la història dels vencedors sinó en la tradició dels vençuts. Davant del relat acumulatiu i autocomplaent de l’historicisme, davant de la falsa totalitat, il·lusió mítica de l’ historicisme, per anar més enllà del relat dels vencedors s'imposa la necessitat d'una altra escriptura de la història, d'una altra història. En aquest sentit, resulta d'especial importància la figura del testimoni, ja que aquesta carrega contra la invisibilitat a la qual es pretén confinar els vençuts, ja que s'entén que el veritable testimoni és aquell que no ha pogut testimoniar. Així s'ha d'entendre l'imperatiu de la representació que s'exposa -doncs en el centre de la representació sempre batega l'absència-, com aquell gest ètic que posa en joc en la llacuna constitutiva del veritable relat històric i que es presenta en la figura del testimoni , així com en l'obra d'art entesa com a espai del testimoni i com a testimoni. En un segon moment, s'estudia com es trasllada a la representació del passat tal concepció i com aquesta intervé en el paisatge del sensible a través de l'obra d'art, en concret en la narrativa de l'escriptor W. G. Sebald. L'escriptor alemany, doncs, es pren com a exemple de l'escriptura de la història com rememoració reivindicada per Benjamin i il·lustra perfectament el marc teòric que presenta el filòsof. / En la presente tesis doctoral se presenta una aportación a los estudios relacionados con la construcción y reivindicación de la memoria colectiva y de los procesos de representación implicados en la escritura de la historia y en particular en la historia de los vencidos. El actual trabajo de investigación expone y estudia, en un primer momento, la concepción de historia presentada por Benjamin. Esta se centra en particular en su última obra, las "Tesis de filosofía de la historia", pero asimismo ofrece las respuestas que el filósofo alemán presenta a lo largo de todo su recorrido intelectual hasta llegar a sus últimas formulaciones, que deben entenderse como una respuesta de carácter ético y profundamente político. La tesis, pues, toma como punto de partida metodológico y conceptual las aportaciones realizadas por Walter Benjamin al debate contemporáneo en torno a la filosofía de la historia. Su interpretación de la historia como discontinuidad y redención sirve para ofrecer una sugerente forma de narrar y recuperar del olvido la voz de los vencidos que rompe con la historiografía clásica, basada en una interpretación lineal y causal de los acontecimientos historicos. Conceptos como “instante”, “tiempo-ahora”, “discontinuidad”, “rememoración” están al servicio de una construcción de la historia que pone el acento en las lagunas y en los intersticios de toda narración histórica con pretensión de totalidad. Esta estrategia permite rescatar el valor del testimonio y romper así con la homogeneidad de la cronología clásica. A lo largo de este estudio, se presenta cómo se articula esta nueva concepción de la historia, a partir de la idea central de rememoración, que pretende presentar un relato histórico que, tal y como propone Benjamin, se centre no ya en la épica de la historia de los vencedores sino en la tradición de los vencidos. Frente al relato acumulativo y autocomplaciente del historicismo, frente a la falsa totalidad, ilusión mítica del historicismo, para ir más allá del relato de los vencedores se impone la necesidad de otra escritura de la historia, de otra historia. En este sentido, resulta de especial importancia la figura del testimonio, pues esta arremete contra la invisibilidad a la que se pretende confinar a los vencidos, pues se entiende que el verdadero testimonio es aquel que no ha podido testimoniar. Así debe entenderse el imperativo de la representación que se expone —pues en el centro de la representación siempre late la ausencia—, como aquel gesto ético que pone en juego en la laguna constitutiva del verdadero relato histórico y que se presenta en la figura del testimonio, así como en la obra de arte entendida como espacio del testimonio y como testimonio. En un segundo momento, se estudia cómo se traslada a la representación del pasado tal concepción y como ésta interviene en el paisaje de lo sensible a través de la obra de arte, en concreto en la narrativa del escritor W. G. Sebald. El escritor alemán, pues, se toma como ejemplo de la escritura de la historia como rememoración reivindicada por Benjamin e ilustra perfectamente el marco teórico que presenta el filósofo. / This thesis presents a contribution to the studies related to the construction and assertion of collective memory and representation of processes involved in the writing of history and in particular in the history of the vanquished. The current research presents and examines, at first, the conception of history presented by Benjamin. This focuses in particular on his latest work, "Theses on the Philosophy of History", but also provides the answers that the German philosopher presents throughout his intellectual journey to reach its final formulation, to be understood as an ethical and deeply political answer. The thesis, then, takes as its starting point the methodological and conceptual contributions made by Walter Benjamin to the contemporary debate about the philosophy of history. His interpretation of history as a discontinuity and redemption is to offer a suggestive way of narrating and retrieve from oblivion the voice of the vanquished that breaks with classical historiography, based on a linear and causal interpretation of historical events. Concepts such as "instant", "now-time", "discontinuity", "remembrance" work in the service of a construction of history that emphasizes the gaps and interstices of any historical narrative with a claim to totality. This strategy allows to recover the value of the testimony and break the homogeneity of the classical chronology. Throughout this study, it is reported how this new conception of history is articulated, from the central idea of remembrance, which aims to present a historical account that, as Benjamin suggests, should focus not only on epic the history of the victors but the tradition of the vanquished. Faced with the cumulative and self-serving account of historicism, against the false totality, mythical illusion of historicism, to go beyond the story of the victors there is a need of another writing of history, another story. In this sense, it is particularly important the figure of the testimony, as this attacks the invisibility to which is to confine the vanquished, since it is understood that the true witness is one who could not testify. So we should understand the imperative of representation, as set out in the center of representation always beats the absence, as a gesture of ethics that comes into play in the lake that constitutes the true historical account and presented in the figure of the witness as well as the work of art understood as a place of witness and testimony. In a second step, we study how such a conception of the past is transferred to the representation and how it intervenes in the landscape of the senses through the art work, specifically in the narrative of writer W. G. Sebald. The German writer, then, is taken as an example of writing history as remembrance as claimed by Benjamin and perfectly illustrates the theoretical framework presented by the philosopher.

The heart is the first functioning organ to develop during embryogenesis to maintain the growing embryo with oxygen and nutrients. However, cardiogenesis is a complex and highly coordinated biological process, and any perturbation to this process can result in detrimental defects to the heart. Haemodynamics is known to play an important role in cardiac growth and vasculature remodelling. Congenital heart defects (CHDs) accounts for 0.4-1.3% of all live birth, whereas cardiomyopathy accounts for 8-11% of cardiovascular disease diagnoses detected in utero. Although the heart defects and cardiomyopathies are known to be attributed by genetic mutations, most cases have unknown etiology. Hence, OFT-banding model was employed to alter the haemodynamic loading via pressure overloading. Upon alteration of haemodynamics, enlargement of the heart with a spectrum of cardiac anomalies were found (e.g ventricular septal defects, thickened epicardium and dysmorphic atrioventricular valves) upon morphological and stereological analysis. A study of global differential expression of OFT-banded hearts by RNA sequencing revealed a number of differentially expressed genes and they were associated with cardioprotection, metabolism, shear stress and valve development; further, a reduction of apoptosis was seen in these banded hearts as well. One of the cardiac phenotypes seen upon OFT-banding, the abnormal primordial atrioventricular valve, was further characterized to provide an insight how the atrioventricular valve is affected upon alteration of haemodynamics. Aberrant expressions of extracellular matrix (ECM) genes such as TBX20, Aggrecan and Periostin alongside with the shear stress responsive genes (KLF2 and EDN1) were found, and a decrease in apoptosis was seen. Moreover, dysregulation of ECM proteins such as fibrillin-2, type III collagen and tenascin were further demonstrated in more mature primordial AV leaflets at HH35, with a concomitant decrease of ECM cross-linking enzyme, transglutaminase-2. In addition, for many years sarcomeric proteins have been associated with a range of cardiomyopathies, but only in recent years they have been linked to congenital heart defects (CHDs). To date, cardiac troponin T (TNNT2) has been associated with cardiomyopathies but not with isolated CHDs. TNNT2 encodes for cTnT regulatory proteins of the thin filament of the sarcomere and is vital for muscle contraction and force generation within cardiomyocytes. To investigate a role of TNNT2 in the early developing heart, targeted manipulation of TNNT2 was performed in embryonic chick to reduce the protein levels of cTNT (protein product of TNNT2) in ovo via translational block. Abnormal atrial septal growth, reduced ventricular trabeculation and ventricular diverticula were found upon TNNT2 morpholino treatment. The abnormal phenotype observed in the TNNT2 morpholino-treated groups was potentially suggested by differential expression of shear stress responsive gene, NOS3 gene.

Die vorliegende Arbeit ist im Rahmen eines Projektes zur Untersuchung der Genexpression bei Tiermodellen neurologischer Erkrankungen entstanden. Mit herkömmlichen Kandidatenansätzen ist eine Genexpressionsanalyse nur in beschränktem Umfang zu realisieren. Ziel war daher die Etablierung eines Verfahrens wie SAGE (serielle Analyse der Genexpression), das die Analyse des gesamten Transkriptoms zuläßt. Wie die Arbeit zeigt, ist SAGE in einem Standardlabor durchführbar. Es wurden geringfügige Abwandlungen der Orginalmethode eingeführt. Zur Sequenzfehlerkorrektur wurde ein spezielles Computerprogramm entwickelt und evaluiert. Zur Evaluierung der statistischen Auswertung von SAGE wurde zusätzlich zu einer Darstellung des gesamten statistischen Entscheidungsprozesses explorativ die Situation statistischer Entscheidungen wie sie im Rahmen üblicher SAGE Experimente auftreten mit vier Tests nachgeahmt. Es wurde eine Testvariante (modifizierter Z-Test) angewandt und evaluiert, die bis dato noch nicht zur Auswertung von SAGE benutzt worden war. Um die Reliabilität von SAGE abschätzen zu können, wurde von vier Mäusegroßhirnen die Gesamt-RNS vereinigt. Diese Transkriptgrundpopulation wurde zweigeteilt und parallel untersucht. Die beiden Gruppen wurden anhand eines statistischen Tests, der die gesamte Verteilungen der beiden Profile prüft, auf Homogenität untersucht. Zusätzlich wurde das Zusammenhangsmaß ermittelt. Dies ergab, daß die Reliabilität von SAGE im vorliegenden Kontext (relativ geringe Stichprobe und ein komplexes Gewebe) nicht optimal ist. Es kann jedoch keine Aussage dazu gemacht werden, ob dies der Methode selbst, das heißt ihrer molekularbiologischen Praxis und der Datenaufbereitung, anzulasten ist oder einer großen Stichprobenvariabilität. Dies bedeutet, daß in der vorliegenden Arbeit keine endgültige Aussage zur Reliabilität von SAGE möglich ist. Es werden Möglichkeiten dargestellt mit einer suboptimale Reliabilität im Rahmen von zukünftigen Projekten umzugehen. / The work presented here evolved within the framework of a project that examines gene expression of neurological conditions in animal models. Using conventional methods (candidate genes study) gene expression analysis is limited. Hence, the aim was to establish a procedure like SAGE (serial analysis of gene expression) that allows for analysis of the entire transcriptome. As shown it is possible to perform SAGE in a standard laboratory. Minor changes to the original version were made. A special computer program was developed and evaluated to reduce sequencing errors. In addition to a description of the entire statistical process, the statistics of SAGE were explored by simulating normal SAGE experiments, using 4 statistical tests. One test version (modified Z-test) that has not been used for statistical analysis of SAGE yet was applied and reviewed. To assess the reliability of SAGE the total-RNA of 4 corteces of mice was extracted and combined. This basic transcript population was divided in two and the parts examined in parallel. Both groups were analysed using a statistical test that tests the entire distribution of both profiles for homogeneity. Additionally the correlation (and its degree) of the profiles was calculated. The result was that the reliability of SAGE is not optimal in the context of this work (relatively small sample and complex tissue). However, no conclusion can be drawn as to whether the method itself (biomolecular practice and data analysis) is responsible for this, or whether it is due to sample variability. This means that in the work presented here no final statement concerning the reliability of SAGE is possible. Possibilities are described to deal with the issue of suboptimal reliability within the framework of future projects.

Während der Evolution entwickelten sich Promotorelemente von Prokaryonten, Eukaryonten und Plastiden in Sequenz und Struktur unterschiedlich. Ein Transfer von Promotorsequenzen zwischen Prokaryonten, Eukaryonten und Plastiden sollte daher zu keiner effizienten Genexpression führen. Mit dieser Arbeit sollte die Spezifität von Promotoren analysiert und ihre Funktionalität über `kingdom´-Grenzen hinaus untersucht werden. Hierfür wurden zwölf pflanzenspezifische Promotoren, welche in der Gentechnik zur Herstellung von transgenen Pflanzen eingesetzt werden, auf Genexpression in fünf Bakterienarten untersucht. Weiterhin wurden drei bakterielle und sechs Plastiden Promotoren auf Genexpression in Nicotiana tabacum untersucht. Die Frequenz, mit der die pflanzenspezifischen Promotoren (P) in den untersuchten Bakterienarten zu einer Expression führten, lag bei 50 % der getesteten Kombinationen. Für den P ST-LS1 aus Solanum tuberosum wurde eine Expression in den fünf untersuchten Bakterienarten nachgewiesen. Zwei der getesteten pflanzenspezifischen Promotoren, P RolC aus Agrobacterium rhizogenes und P 247 aus N. tabacum zeigten keine Expression in den untersuchten Bakterienarten. Die Charakterisierung der mRNA-Transkripte ausgewählter Fusionen der pflanzenspezifischen Promotoren mit den Reportergenen zeigten eindeutig, dass für die Transkriptionsinitiation durch die bakterielle RNA-Polymerase Sequenzelemente der pflanzenspezifischen Promotoren genutzt wurden. Mit einer ortsspezifischen Mutagenese des P ST-LS1 konnte die von der bakteriellen RNA-Polymerase genutzte -10-Region in Escherichia coli und Acinetobacter sp. ermittelt werden. Die `down-Mutanten´ führten in E. coli und Acinetobacter sp. zu einer Reduktion der Expression, wohingegen sie nach stabiler Integration in das Pflanzengenom von N. tabacum eine unverminderte Expression in bezug auf den Wildtyp-Promotor zeigten. Die Untersuchung der bakteriellen und Plastiden Promotoren ergab nur in einem einzigen Fall von neun getesteten Promotoren eine sehr geringe transiente Expression. / During evolution the promoter elements from prokaryotes, eukaryotes and plastids have developed differently with regard to their sequence and structure, implying that in general a transfer of promoter sequences between prokaryotes, eukaryotes and plastids will not cause an efficient gene expression. The aim of this study was to investigate the specificity of promoter sequences and their functionality in different kingdoms. Therefore 12 different plant-specific promoters, all used for the construction of genetically modified plants, were tested for their capability to direct a gene expression in various bacteria. Furthermore three bacterial and six plastid promoters were tested with respect to their ability to direct a gene expression in Nicotiana tabacum. The frequency of plant-specific promoters (P) directing gene expression in bacteria was 50 % of the combinations analysed. The promoter P ST-LS1 of Solanum tuberosum was functional in all bacteria tested. Two of the plant-specific promoters, P RolC of Agrobacterium rhizogenes and P 247 of N. tabacum, caused no gene expression in the bacteria tested. The characterisation of mRNA-transcripts of fusions between the plant-specific promoter sequences and the reporter genes proved that sequence elements of the plant-specific promoters themselves were used for transcription initiation by the bacterial RNA polymerase. By site-directed mutagenesis of the P ST-LS1 the -10 region used by the bacterial RNA polymerase of E. coli and Acinetobacter sp. was identified. The generated `down-mutants´ of the P ST-LS1 promoter showed a reduction of expression in E. coli and Acinetobacter sp. while these mutants were still fully active after stable integration in the genome of N. tabacum compared to the wildtype promoter. The investigation of the bacterial and the plastid promoters showed a very low transient expression of one out of nine promoters tested.

Mein Ziel war es, genetische Faktoren in C. elegans zu identifizieren, die eine Rolle bei induzierter Transdifferenzierung durch Missexpression des Transkriptionsfaktors (TF) HLH-1, welcher das Wurmhomolog des myogenen bHLH TF MyoD ist, spielen. Ich entwickelte hierzu einen semiautomatischen Hochdurchsatz-Vorwärtsgenetik-Screen, indem ich EMS Mutagenese mit dem Biosorter-System (Union Biometrica) kombinierte. Mit diesem Ansatz ist es mir gelungen, die Mutante bar18 zu isolieren, die eine Anhäufung an Muskelzellkernen um den posterioren Teil des Pharynx zeigt. Ich identifizierte den mutierten Lokus, indem ich das gesamte Genom sequenzierte und charakterisierte den mutanten Phänotyp im Detail. Zusätzlich war ich bei der Charakterisierung von Faktoren, die das Umprogrammieren zu neuronalen Zellen in C. elegans verhindern, beteiligt. Dabei stand der sogenannte FACT-Komplex im Focus, welcher mittels eines genom-weiten RNAi-Screen in unserer Arbeitsgruppe identifiziert wurde. Interessanterweise ist eine der FACT-Komplex-Untereinheiten, spt-16, das parentale Gen zu dem bislang nicht charakterisierten Pseudogen F55A3.7. Eine putative Null-Mutante von F55A3.7, in Kombination mit ubiquitärer Überexpression von CHE-1, zeigte einen Keimzellen-zu-Neuronen Transdifferenzierungsphänotyp ähnlich dem Phänotypen, der nach dem Knock-down der FACT-Komponente hmg-3 beobachtet wird. Unseres Wissens nach ist dies das erste Beispiel einen Pseudogens, dessen Knock-down dazu führt, dass ein bestimmtes Gewebe durch einen terminalen Selektor-TF reprogrammiert werden kann, dessen Expression unter normalen Konditionen dies nicht zur Folge hätte. Aufgrund dieser Einzigartigkeit, habe ich das Pseudogen F55A3.7 charakterisiert und außerdem versucht, einen Mechanismus zu finden, wie F55A3.7 die Keimbahnidentität schützt. / My aim was to identify and characterize genetic factors in C. elegans that play a role in induced transdifferentiation by mis-expressing the transcription factor (TF) HLH-1, which is the worm homolog of the myogenic bHLH TF MyoD. For this, I developed a semi-automated high-throughput forward genetic screen combining EMS mutagenesis with the Biosorter system (Union Biometrica). When mis-expressed, HLH-1 induces muscle fate in early embryonic cells, but terminally differentiated cells are resistant to HLH-1-induced direct reprogramming. In order to identify mechanisms that antagonize HLH-1-induced reprogramming, I used a transgenic line allowing ectopic expression of hlh-1 in combination with a reporter for muscle fate. Using this approach, I isolated the mutant bar18, showing an accumulation of muscle cell nuclei around the posterior pharyngeal bulb. I identified the mutated locus using whole genome sequencing and characterized the identified gene and the mutant phenotype further. Additionally, I was also involved in characterizing the FACT complex, which was identified through a whole-genome RNAi screen conducted by my colleague Ena Kolundžić. This reverse genetic screen aimed at identifying factors that play a role in induced transdifferentiation by mis-expressing the TF CHE-1, a Zn-finger TF essential for terminal differentiation of glutamatergic ASE neurons. Interestingly, one of the FACT complex members, spt-16, is the parental gene of a previously uncharacterized pseudogene named F55A3.7. A putative null mutant of F55A3.7, combined with broad overexpression of CHE-1, showed a germ cells to neurons transdifferentiation phenotype. To our knowledge, this is the first example of a pseudogene whose depletion leads to the permissiveness of a certain tissue to be reprogrammed when challenged by a terminal selector TF. Due to this uniqueness, I characterized the pseudogene F55A3.7 and tried to find a potential mechanism for how F55A3.7 safeguards germline identity.

Condensine sind essentiell für die Faltung von Chromatin und wurden auch mit der Transkriptionsregulation in Verbindung gebracht. Der zugrunde liegende Mechanismus für die Transkriptionsregulation ist jedoch unklar. Condensin DC in C. elegans ist ein gutes Modell zur Erforschung der Transkriptionsregulation durch Condensine, da es spezifisch für die Dosiskompensation der Gene auf dem X Chromosom benutzt wird. Condensin DC bindet an beide X Chromosome in C. elegans Hermaphroditen und reduziert deren Transkription um die Hälfte. In meiner Dissertation habe ich untersucht, welche Rolle ein dynamisches Binden von Condensin DC an Chromatin spielt und wie dies die Transkription während der Embryogenese reguliert. Condensine binden dynamisch an Chromatin, um es zu komprimieren und durch Bildung von Schlaufen die Transkription zu regulieren. Mit Hilfe von „fluorescence recovery after photobleaching“ (FRAP) habe ich in adulten Darmzellen von C. elegans untersucht, welche Faktoren das dynamische Binden von Condensin DC an die X Chromosomen beeinflussen. Meine Daten zeigen, dass sowohl die ATPase-Domäne von Condensin DC, als auch eine nicht-katalytische Aktivität einer Histon-Demethylase die Bindedynamik von Condensin DC beeinflussen und damit Transkription regulieren. Zusätzlich habe ich mit einem Mikroskopieansatz, der auf dem Nachweis von einzelnen RNA Molekülen beruht (smFISH), die Transkription von mehreren Genen untersucht, die durch Condensin DC während der Embryonalentwicklung reguliert werden. Die aus diesen Daten ermittelten Transkriptionskinetiken deuten darauf hin, dass Condensin DC vorrangig die Häufigkeit der Transkriptionsinitiation reguliert. Zusammenfassend liefert meine Forschung neue Einblicke in die Transkriptionsregulation durch Condensine und kann als Basis für detailliertere, mechanistische Studien der Rolle von Condensinen in der Transkriptionsregulation in C. elegans und auch in anderen Organismen dienen. / Condensins are essential for chromosome compaction and have been implicated in transcription regulation. The mechanistic foundation of this regulatory function is poorly understood. A clear paradigm to address this question is the X-specific condensin DC in C. elegans, which specifically binds to and transcriptionally represses X chromosomes in XX hermaphrodites by 2-fold. In my thesis, I studied condensin DC binding dynamics to the X chromosome and how condensin DC affects transcription kinetics in single embryos. The binding of condensins to chromatin has been described in recent microscopy-based studies as dynamic in processes including loop formation, chromatin compaction and transcription regulation. To study the dynamics of condensin DC binding, I established fluorescence recovery after photobleaching (FRAP) in C. elegans adult intestinal cells. With this method, I studied how the ATPase domain and different histone modifiers regulate the dynamic binding of condensin DC. I found that the ATPase domain is critical for binding of the complex and that the noncatalytic activity of a histone demethylase increases the dynamics of condensin DC binding, which is crucial for its role in transcription regulation. To further study the mechanism of condensin DC in transcription regulation, I used an imaging approach based on widefield single-molecule RNA fluorescence in situ hybridization (smFISH). I obtained thousands of smFISH images for a set of condensin DC-regulated genes and extracted mature and nascent RNA counts in 3D, which I used to determine transcription burst characteristics throughout embryonic development. My data show that condensin DC regulates the frequency of transcription initiation to down-regulate X-chromosomal genes. Taken together, my results provide new insight into condensin-mediated transcription regulation, which can be used to inform future studies on the mechanism of condensins in transcription regulation in C. elegans and other organisms.

Der Transkriptionsfaktor CEBPA reguliert Differenzierung und Proliferation in verschiedenen Zelltypen und spielt eine herausragende Rolle in der Hämatopoese. Die CEBPA RNA kann in die lange P42-Isoform oder die N-terminal verkürzte P30-Isoform translatiert werden. Während P42-CEBPA differenzierungsinduzierend wirkt, ist P30 als Inhibitor von P42 und als Onkogen in akuter myeloider Leukämie beschrieben. Die Modularität und Multifunktionalität von CEBPA, die ihn zahlreichen Studien beobachtet wurde, lässt sich möglicherweise durch differentielle Protein–Protein-Interaktionen erklären. Zahlreiche post-translationale Modifikationen (PTMs) und die intrinsisch ungeordnete, flexible Struktur von CEBPA stellen jedoch eine Herausforderung für traditionelle Ansätze in Proteininteraktionsstudien dar. In der vorliegenden Arbeit wird ein neuer, alternativer Ansatz präsentiert, der auf einem in vitro Proteininteraktions-screen auf einer Peptidmatrix (PRISMA) und Biotinligase proximity labelling (BioID) in lebenden Zellen basiert. In einem PRISMA-screen wurden 120 CEBPA Peptide auf Proteininteraktionen mit Proteinextrakt aus myeloiden Zellen untersucht. Im Screen wurden 40 verschiedene CEBPA PTMs inkludiert, unter anderem auch die hier erstmals neu beschriebenen Methylierungen der CEBPA Argininreste R12 und R142. Daten aus dem PRISMA-screen wurden mit BioID Experimenten in myeloiden Zellen validiert, um eine Proteininteraktionslandkarte von CEBPA zu generieren, die 52 bekannte und 68 neue CEBPA Proteininteraktoren umfasst. Hotspots für Proteininteraktionen fallen in evolutionär konservierte CEBPA Regionen und der Vergleich des Bindungsprofils mit publizierten Daten zeigt Ähnlichkeiten zu verwandten Transkriptionsfaktoren der CEBP Familie. Die Ergebnisse legen nahe, dass die Multifunktionalität von CEBPA von multivalenten Proteininteraktionen in Abhängigkeit von PTMs koordiniert wird, um CEBPA mit dem epigenetischen und transkriptionellen Apparat der Zelle verknüpfen. / The pioneering transcription factor CEBPA plays a lineage-instructing role during haematopoiesis and also regulates proliferation and differentiation in many other cell types. The CEBPA RNA can be translated into a full length (P42-CEBPA) or N-terminally truncated isoform (P30-CEBPA). While P42 induces differentiation in various cell types, the P30 isoform is mostly regarded as a dominant inhibitor of P42-CEBPA and acts as an oncogene in acute myeloid leukaemia. Protein interactions may be the key to explaining the functional plasticity and modularity of CEBPA that has been demonstrated in diverse experimental settings. However, the disordered structure and the numerous post-translational modification sites (PTMs) of CEBPA pose a challenge to traditional protein interaction studies. In the present work, a novel alternative approach is presented that combines an in vitro protein interaction screen on a peptide matrix (PRISMA) with biotin ligase proximity labelling (BioID) in living cells. To this end, 120 CEBPA peptides were probed for protein interactions with PRISMA. The screen comprised 40 different PTMs, including newly identified CEBPA arginine methylation sites. PRISMA data was validated with BioID experiments and generated a detailed CEBPA protein interaction map in myeloid cells. The interactome presented here contains 52 known and 68 novel CEBPA interactors that can now be mapped across the CEBPA sequence in a PTM dependent fashion. Hotspots of protein interaction correlated with conserved regions and comparison with previously published data revealed related binding profiles of homologous CEBP regions. Taken together, the data indicates that the functional plasticity of CEBPs is orchestrated by multivalent protein interactions and PTMs to configure a dynamic CEBP hub that interacts with many partners of the transcriptional and epigenetic machinery.