Lobbying: economic and legal aspects Abstract The aim of this thesis is to determine lobbying as a legitimate part of democratic processes and to propose recommendations for the regulation of lobbying in the Czech Republic. To achieve this aim, an analysis of the regulation of lobbying in the USA, EU and neighbour states, is conducted. Emphasis is also put on the analysis of regulation attempts in the Czech Republic with regard to influence of Non-Governmental Organizations. The first chapter is dedicated to a comparison of various lobbying definitions which are presenting main features and similarities across definitions. Among the main features are definitions of lobbying activity, lobbyist and public officials. The second chapter is dedicated to a comparison of government regulation and self-regulation, and the positives and negatives are assessed. The difference between lobbying and corruption is the main focus of the third chapter. Next three chapters are dedicated to the historical and legal analysis of lobbying in the USA, EU and neighbour states. The regulation of lobbying in the USA has the longest history. The regulation of lobbying in EU is an example of different terminology which is used in communication with the European Parliament and the European Commission. The seventh chapter is dedicated...

The respiratory system forms a major mucosal interface with the external environment. Consequently, the respiratory tract is constantly exposed to inhaled foreign antigens, commensal microorganisms, and potential pathogens. The respiratory system has evolved a complex regulatory network designed to prevent unnecessary inflammation to harmless antigens and to limit immune-mediated damage to the fragile lung epithelium in response to infection. The lung maintains a default anti-inflammatory state that is coordinated by the respiratory epithelium, alveolar macrophages, dendritic cells, and regulatory Foxp3+ CD4 T cells (Tregs). It is likely that all of these cells influence the development of pathogen-specific T cell responses in the lung. Following infection with a respiratory virus, virus-specific CD8 T cells in the lung are inhibited in their ability to produce cytokines. Current studies suggest that this functional inactivation occurs following infection with respiratory viruses within the Paramyxoviridae family. The data presented here demonstrate that suppression of effector functions of virus-specific CD8 T cells in the lungs occurs following infection with several unrelated respiratory viruses. These results indicate that the functional inhibition of virus-specific T cell responses is not restricted to infection with viruses from the Paramyxoviridae family. Furthermore, I show data indicating that the functional inactivation of virus-specific CD8 T cells in the lungs occurs in the absence of infection. I also demonstrate for the first time that the lung environment also regulates the effector functions of virus-specific CD4 T cells. Inhibition of cytokine production by pulmonary T cells is reversible as stimulation with exogenous peptide-pulsed antigen-presenting cells rescues IFN-gamma production. The inhibition of IFN-gamma production by virus-specific T cells occurs in other organs such as the kidney. These data suggest that regulation of T cell cytokine production by peripheral tissues may serve as an important mechanism to prevent immunopathology and preserve normal tissue function. Foxp3+ Tregs have been shown to inhibit conventional effector T cell responses in a large number of chronic infection models. However, their role during acute infections remains unclear. Examination of Foxp3+ Tregs during RSV infection showed that Tregs are rapidly recruited into the lungs and acquire an activated phenotype. Depletion of Foxp3+ Tregs prior to RSV infection revealed that Tregs facilitate the early recruitment of RSV-specific CD8 T cells from the draining lymph nodes to the lung and later limit the overall magnitude of the virus-specific CD8 T cell response. Depletion of Tregs increased TNF-αa production by RSV-specific CD8 T cells and enhanced T-cell-mediated immunopathology. These data demonstrate that Foxp3+ Tregs play a major role in regulating CD8 T cell responses to respiratory virus infections. Collectively, the data presented here demonstrate that CD8 T cell responses to respiratory pathogens are tightly regulated within the lung environment.

F. tularensis is an intracellular pathogen, and is the causative agent of tularemia in humans. The ability of F. tularensis to parasitize host cells is largely dependent upon genes within a pathogenicity island (FPI), including those in the iglABCD operon. Specific mechanisms and gene products involved in regulation of the FPI are not well understood. I initiate the study of this regulatory system by creating an efficient Tn5-based mutagenesis system optimized for use in F. tularensis, and utilize this system to construct a lacZ reporter library. I identify genes differentially regulated in response to growth on two different media, including those in the iglABCD and fslABCD operons, and identify iron availability as a factor contributing to the differential regulation. One of these reporter strains, carrying a chromosomal iglB-lacZ fusion, is used as the basis for a secondary transposon mutagenesis to identify mutations that affect iglABCD expression. One such mutation is in FTL_1542 (migR), a hypothetical protein, and reduces expression of the iglABCD approximately 8-fold. The effect of this mutation on igl expression is likely through its effect on another known virulence regulator, fevR, as demonstrated by data from RT-PCR experiments. I compare the phenotypes of LVS fevR and migR mutant strains in primary macrophage and epithelial cell lines and in neutrophils. The mutation in migR effects growth and intracellular trafficking in macrophages but not epithelial cells, and reverses the ability of wild type F. tularensis to block the respiratory burst in neutrophils. When similar mutations were examined in the human virulent F. tularensis strain Schu S4, migR retained its regulatory role, but did not impair replication in macrophages. The migR mutation in Schu S4 did however have an attenuating effect when administered to mice intranasally. Comparison of LVS and Schu S4 in primary human airway epithelial cell infections revealed an inability of LVS to replicate within these cells, which is in contrast to the robust replication of LVS in cultured epithelial cell lines. Together, this work contributes to the understanding of regulatory mechanisms governing virulence gene expression in F. tularensis and highlights differences between LVS and Schu S4 strains.

Francisella tularensis is a facultative intracellular pathogen and is the etiological agent of tularemia. One key aspect to the success of Francisella as a pathogen is ability of the organism to establish infection with a low inoculum, as few as 10 colony forming units (cfu). Essential to this process is the Francisella pathogenicity island (FPI). Several studies have been performed to understand how the FPI is regulated; however, the working model is not complete, as the signals important for regulation are unknown. Additionally, the mechanisms of the proteins MigR, TrmE, and CphA, which are important for activation of the FPI, are unknown. I initiated the study of this regulatory system by measuring the ability of various cellular stresses to activate an iglA-lacZ reporter. I identified that amino acid starvation and growth in basic pH activated expression of the reporter in both LVS and Schu S4. By combining these two stresses I was able to induce iglA-lacZ reporter expression in an additive manner. As it was previously demonstrated that ppGpp is important for stabilization of the regulatory complex that transcribes FPI genes, I demonstrated by TLC that both amino acid starvation and basic pH effected iglA-lacZ expression by increasing ppGpp. Due to the importance of ppGpp in FPI expression and because MigR, TrmE, and CphA each appear to be involved in a metabolic process: fatty acid metabolism (migR) t-RNA modification (trmE) and amino acid storage (cphA), I had hypothesized that the effect on these mutations were due to decreased levels of the small alarmone ppGpp. I compared ppGpp accumulation of LVS mutants in migR, trmE, and cphA to the parent strain and observed that loss of these genes resulted in reduced ppGpp. To better understand the importance of ppGpp synthesis in F. tularensis pathogenesis, I compared the phenotypes of these strains in primary human macrophages and two immortalized epithelial cell lines. These experiments demonstrated that although each of these strains had reduced ppGpp, there were cell line specific growth phenotypes. Mice infected with these strains survived suggesting tight regulation of the FPI is required for virulence. When similar mutations were characterized in the Schu S4 background these mutations retained their regulatory role; however, mutation of migR did not significantly decrease virulence in mice. As my data demonstrated that there are different challenges that Francisella must overcome to successfully replicate within cells, I developed an in vitro model to study the interactions of F. tularensis with human alveolar type II cells (AT-II). Interestingly, Schu S4 internalizes and replicates in these recently immortalized human AT-II cells whereas, LVS internalizes, but replicates poorly within these cells. Finally, to better understand the role of AT-II cells in vivo, I performed Transmission Electron Microscopy (TEM) of infected mice. These data confirmed that Schu S4 infected both alveolar macrophages and AT-II cells. Together, this work contributes to the understanding of how Francisella adapts to various environments by modulating virulence gene expression and highlights differences between virulent Schu S4 and LVS, which may partially contribute to virulence differences observed between strains.

This study evaluated one important dimension of nonprofit self-regulation, the relationship between the Iowa Register of Accountability and voluntary website disclosure by Iowa nonprofits. The purpose of this study was to assess the effectiveness of self-regulation in Iowa to improve accountability as measured by voluntary website disclosure and transparency. As part of the study, an instrument was developed to reliably measure nonprofit website disclosure and transparency. The disclosure score ratings from Iowa Register of Accountability nonprofit organizations were compared to those not listed on the Register. Other factors relevant to disclosure and transparency (e.g. the method to become listed on the Iowa Register of Accountability and the type and number of organization staff members who received training) were also tested. Results indicated that nonprofit organizations listed on the Iowa Register of Accountability were more likely to have active websites and to voluntarily disclose recommended information on their websites than those Iowa nonprofits that have not sought or achieved listing on the Register. In particular, the Register group had statistically higher mean disclosure scores in four areas (Key Staff, Strategic Plan, Annual Reports, and Audit and Financial Statements) compared to the Non-Register group. Contrary to expectations, the method to become listed on the Iowa register of Accountability and the type and number of staff members who received training were not related to higher disclosure scores. The important finding of this research is that nonprofit organizations listed on the Iowa Register of Accountability were more likely to voluntarily disclose recommended information on their websites than those Iowa nonprofits that have not sought or achieved listing on the Register. Recommendations are made in terms of ways to improve nonprofit website disclosure and transparency by enhancing and expanding training opportunities. A major contribution of this study for future research in the field was the development of a disclosure scoring instrument to assess and compare website disclosure and transparency.

Herpes simplex virus type 1 (HSV-1) is a virus commonly causing cold sores in humans, however, virulent infections are known to produce debilitating encephalitis and death. HSV-1 transcription is carried out by the host cell RNA polymerase II in a tightly regulated temporal cascade. The first genes transcribed, the a genes, are activated in the absence of viral DNA synthesis. Transcription of the other temporal classes, the β, βγ, and γ genes is dependent upon the protein products of the a genes for activation. The purpose of this study was to investigate the factors that contribute to this rigid regulation of HSV-1 transcription. This investigation sought to identify some of the cellular and viral transcription factors that activate transcription of genes of the later kinetic classes. Two separate approaches were utilized in these investigations. 1) In vitro transcription using a soluble, cell free system to study the transcriptional regulation of the VP5 gene, and 2) DNA competition binding assays to identify and characterize the protein-DNA complexes resulting from interaction between the cisacting DNA sequences of the VP5 gene, other viral genes, and the proteins that bind to them. Attempts at in vitro transcription of β, βγ, and γ genes were unsuccessful. Because these genes require a products for activation, it was necessary to prepare nuclear extracts from infected cells. However, HSV-1 contains endogenous RNase activities which are components of the biochemical machinery by which the virus directs host transcription to the synthesis of viral molecules. The uses of virus deficient in the host shut-off function and various drugs were unsuccessful. Previous work in the Millette laboratory demonstrated a sequence in the VP5 promoter that played a significant role in the up regulation of expression of that gene. DNA binding competition studies using a number of HSV-1 sequences exhibiting partial homology to this sequence demonstrated that these sequences all compete for the binding of the same protein factor. Similarly, a piece of the human immunodeficiency virus (HIV) exhibiting a seven base pair homology also exhibited weak competition.

Background: Nicotine addiction is the most prevalent type of drug addiction that has been described as a cycle of spiraling dysregulation of the brain reward systems. Imaging studies have shown that nicotine addiction is associated with abnormal function in prefrontal brain regions that are important for cognitive emotion regulation. It was assumed that addicts may perform less well than healthy nonsmokers in cognitive emotion regulation tasks. The primary aims of this thesis were to investigate emotional responses to natural rewards among smokers and nonsmokers and to determine whether smokers differ from nonsmokers in cognitive regulation of positive and negative emotions. To address these aims, two forms of appraisal paradigms (i.e., appraisal frame and reappraisal) were applied to compare changes in emotional responses of smokers with that of nonsmokers as a function of appraisal strategies. Experiment 1: The aim of the first experiment was to evaluate whether and how appraisal frames preceding positive and negative picture stimuli affect emotional experience and facial expression of individuals. Twenty participants were exposed to 125 pairs of auditory appraisal frames (either neutral or emotional) followed by picture stimuli reflecting five conditions: unpleasant-negative, unpleasant-neutral, pleasant-positive, pleasant-neutral and neutral-neutral. Ratings of valence and arousal as well as facial EMG activity over the corrugator supercilii and the zygomaticus major were measured simultaneously. The results indicated that appraisal frames could alter both subjective emotional experience and facial expressions, irrespective of the valence of the pictorial stimuli. These results suggest and support that appraisal frame is an efficient paradigm in regulation of multi-level emotional responses. 8 Experiment 2: The second experiment applied the appraisal frame paradigm to investigate how smokers differ from nonsmokers on cognitive emotion regulation. Sixty participants (22 nonsmokers, 19 nondeprived smokers and 19 12-h deprived smokers) completed emotion regulation tasks as described in Experiment 1 while emotional responses were concurrently recorded as reflected by self-ratings and psychophysiological measures (i.e., facial EMG and EEG). The results indicated that there was no group difference on emotional responses to natural rewards. Moreover, nondeprived smokers and deprived smokers performed as well as nonsmokers on the emotion regulation task. The lack of group differences in multiple emotional responses (i.e., self-reports, facial EMG activity and brain EEG activity) suggests that nicotine addicts have no deficit in cognitive emotion regulation of natural rewards via appraisal frames. Experiment 3: The third experiment aimed to further evaluate smokers’ emotion regulation ability by comparing performances of smokers and nonsmokers in a more challenging cognitive task (i.e., reappraisal task). Sixty-five participants (23 nonsmokers, 22 nondeprived smokers and 20 12-h deprived smokers) were instructed to regulate emotions by imagining that the depicted negative or positive scenario would become less negative or less positive over time, respectively. The results showed that nondeprived smokers and deprived smokers responded similarly to emotional pictures and performed as well as nonsmokers in down-regulating positive and negative emotions via the reappraisal strategy. These results indicated that nicotine addicts do not have deficit in emotion regulation using cognitive appraisal strategies. In sum, the three studies consistently revealed that addicted smokers were capable to regulate emotions via appraisal strategies. This thesis establishes the groundwork for therapeutic use of appraisal instructions to cope with potential self-regulation failures in nicotine addicts. / Hintergrund: Nikotinsucht ist die am weitesten verbreitete Form von Drogenabhängigkeit und wird beschrieben als eine immer stärker werdende Dysregulation des Belohnungssystems im Gehirn. Bildgebende Studien zeigten, dass Nikotinabhängige eine abnormale Funktion der präfrontalen Gehirnregionen aufweisen, die für die kognitive Emotionsregulation von entscheidender Bedeutung sind. Es wurde angenommen, dass Süchtige bei kognitiven Aufgaben zur Emotionsregulation schlechter abschneiden als gesunde Nichtraucher. Vorrangige Ziele dieser Thesis waren die Untersuchung emotionaler Reaktionen auf natürliche, Raucher-irrelevante Stimuli bei Rauchern und Nichtrauchern. Außerdem sollte herausgefunden werden, ob sich Raucher von Nichtrauchern bezüglich ihrer kognitiven Regulation von positiven und negativen Emotionen unterscheiden. Um diese Veränderungen in der emotionalen Reaktion in Abhängigkeit der Interpretationsstrategie vergleichen zu können, wurden zwei Paradigmen zur Einschätzung emotionaler Stimuli eingesetzt: Eine prospektive Interpretationsstrategie des kommenden Stimulus (appraisal frame) und eine retrospektive Interpretationsstrategie nach der Stimuluspräsentation (reappraisal). Experiment 1: Ziel des ersten Experiments war die Evaluierung ob und wie Interpretationen vor positiven oder negativen Stimulusbildern die emotionale Erfahrung und den Gesichtsausdruck von Personen beeinflussen. 20 Versuchspersonen wurden 125 Paare auditiver Beschreibungen (entweder neutral oder emotional) präsentiert, gefolgt von Stimulusbildern, die zusammen fünf Stimulus-Kategorien bildeten: unangenehm – negativ, unangenehm – neutral, angenehm – positiv, angenehm – neutral und neutral – neutral. Valenz- und Arousal-Ratings wurden abgefragt und die EMG-Aktivität der Gesichtsmuskeln corrugator supercilii und zygomaticus 10 major wurden zeitgleich aufgenommen. Die Ergebnisse zeigten, dass appraisal frames sowohl emotionale Reaktionen einschließlich subjektiver emotionaler Erfahrungen beeinflussen als auch den Gesichtsausdruck verändern können, unabhängig von der Valenz des Bildstimulus. Dies zeigt und beweist die Effizienz des appraisal frame Paradigmas bei der Regulation von emotionalen Reaktionen auf mehreren Verarbeitungsebenen. Experiment 2: Das zweite Experiment bezog sich auf das appraisal frame Paradigma und sollte untersuchen wie sich Raucher von Nichtrauchern in ihrer kognitiven Emotionsregulation unterscheiden. 60 Probanden (22 Nichtraucher, 19 Raucher ohne Entzug und 19 Raucher mit 12 Stunden Zigarettenentzug) führten Emotionsregulationsaufgaben wie in Experiment 1 beschrieben aus, während ihre emotionalen Reaktionen ständig über Selbsteinschätzungen und psychophysiologische Messungen aufgenommen wurden (faziales EMG und EEG). Die Ergebnisse zeigten keine Gruppenunterschieden bei den emotionalen Reaktionen auf natürliche Stimuli, ohne Bezug zum Rauchen; Außerdem schnitten Raucher mit und ohne Zigarettenentzug in der Emotionsregulationsaufgabe genauso gut ab wie Nichtraucher. Die gleichen Ergebnisse in allen Gruppen hinsichtlich emotionaler Reaktionen (Selbsteinschätzung, faziale EMG Aktivität und EEG Aktivität) machten deutlich, dass Nikotinabhängige keine Einschränkungen in der kognitiven Emotionsregulation auf natürliche Stimuli mittels Vorbeurteilungen haben. Experiment 3: Der dritte Versuch wurde durchgeführt, um die Fähigkeiten von Rauchern bei der Emotionsregulation zu untersuchen, indem die Erfolge von Rauchern und Nichtrauchern in einer schwierigeren kognitiven Aufgabe (reappraisal task) verglichen wurden. 65 Versuchspersonen (23 Nichtraucher, 22 Raucher ohne Entzug und 20 Raucher mit 12 Stunden Zigarettenentzug) wurden instruiert ihre Emotionen zu regulieren, indem sie emotionale Bilder 11 mit neutralem Gefühl interpretieren. Die Probanden sollten sich vorstellen, dass die negativen oder positiven Syenarios immer weniger negativ oder weniger positiv werden. Die Ergebnisse stellen heraus, dass Raucher mit und ohne Zigarettenentzug ähnlich auf emotionale Bilder reagierten und ihre positiven und negativen Emotionen mit der reappraisal Strategie genauso gut herunterregulierten wie Nichtraucher. Zusammenfassend machen die drei Studien deutlich, dass Nikotinabhängige mittels Interpretationsstrategien ihre Emotionen regulieren können. Diese Thesis bilden das Fundament für den therapeutischen Nutzen von Interpretationsstrategien, damit Nikotinabhängige mit potenziellen Selbstregulationsstörungen umgehen können.

GAS2L3 was identified recently as a target gene of the DREAM complex (Reichert et al., 2010; Wolter et al., 2012). It was shown that GAS2L3 is expressed in a cell cycle specific manner and that depletion of the protein leads to defects in cytokinesis and genomic instability (Wolter et al., 2012). Major aim of this thesis was, to further characterize the biochemical properties and physiological function of GAS2L3. By in vitro co-sedimentation and bundling assays, GAS2L3 was identified as a cytoskeleton associated protein which bundles, binds and crosslinks F-actin and MTs. GST pulldown assays and co-immunoprecipitation experiments revealed that GAS2L3 interacts in vitro and in vivo with the chromosomal passenger complex (CPC), a very important regulator of mitosis and cytokinesis, and that the interaction is mediated by the GAR domain of GAS2L3 and the C-terminal part of Borealin and the N-terminal part of Survivin. Kinase assays showed that GAS2L3 is not a substrate of the CPC but is strongly phosphorylated by CDK1 in vitro. Depletion of GAS2L3 by shRNA influenced protein stability and activity of the CPC. However pharmacological studies showed that the decreased CPC activity is not responsible for the observed cytokinesis defects upon GAS2L3 depletion. Immunofluorescence experiments revealed that GAS2L3 is localized to the constriction zone by the CPC in a GAR dependent manner and that the GAR domain is important for proper protein function. New interacting proteins of GAS2L3 were identified by stable isotope labelling by amino acids in cell culture (SILAC) in combination with tandem affinity purification and subsequent mass spectrometrical analysis. Co-immunoprecipitation experiments further confirmed the obtained mass spectrometrical data. To address the physiological function of GAS2L3 in vivo, a conditional and a non-conditional knockout mouse strain was established. The non-conditional mouse strain showed a highly increased mortality rate before weaning age probably due to heart failure. The physiological function of GAS2L3 in vivo as well as the exact reason for the observed heart phenotype is not known at the moment. / GAS2L3 wurde vor kurzem als Zielgen des DREAM Komplex identifiziert (Reichert et al., 2010; Wolter et al., 2012). Es konnte gezeigt werden, dass die Expression von GAS2L3 Zellzyklus abhängig reguliert wird und dass Depletion des Proteins zu Fehlern in der Zytokinese und genomischer Instabilität führt (Wolter et al., 2012). Hauptziel dieser Doktorarbeit war es, GAS2L3 hinsichtlich seiner biochemischen Eigenschaften und physiologischer Funktion näher zu charakterisieren. Unter Verwendung verschiedener in vitro Experimente konnte gezeigt werden, dass GAS2L3 sowohl F-Aktin als auch Mikrotubuli binden, bündeln und quervernetzen kann. In vitro und in vivo Protein-Protein Interaktionsexperimente zeigten, dass GAS2L3 mit dem „chromosomal passenger complex“ (CPC), einem wichtigen Mitose- und Zytokineseregulator, interagiert und dass diese Interaktion durch die GAR Domäne von GAS2L3 und den C-Terminus von Borealin beziehungsweise den N-terminus von Survivin vermittelt wird. Phosphorylierungsexperimente zeigten deutlich, dass GAS2L3 kein Substrat des CPC ist, jedoch von CDK1 phosphoryliert wird. Zellbiologische Experimente belegten, dass Depletion von GAS2L3 mittels shRNA die Proteinstabilität und Aktivität des CPC beeinflusst. Experimente mit einem chemischen Aurora B Inhibitor dokumentierten, dass die verringerte CPC Aktivität nicht die Ursache der beobachteten Zytokinesefehler nach GAS2L3 Depletion ist. Immunfluoreszenzexperimente machten deutlich, dass GAS2L3 mit Hilfe des CPC an der Abschnürungszone lokalisiert wird und dass die Lokalisation abhängig von der GAR Domäne erfolgt. Mit Hilfe von SILAC in Kombination mit Tandem-Affinitätsaufreinigung und anschließender massenspektrometrischer Auswertung wurden neue Proteininteraktoren von GAS2L3 identifiziert. Protein-Protein Interaktionsexperimente bestätigten die massenspektrometrisch ermittelten Daten. Um die physiologische Funktion von GAS2L3 in vivo näher analysieren zu können, wurden verschiedene Knockout Mauslinien etabliert. Die nicht-konditionelle Mauslinie zeigte erhöhte Sterblichkeit vor dem Absetzalter wahrscheinlich verursacht durch Herzversagen. Die genaue physiologische Funktion von GAS2L3 und der Grund für den beobachteten Herzphänotyp sind momentan noch unbekannt.

In mammals, KSR1 functions as an essential scaffold that coordinates the assembly of RAF/MEK/ERK complexes and regulates intracellular signal transduction upon extracellular stimulation. Aberrant activation of the equivalent MAPK signaling pathway has been implicated in multiple human cancers and some developmental disorders. The mechanism of KSR1 regulation is highly complex and involves several phosphorylation/dephosphorylation steps. In the present study, a number of novel in vivo phosphorylation sites were detected in mKSR1 by use of mass spectrometry analysis. Among others, Tyr728 was identified as a unique regulatory residue phosphorylated by LCK, a Src kinase family member. To understand how phosphorylation of Tyr728 may regulate the function of KSR1 in signal transduction and cellular processes, structural modeling and biochemical studies were integrated in this work. Computational modeling of the mKSR1(KD) protein structure revealed strong hydrogen bonding between phospho-Tyr728 and the residues surrounding Arg649. Remarkably, this pattern was altered when Tyr728 was non-phosphorylated or substituted. As confirmed by biochemical analysis, Arg649 may serve as a major anchor point for phospho-Tyr728 in order to stabilize internal structures of KSR1. In line with the protein modeling results, mutational studies revealed that substitution of Tyr728 by phenylalanine leads to a less compact interaction between KSR1 and MEK, a facilitated KSR1/B-RAF binding and an increased phosphorylation of MEK in complex with KSR1. From these findings it can be concluded that phospho-Tyr728 is involved in tightening the KSR1/MEK interaction interface and in regulating the phosphorylation of KSR1-bound MEK by either RAF or KSR1 kinases. Beside the Tyr728, Ser722 was identified as a novel regulatory phosphorylation site. Amino acid exchanges at the relevant position demonstrated that Ser722 regulates KSR1-bound MEK phosphorylation without affecting KSR1/MEK binding per se. Due to its localization, Ser722 might consequently control the catalytic activity of KSR1 by interfering with the access of substrate (possibly MEK) to the active site of KSR1 kinase. Together with Ser722, phosphorylated Tyr728 may further positively affect the kinase activity of KSR1 as a consequence of its vicinity to the activation and catalytic loop in the KSR1(KD). As revealed by structural modeling, phospho-Tyr728 builds a hydrogen bond with the highly conserved Lys685. Consequently, phospho-Tyr728 has a stabilizing effect on internal structures involved in the catalytic reaction and possibly enhances the phosphate transfer within the catalytic cleft in KSR1. Considering these facts, it seems very likely that the LCK-dependent phosphorylation of Tyr728 plays a crucial role in the regulation of KSR1 catalytic activity. Results of fractionation and morphology analyses revealed that KSR1 recruits LCK to cytoskeleton for its phosphorylation at Tyr728 suggesting that this residue may regulate cytoskeleton dynamics and, consequently, cell motility. Beside that, phosphorylation of Tyr728 is involved in the regulation of cell proliferation, as shown by a significantly reduced population doubling time of KSR1-Y728F cells compared to cells expressing wild type KSR1. Taken together, tyrosine phosphorylation in KSR1 uncovers a new link between Src family kinases and MAPK signaling. Tyr728, the novel regulatory phosphorylation site in murine KSR1, may coordinate the transition between the scaffolding and the catalytic function of KSR1 serving as a control point used to fine-tune cellular responses. / KSR1 fungiert bei Säugetieren als zentrales Gerüstprotein, welches die Anordnung von RAF/MEK/ERK-Komplexen koordiniert und die intrazelluläre Signalweiterleitung nach extrazellulärer Stimulation reguliert. Eine abweichende Aktivierung des entsprechenden MAPK-Signalwegs wurde mit vielen humanen Krebsformen und einigen Entwicklungsstörungen in Verbindung gebracht. Der Mechanismus der KSR1-Regulierung ist hochgradig komplex und involviert mehrfach Schritte der Phosphorylierung/Dephosphorylierung. In der vorliegenden Studie wurden etliche neue in-vivo-Phosphorylierungsstellen in mKSR1 mittels massenspektrometrischer Analyse entdeckt. Neben anderen wurde Tyr728 als besonderer regulatorischer Rest identifiziert, welcher durch LCK, einem Mitglied der Src-Kinase-Familie, phosphoryliert wird. Um zu verstehen wie die Phosphorylierung von Tyr728 die Funktion von KSR1 innerhalb der Signalweiterleitung und zellulärer Prozesse regulieren könnte, wurden strukturelle Modellierungen und biochemische Untersuchungen in diese Arbeit integriert. Die Computermodellierung der mKSR1(KD)-Proteinstruktur zeigte starke Wasserstoff- brückenbindungen zwischen Phospho-Tyr728 und den Resten in der Umgebung von Arg649 auf. Dieses Muster war auffällig verändert, wenn Tyr728 nicht phosphoryliert oder substituiert war. Wie anhand biochemischer Analyse untermauert wurde, könnte Arg649 für phospho-Tyr728 als Hauptankerpunkt dienen, um interne Strukturen in KSR1 zu stabilisieren. In Übereinstimmung mit den Ergebnissen der Proteinmodellierung enthüllten die Mutationsstudien, dass die Substitution von Tyr728 mit Phenylalanin zu einer weniger kompakten Interaktion zwischen KSR1 und MEK, einer erleichterten KSR1/B-RAF-Bindung und einer ansteigenden Phosphorylierung von MEK im Komplex mit KSR1 führt. Anhand dieser Erkenntnisse kann man rückschließen, dass Phospho-Tyr728 in die Verstärkung der Interaktionen innerhalb der KSR1/MEK-Grenzfläche und in die Regulierung der Phosphorylierung von KSR1-gebundenem MEK durch entweder RAF- oder KSR1-Kinasen involviert ist. Neben Tyr728 wurde Ser722 als eine neuartige regulatorische Phosphorylierungsstelle identifiziert. Aminosäureaustausche an der betreffenden Position demonstrierten, dass Ser722 die Phosphorylierung von KSR1-gebundenem MEK reguliert ohne die KSR1/MEK-Bindung selbst zu beeinträchtigen. Bedingt durch seine Lokalisierung könnte Ser722 folglich die katalytische Aktivität von KSR1 kontrollieren, indem es den Zugang des Substrates (möglicherweise MEK) zur aktiven Seite der KSR1-Kinase behindert. Zusammen mit Ser722 könnte phosphoryliertes Tyr728 ferner die Kinaseaktivität von KSR1 positiv beeinflussen, infolge von dessen Nähe zur Aktivierungs- und katalytischen Schleife in der KSR1(KD). Wie mittels Strukturmodellierung offengelegt wurde, bildet Phospho-Tyr728 eine Wasserstoffbrücke mit dem hochgradig konservierten Lys685 aus. Folglich hat Phospho-Tyr728 einen stabilisierenden Effekt auf interne Strukturen, welche in die katalytische Reaktion involviert sind, und erleichtert möglicherweise den Phosphattransfer innerhalb der katalytischen Spalte in KSR1. In Anbetracht dieser Fakten scheint es sehr wahrscheinlich, dass die LCK-abhängige Phosphorylierung von Tyr728 eine äußerst wichtige Rolle in der Regulierung der katalytischen Aktivität von KSR1 spielt. Die Ergebnisse der Fraktionierungs- und Morphologieanalysen enthüllten, dass KSR1 für die Phosphorylierung an Tyr728 LCK zum Zytoskelett rekrutiert, was darauf hindeutet, dass dieser Rest die Dynamik des Zytoskeletts und folglich Zellmotilität regulieren könnte. Darüber hinaus ist die Phosphorylierung von Tyr728 in die Regulierung der Zellproliferation involviert, wie anhand einer bedeutend reduzierten Populationsverdopplungszeit von KSR1-Y728F-Zellen im Vergleich zu Zellen, welche wildtypisches KSR1 exprimieren, gezeigt wurde. Zusammenfassend lässt sich sagen, dass die Tyrosin-Phosphorylierung in KSR1 eine neue Verknüpfung zwischen Kinasen der Src-Familie und der MAPK-Signalwirkung enthüllt. Tyr728, die neuartige regulatorische Phosphorylierungsstelle in Maus-KSR1, könnte den Übergang zwischen der Gerüst- und der katalytischen Funktion von KSR1 koordinieren und damit als Kontrollpunkt dienen, um zelluläre Reaktionen fein abzustimmen.

The protozoan parasite Trypanosoma brucei is the causal agent of sleeping sickness and besides its epidemiological importance it has been used as model organism for the study of many aspects of cellular and molecular biology especially the post-transcriptional control of gene expression. Several studies in the last 30 years have shown the importance of mRNA processing and stability for gene regulation. In T. brucei genes are unusually arranged in polycistronic transcription units (PTUs) and a coupled process of trans-splicing and polyadenylation produces the mature mRNAs. Both processes, mRNA processing and stability, cannot completely explain the control of gene expression in the different life cycle stages analyzed in T. brucei so far. In recent years, the relevance of expression regulation at the level of translation has become evident in other eukaryotes. Therefore, in the first part of my thesis I studied the impact of translational regulation by means of a genome-wide ribosome profiling approach. My data suggest that translational efficiencies vary between life cycle stages of the parasite as well as between genes within one life cycle stage. Furthermore, using ribosome profiling I was able to identify many new putative un-annotated coding sequences and to evaluate the coding potential of upstream open reading frames (uORF). Comparing my results with previously published proteomic and RNA interference (RNAi) target sequencing (RIT-seq) datasets allowed me to validate some of the new coding sequences and to evaluate their relevance for the fitness of the parasite. In the second part of my thesis I used the transcriptomic and translatomic profiles obtained from the ribosome profiling analysis for the identification of putative non-coding RNAs (ncRNAs). These results led to the analysis of the coding potential in the regions upstream and downstream of the expressed variant surface glycoprotein (VSG), which is outlined in the third part of the results section. The region upstream of the VSG, the co-transposed region (CTR), has been implicated in an increase of the in situ switching rate upon its deletion. The ribosome profiling results indicated moderate transcription but not translation in this region. These results raised the possibility that the CTR may be transcribed into ncRNA. Therefore, in the third part of my thesis, I performed a primary characterization of the CTR-derived transcripts based on northern blotting and RACE. The results suggested the presence of a unique transcript species of about 1,200 nucleotides (nt) and polyadenylated at the 3’-end of the sequence. The deletion of the CTR sequence promoting and increase of the in situ switching rates was performed around 20 years ago by means of inserting reporter genes. With the recent development of endonuclease-based tools for genome editing, it is now possible to delete sequences in a marker-free way. In the fourth part of my thesis, I show the results on the implementation of the highly efficient genome-editing CRISPR-Cas9 system in T. brucei using episomes. As a proof of principle, I inserted the sequence coding for the enhanced green fluorescent protein (eGFP) at the end of the SCD6 coding sequence (CDS). Fluorescent cells were observed as early as two days after transfection. Therefore, after the successful set up of the CRISPR-Cas9 system it will be possible to modify genomic regions with more relevance for the biology of the parasite, such as the substitution of codons present in gene tandem arrays. The implementation of ribosome profiling in T. brucei opens the opportunity for the study of translational regulation in a genome-wide scale, the re-annotation of the currently available genome, the search for new putative coding sequences, the detection of putative ncRNAs, the evaluation of the coding potential in uORFs and the role of unstranslated regions (UTRs) in the regulation of translation. In turn, the implementation of the CRISPR-Cas9 system offers the possibility to manipulate the genome of the parasite at a nucleotide resolution and without the need of including resistant makers. The CRISPR-Cas9 system is a powerful tool for editing ncRNAs, UTRs, multicopy gene families and CDSs keeping their endogenous UTRs. Moreover, the system can be used for the modification of both alleles after just one round of transfection and of codons coding for amino acids carrying post-translational modifications (PTMs) among other possibilities. / Trypanosoma brucei ist nicht nur als Erreger der Schlafkrankheit von großer epidemiologischer Bedeutung, sondern dient auch der Zell-­‐ und Molekularbiologie – insbesondere zur Erforschung der Genregulation auf posttranskriptionaler Ebene – als wichtiger Modellorganismus. In den vergangenen 30 Jahren konnten mehrere Forschungsarbeiten zeigen, dass mRNA-­‐Stabilität und –Prozessierung maßgeblich zur Regulation der Genexpression beitragen. Anders als in den meisten Eukaryoten sind die Gene in T. brucei in polycistronischen Transkriptionseinheiten (PTUs) angeordnet. Die reife mRNA entsteht aus dem polycistronischen Transkript in einem gekoppelten Prozess aus Trans-­‐splicing und paralleler Polyadenylierung. Beide Vorgänge allein, mRNA-­‐Stabilität und –Prozessierung, reichen nicht aus, um die Regulation der Genexpression in T. brucei vollständing zu erklären und zusätzliche Mechanismen müssen wirksam sein. Daher habe ich im ersten Teil meiner hier vorliegenden Doktorarbeit die Genregulation auf Ebene der Translation mittels genomweitem Ribosome Profiling untersucht. Die dabei gewonnen Daten deuten darauf hin, dass die Translationseffizienzen nicht nur zwischen prozyklischen-­‐ und Blutstromformen des Parasiten differieren, sondern auch die Gene innerhalb eines Stadiums verschieden effizient translatiert werden. Zudem war es mir mit diesem Ansatz möglich, neue, noch nicht annotierte kodierende Sequenzen zu identifizieren und das Kodierungspotenzial der jeweils vorgelagerten offenen Leseraster (ORFs) zu evaluieren. Mithilfe bereits veröffentlichter Proteom-­‐ und RNA Interferenz-­‐ Studien (RIT-­‐seq) konnte ich einige der neu identifizierten kodierenden Sequenzen validieren und deren Bedeutung für die Fitness des Parasiten bestimmen. Im zweiten Teil der Arbeit wurden die ermittelten Translations-­‐ und Transkriptionsprofile miteinander verglichen, um auf diese Weise mögliche nicht-­‐kodierende RNAs (ncRNAs) zu identifizieren. Dies führte zu einer eingehenderen Betrachtung der Kodierungspotenziale der dem exprimierten variablen Oberflächenproteins (VSG) vor-­‐ und nachgeschalteten Regionen. In früheren Arbeiten wurde bereits beschrieben, dass eine Deletion der dem VSG vorgelagerten, sogenannten co-­‐transposed region (CTR), vermehrt zu einer Aktivierung einer alternativen VSG Expressionsseite (in situ switches) führt. Ribosome Profiling zeigte, dass eben jede Regionen zwar moderat transkribiert, jedoch nicht translatiert werden. Da diese Ergebnisse vermuten ließen, dass die CTR für eine ncRNA kodiert, hab ich im dritten Teil meiner Arbeit die CTR Transkripte mittels Northern Blot und RACE weiter charakterisiert. Auf diese Weise konnte ich spezifische, 1200 Nukleotide (nt) lange und am 3`-­‐Ende polyadenylierte Transkripte nachweisen. Die bereits erwähnte Deletion der CTR verbunden mit einer erhöhten Rate an in situ switches wurde vor etwa 20 Jahren durch Insertion von Reportergenen durchgeführt. Heute ist es möglich mithilfe von Endonukleasen Genome ohne solche Marker zu editieren. So beschreibt der vierte Teil der Arbeit die Konstruktion von Episomen zur Etablierung und Anwendung des CRISPR-­‐ Cas9 Systems in T. brucei. Als Machbarkeitsnachweis wurde die kodierende Sequenz des grün fluoreszierenden Proteins (eGFP) am Ende des SCD6 Gens als Fusionsprotein inseriert. Grün fluoreszierende Zellen konnten bereits zwei Tage nach der Transfektion nachgewiesen werden. Nachdem CRISPR-­‐Cas9 erfolgreich in T. brucei etabliert werden konnte, werde ich im Folgenden weitere relevante Regionen im Genom modifizieren und beispielsweise die Deletion zweier Histonvarianten durchführen. Die Ribosome Profiling Studie in T. brucei erlaubt es uns, genomweit Genregulation auf Ebene der Translation zu analysieren, das uns zurzeit vorliegende Genom zu re-­‐annotieren, neue kodierende Sequenzen wie auch ncRNAs zu identifizieren und den Einfluss nicht-­‐kodierender Sequenzen auf die Translation zu untersuchen. Gleichzeitig ermöglicht die Etablierung des CRISPR-­‐ Cas9 Systems in T. brucei eine hochpräzise Manipulation des Genoms ohne den Einsatz von Resistenzmarkern. Auf diese Weise ist es möglich, Gene zu modifizieren und dabei die zugehörigen untranslatierten Bereiche (UTRs) zu erhalten, aber auch ncRNAs, UTRs und mehrfache Kopien eines Gens (gleichzeitig) zu editieren. Ebenso können einzelne Kodons in der Sequenz und somit posttranslational modifizierte Aminosäuren im Genprodukt verändert werden, was uns weitere Möglichkeiten zur Erforschung der Genregulation eröffnet.