The breakdown of glucose to pyruvate, known as glycolysis, is a central biochemical pathway, critically important for energy production and biosynthesis. Phosphofructokinase (PFK), the third enzyme in the pathway, is a crucial regulator of glycolytic flux, being the first committed step of glycolysis and modulating entry into the pentose-phosphate-pathway. Alterations in PFK activity have been implicated in many neurological conditions, including Tarui's disease, epilepsy, Alzheimer's disease, Down's syndrome, and cancers. There are three isoforms of human PFK; it is assumed that these evolved to fulfil specific metabolic niches both within cells and between tissue types. However, the differences between isoforms have never been systematically compared. Understanding these differences is an essential prerequisite for developing novel therapeutic agents targeting human PFK. Trypanosomatid parasites are a major global cause of neurological morbidity and mortality. Neglected tropical diseases caused by trypanosomatid parasites include African Sleeping Sickness (Trypanosoma brucei), Chagas disease (Trypanosoma cruzi), and leishmaniasis (Leishmania spp.). There is increasing interest in targeting the metabolic enzymes of these parasites, including PFK, which greatly differ from mammalian counterparts. This thesis describes biochemical, bio-physical, and bio-informatic studies on the three human PFK isoforms (PFK-M, PFK-L, and PFK-P), expressed in S. cerevisiae. Biophysical studies showed that the active conformation was tetrameric, with activity regulated by time and concentration dependent dissociation into smaller inactive species. The propensity to dissociate differed between isoforms, with PFK-M being most stable and PFK-P least stable. Dissociation was synergistically slowed by the addition of substrates and reducing agents, indicating different mechanisms of action. Kinetic studies were performed with respect to both substrates (ATP and F6P) in the presence of natural metabolites hypothesised to act as modulators of enzyme activity. Each isoform conformed to an allosteric sigmoidal kinetic model and had differing kinetic properties, with PFK-M being the most active and PFK-P the least active. ATP was found to act as both substrate and allosteric inhibitor, with activity showing a biphasic response to ATP concentration. Each isoform showed different susceptibilities to both ATP inhibition and regulation by allosteric modulators. The reverse reaction was shown to be possible under certain conditions. Bio-informatic data on intra-cellular and inter-cellular locations were determined using the Human Protein Atlas and the FANTOM5 datasets. PFK-M localises to the cytosol and may co-localise with endoplasmic reticulum; PFK-L associates with nucleoli and mitochondria; and PFK-P is cytosolic. Splice variants were not shown to be physiologically significant. Each isoform had different tissue expression levels, with overall PFK expression varying by tissue type. PFK-P was the principal isoform in cancers, whereas PFK-L was dominantly expressed in immune cells. Activated macrophages switched rapidly from PFK-L to PFK-P. PFK-M and PFK-P were the dominant isoforms in the brain, although there were differences between brain areas. Neurons expressed less PFK than astrocytes, in keeping with the lactate shuttle theory. PFK from each of the three main pathological trypanosomatid species were compared (T. brucei, TbPFK; T. cruzi, TcPFK; L. infantum, LmPFK); expressed in E. coli. Biophysical analysis showed each PFK to be tetrameric; no evidence of time or concentration dependent dissociation or inactivation was found. Kinetic properties differed between isoforms, with TcPFK being most active and LmPFK being least active. LmPFK was very poorly active with regard to F6P titrations unless AMP was present. No other modulators were shown to affect activity, although GTP was an alternate substrate. The reverse reaction was shown to be possible and may be compatible with physiological concentrations of ADP and F16BP in the trypanosomatid glycosome.

Orientador: Regina Maria Barretto Cicarelli / Banca: João Aristeu da Rosa / Banca: Maurício Bacci Junior / Resumo: No momento há apenas dois fármacos disponíveis para o tratamento da doença de Chagas, nifurtimox e benzonidazol, ambos de ações limitadas. Assim, a compreensão dos mecanismos utilizados por T. cruzi para diminuir os efeitos citotóxicos de fármacos e a busca de potenciais moléculas para quimioterapia, bem como os alvos para elas, são de extrema importância. O aumento na produção de certas enzimas do metabolismo oxidativo seria responsável pela resistência das cepas do parasito ao benzonidazol (BZ). Tais enzimas são: triparedoxina peroxidase (mTcTXNPx) que catalisa a redução de peróxidos, superóxido dismutase (mTcSOD) que realiza a dismutação do ânion superóxido e old yellow enzyme (cTcOYE) que está envolvida na redução de substâncias tripanossomicidas, como naftoquinonas e compostos nitroheterocíclicos. Estudos demonstram que a atividade antimalarial da cloroquina foi aumentada com a adição de ferroceno. Neste trabalho, foram avaliadas as diferenças de suscetibilidade entre seis cepas de T. cruzi (Y, Bolívia, SI1, SIGR3, SI8 e QMII) em relação a três estruturas derivadas de diaminas do ferroceno e a expressão das proteínas antioxidativas. A substância AAC09 mostrou maior citotoxicidade, com CI50 de 2,21 μM para a cepa menos resistente (cepa Y) e 15,20 μM (cepa QMII) para a mais resistente, enquanto que para BZ foi 27,28 μM (cepa SI1) e 105,28 μM (cepa SIGR3), respectivamente. Após tratamento, a expressão das enzimas mTcTXNP (25,5 kDa) e cTcOYE (43 kDa) não aumentaram em cepas mais resistentes aos derivados de diaminas do ferroceno e BZ. Por outro lado, somente ocorreu aumento de expressão de mTcTXNPx na cepa Y tratada com BZ. Os resultados obtidos mostraram que cada cepa apresenta distinta suscetibilidade a um agente oxidativo e as cepas mais resistentes não aumentaram a expressão das... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: At the moment there are only two drugs available for the treatment of Chagas disease, nifurtimox and benznidazole, both of use restricted. Understanding the mechanisms used by T. cruzi to reduce de cytotoxic effects of drugs and finding potential molecules for chemotherapy, as well as targets for them, are extremely important. Resistance to benznidazole (BZ) in T. cruzi strains may be caused by increased production of enzymes that act on oxidative metabolism. Such enzymes are: tryparedoxin peroxidase (mTcTXNPx), which catalyzes the reduction of peroxides, superoxide dismutase (mTcSOD), which performs the dismutation of superoxide anion and old yellow enzyme (cTcOYE), which is related to the reduction of trypanocidal substances, such as naphthoquinones and nitroheterocyclic compounds. Studies showed that the antimalarial activity of chloroquine was increased with the addition of ferroceno. Herein the susceptibilities in six T. cruzi strains (Y, Bolivia, SI1, SIGR3, SI8 and QMII) were analyzed in relation to three structures of ferrocene diamine derivatives and the expression of antioxidant proteins. The AAC09 substance showed higher cytotoxicity, with IC50 of 2.21 μM for the less resistant strain (Y strain) and 15.20 μM (QMII strain) for more resistant, whereas to BZ was 27.28 μM (strain SI1) and 105.28 μM (SIGR3), respectively. After treatment, the expression of the enzymes mTcTXNP (25.5 kDa) and cTcOYE (43 kDa) did not increase in more resistant strains to ferrocene diamine derivatives and BZ. However, there was increased expression of mTcTXNPx only in the Y strain treated with BZ. The results showed that each strain exhibited different susceptibility to oxidative agent and the most resistant strains did not enhance expression of these enzymes. Based on these findings, probably other mechanisms may be... (Complete abstract click electronic access below) / Mestre

The parasitic protist Trypanosoma brucei is the causative agent of human African trypanosomiasis. The parasite undergoes a complex life cycle involving stages within the mammalian bloodstream and its tsetse fly vector. The fundamental differences between energy metabolism in the procyclic insect form (PCF) and long slender bloodstream form (BSF) T. brucei involve a switch in the directionality of the F1Fo- ATPase. In PCF, the need for oxidative phosphorylation in low glucose conditions requires the enzyme to generate ATP. In the slender BSF, the enzyme uses ATP from glycolysis to drive proton pumping to maintain the essential mitochondrial membrane potential. Fo-ATPase subunit 6 (A6) is critical for proton translocation in either direction and is encoded in the mitochondrial DNA (kDNA). The parasite’s kDNA is therefore essential in the slender BSF, and also in PCF where it encodes multiple subunits of the respiratory chain complexes that constitute the oxidative phosphorylation pathway. Specific point mutations in the nuclearly encoded γ subunit of the mitochondrial F1Fo-ATPase allow survival in the absence of kDNA in the slender BSF T. brucei (Dean et al., 2013). These mutations, even in the heterozygous genotype, cause an increase in resistance to multiple drugs in vitro (Gould and Schnaufer, 2014). This thesis investigates two questions: (1) What is the molecular mechanism of compensation for kDNA loss? (2) Are kDNA and a functional FoF1-ATPase required for life cycle progression? Slender BSF T. brucei were generated expressing ATPase L262Pγ. The effects of this γ mutation and kDNA loss, respectively, on structure/function of the F1Fo- ATPase were probed. Cells expressing L262Pγ show decreased sensitivity to Fo inhibitor oligomycin compared to WT cells, suggesting that the L262Pγ mutation functionally uncouples the enzyme. The impact of the L262Pγ mutation on the structure of the enzyme was probed by high resolution clear native electrophoresis. This shows there are dramatic consequences to F1Fo structure in the presence of the L262Pγ mutation. The apparent selection for cells that no longer express intact F1Fo suggests that L262Pγ uncouples the enzyme, resulting in a lethal proton leak. Pleomorphic T. brucei with and without kDNA were also generated by expressing mutant γ in strain AnTat1.1 90:13. Differentiation studies demonstrate kDNA0 cells can differentiate to insect-transmissible stumpy forms. These cells show a decreased lifespan, suggesting a critical role for a kDNA-encoded product in the stumpy form. Tsetse fly infections show kDNA is indispensable for progression to the PCF. Unexpectedly, parasites homozygous for L262Pγ can establish a midgut infection, while they do not infect the salivary glands. Heterozygous parasites, on the other hand, can form animal-transmissible metacyclics in the salivary glands, providing a potential mechanism for spreading decreased sensitivity to multiple drugs.

Contrasting physiological requirements for T. brucei survival between procyclic (vector) and bloodstream (mammal) forms necessitate different molecular processes and therefore changes in protein expression. Transcriptional regulation is unusual in T. brucei because the arrangement of genes is polycistronic; however, genes which are transcribed together are subsequently cleaved into separate mRNAs by trans-splicing and are individually regulated. During the process of trans-splicing, a 39-nucleotide splice-leader RNA is added to the 5´ end of mRNA. In this study, gene regulation in trypanosomes will be examined in the context of the 7-methylguanosine cap attached to the 5´ end of the splice-leader. Interestingly, in addition to the capping enzymes identified in other eukaryotes, trypanosomatids have an additional guanylyltransferase and methyltransferase in the form of a bifunctional enzyme (TbCGM1). TbCGM1 was found to be essential in bloodstream form T. brucei, although the purpose of this bifunctional capping enzyme remains unclear. Null mutants of a related enzyme, monomeric methyltransferase TbCMT1, did not show an effect on cell viability in culture, however, the enzyme proved to be important for virulence in vivo. Complementary to the study of T. brucei capping enzymes, we worked to develop a method to allow structural analysis of the 5´mRNA cap by mass spectrometry. Following pre-mRNA processing, regulation of the mature mRNAs is a tightly controlled cellular process. While multiple stage-specific transcripts have been identified, previous studies using RNA-seq found that the changes in overall transcript level do not necessarily reflect the abundance of the corresponding proteins. We hypothesized that in addition to mRNA stability, mRNA recruitment to ribosomes may play a significant role in the regulation of gene expression in T. brucei. To approach this question, we performed RNA-seq of total, subpolysomal, and polysomal mRNA. This transcriptomic data was then correlated with published proteomic studies to obtain a global picture of the relative translation efficiencies and their relationship to steady-state protein levels between bloodstream and procyclic form T. brucei.

Die Schlafkrankheit hat ihren Schrecken seit den Zeiten Robert Kochs und Paul Ehrlichs nicht verloren. Die zielgerichtete Entwicklung neuer Medikamente ist für die Menschen in den Endemiegebieten damals wie heute von elementarer Bedeutung. Die Naphtylisochinolin-Alkaloide stellen eine neue chemische Substanzklasse dar, die gute Kandidaten für die Entwicklung neuer Medikamente enthält. Mit GBAP 94 im speziellen liegt eine Substanz vor, die gute Startvorrausetzungen hierfür mitbringt. Diese sind eine sehr gute Wirksamkeit gegen Trypanosomen, gepaart mit einer hohen Selektivität durch einen sehr wahrscheinlich relativ spezifisch anti-trypanosomalen Wirkmechanismus. Die verwendeten Naphtylisochinolin-Alkaloide GBAP 94 und GBAP 146 wurden nach unterschiedlichen Gesichtspunkten ausgewählt. GBAP 94 wurde aufgrund seiner guten antitrypanosomalen Wirkung und seiner hohen Selektivität für Trypanosomen ausgewählt. Die IC50 liegt mit 0,383 µmol/l im Vergleich zu den aktuell verwendeten Medikamenten sehr niedrig. Die Selektivitätsindices (IC50 Trypanosoma brucei brucei / IC50 Makrophagen J774.1) mit 85,6 und (IC50 Try-panosoma brucei brucei / IC50 Leishmania major) mit 15,1 liegen in einem sehr günstigen Bereich. GBAP 146 wurde hauptsächlich wegen seiner guten Fluoreszenz-Eigenschaften ausgewählt. Die antitrypanosomale Aktivität ist mit einer IC50 von 0,289 µmol/l zwar sehr gut, eine große Selektivität ist aber nicht gegeben. Die beiden Alkaloide waren aufgrund ihrer Eigenfluoreszenz gut fluoreszenz-mikroskopisch in den Parasiten zu detektieren. Nach 10 min war in den ersten Trypanosomen die Anreicherung der Wirkstoffe erkennbar. Nach 30 min war bei fast allen Parasiten eine Färbung erkennbar. Die Wirkstoffe reicherten sich zunächst in mehreren kleinen Vakuolen an. Bei längeren Inkubationszeiten zeigte sich eine fast homogene Verteilung innerhalb des kompletten Parasiten. Durch-gängig ausgespart blieb eine vakuolische Struktur. Diese entwickelte oder vergrößerte sich im Verlauf der Inkubationszeit im vorderen Drittel des Parasiten, etwa im Bereich des Kinetoplasten. Diese Vakuole konnte auch lichtmikroskopisch in der Giemsa-Färbung nachgewiesen werden. Der Anteil der veränderten Trypanosomen lag bei diesen Untersuchungen nach 1 h bei 25,4%, stieg bis zum Zeitpunkt 2 h auf 46,6% und stabilisierte sich nach 4 h bei 44,8%. Die vakuolische Struktur führte durch ihre Vergrößerung zur zunehmenden Verplumpung der Trypanosomen bis zu einer kugelförmigen Zellform mit geisselartig-wirkender Flagelle. Aufgrund der veränderten Form wurden die Zellorganellen verdrängt. Dies konnte durch die Fluoreszenzmarkierung des Mitochondriums mit Rodamine B Hexylester und der sauren Kompartimente, besonders des Lysosoms, mit LysoTracker® gezeigt werden. Die Vakuolisierung von Trypanosomen im Zusammenhang mit Apoptose ist bekannt. Die neu entstehende Vakuole konnte weder mit LysoTracker® green, noch mit dem endosomalen Farbstoff FM 4-64 angefärbt werden. Damit können eine lysosomale und eine endosomale Herkunft der Vakuole ausgeschlossen werden. Eine genaue Klärung der Genese der Vakuole steht noch aus. In den Untersuchungen mit Annexin V und Propidium-Jodid im FACS® konnte gezeigt werden, dass die Wirkung der NIQs sehr wahrscheinlich Apoptose induziert. Annexin V ist auch bei Trypanosomen als Marker für Apoptose etabliert. Zudem zeigte sich ein Anstieg der Anzahl apoptotischer Trypanosomen mit Periode von 6 h – 8 h. Diese Dauer entspricht ungefähr der Dauer des trypanosomalen Zellzyklus. Ein Eingriff der NIQs in den Zellzyklus ist somit sehr wahrscheinlich. Eine Hemmung von Teilen des Zellzyklus ist als Auslöser für Apoptose bekannt. Über die genaue Zielstruktur der NIQs kann allerdings nur spekuliert werden. Die apotose-induzierende Wirkung anderer Alkaloide auf Trypanosomen ist inzwischen nachgewiesen. Ein weiteres Indiz ist, dass die Ergebnisse von Ponte-Sucre mit den NIQs bei Leishmanien ebenfalls in Richtung Apoptose weisen. / The trypanosomiasis is still an emerging problem in sub-Saharan Africa. Due to the limitations of the currently used drugs and emerging drug resistance, there is an urgent need for the target-oriented development of novel therapies. Naturally occurring naphthylisoquinoline alkaloids (NIQs), axially chiral acetogenic products derived from tropical plants, have been investigated for their activity against Trypanosoma brucei brucei TC 221. The NIQ N-(3'-Methoxyphenyl)-6,8-dimethoxy-1,3-dimethylisochinoliniumtetrafluoroborate seems to be quite specific antitrypanosomal agent. This compound shows a low IC50-value of 0.383 µmol/l against Trypanosoma brucei brucei TC 221 in comparison to the current drugs. For controls another NIQ, N-(4'-N'-Dansylaminophenyl)-6,8-dimethoxy-1,3-dimethylisochinoliniumtrifluoro-acetate, eflornithine an amphotericin B, witch is described to induce apoptosis in trypanosomes, were used. Both NIQ could be detected directly because of their self-fluorescence in the fluorescence-microscopy. After 10 min an accumulation in the first parasites could be detected. After 30 min almost all parasites show the compounds. After an initial accumulation in small vesicles the NIQ spread homogeneous over nearly the whole parasite. Only a vacuole was spared. This structure developed or increased during incubation time. It was located in the front part of the parasite near the kinetoplast. This vacuole could also be detected in light-microscopy of Giemsa-stained parasites. The fraction of the affected trypanosomes was after 1 h 25.4% and increased up to 46.6% after 2 h and stayed almost in this level (44.8% after 4 h). The increase of the vacuole induced a dumpier up to spherical shape. The organelles were displaced. This could be shown by fluorescence-labelled mitochondria, stained with rodamine-B-hexylester, and the acidic compartments, especially the lysosome stained with LysoTracker®. The vacuolisation of trypanosoma brucei is described during apoptosis. The staining of the developing vacuole wasn’t possible neither with LysoTracker® nor with the endosomal staining FM 4-64®. A lysosomal or endosomal origin of this vacuole could be excluded. The genesis of this vacuole needs further investigation. In the FACS®-investigations with annexin V and propidium-iodide staining we got strong hints that the NIQs induce apoptosis. Annexin V is established as a marker for apoptosis in trypanosome. We found an increase of apoptotic parasites in a 6 h – 8 h period. This is also the time for the trypanosomal cell cycle. NIQs seem to interfere with the cell cycle. This is descried from various authors as a trigger for apoptosis. The target structure is however still unknown. Results of other groups indicate an apoptosis-inducing effect of alkaloids in trypanosoma or leishmania.

Post-translational histone modifications (PTMs) such as methylation of lysine residues influence chromatin structure and function. PTMs are involved in different cellular processes such as DNA replication, transcription and cell differentiation. Deregulations of PTM patterns are responsible for a variety of human diseases including acute leukemia. DOT1 enzymes are highly conserved histone methyltransferases that are responsible for methylation of lysine 79 on histone H3 (H3K79). Most eukaryotes contain one single DOT1 enzyme, whereas African trypanosomes have two homologues, DOT1A and DOT1B, which methylate H3K76 (H3K76 is homologous to H3K79 in other organisms). DOT1A is essential and mediates mono- and di-methylations, whereas DOT1B additionally catalyzes tri-methylation of H3K76. However, a mechanistic understanding how these different enzymatic activities are achieved is lacking. This thesis exploits the fact that trypanosomes possess two DOT1 enzymes with different catalytic properties to understand the molecular basis for the differential product-specificity of DOT1 enzymes. A trypanosomal nucleosome reconstitution system was established to analyze methyltransferase activity under defined in vitro conditions. Homology modeling allowed the identification of critical residues within and outside the catalytic center that modulate product-specificity. Exchange of these residues transferred the product-specificity from one enzyme to the other and revealed regulatory domains adjacent to the catalytic center. This work provides the first evidence that few specific residues in DOT1 enzymes are crucial to catalyze methyl-state-specific reactions. These results have also consequences for the functional understanding of homologous enzymes in other eukaryotes. / Posttranslationale Histonmodifizierungen (PTMs), wie beispielsweise die Methylierung von Lysinseitenketten, beeinflussen maßgeblich die Struktur und Funktion von Chromatin. PTMs spielen eine wichtige Rolle in verschiedensten zellulären Prozessen, darunter DNA Replikation, Transkription oder Zelldifferenzierung. Darüber hinaus liegt ein verändertes PTM-Muster einer Vielzahl humaner Erkrankungen zugrunde, wie z.B. der akuten myeloischen Leukämie. DOT1-Enzyme sind hochkonservierte Histonmethyltransferasen, die für die Methylierung von Lysin 79 in Histon H3 (H3K79) verantwortlich sind. Im Gegensatz zu den meisten Eukaryoten, die lediglich ein einziges DOT1-Enzym besitzen, finden sich zwei homologe Proteine in afrikanischen Trypanosomen (DOT1A und DOT1B), die Lysin 76 in Histon H3 (H3K76) methylieren (H3K76 ist homolog zu H3K79 in anderen Organismen). DOT1A ist essentiell und katalysiert Mono- und Di-Methylierungen, wohin gegen DOT1B darüber hinaus eine Trimethylierung an H3K76 setzen kann. Derzeit fehlt jegliches mechanistische Verständnis darüber, wie beide Enzyme diese unterschiedliche Produktspezifität erreichen. Die vorliegende Dissertation macht sich den Umstand zunutze, dass Trypanosomen zwei DOT1-Methyltransferasen mit unterschiedlichen katalytischen Eigenschaften besitzen, um Einblicke in die molekulare Grundlage der unterschiedlichen Produktspezifität zu erlangen. Zunächst wurde ein Rekonstitutionssystem für Nukleosomen aus Trypanosomen etabliert, das es ermöglichte die Methyltransferase-Aktivitäten unter definierten in vitro Bedingungen zu analysieren. Homologiemodelle erlaubten die Identifikation von wichtigen Aminosäurepositionen innerhalb und außerhalb des katalytischen Zentrums der Enzyme, die einen Einfluss auf die Produktspezifität haben. Ein Austausch der Aminosäuren an diesen Positionen führte zu einer Umwandlung der Produktspezifität und offenbarte gleichzeitig DOT1A- und DOT1B-spezifische regulatorische Domänen, die an das katalytische Zentrum angrenzen. Diese Arbeit liefert erste Hinweise, dass wenige maßgebliche Aminosäuren in DOT1-Enzymen für den H3K76-Methylierungsgrad während der Katalyse entscheidend sind. Darüber hinaus haben die hier dargestellten Ergebnisse ebenfalls Konsequenzen für das funktionale Verständnis der homologen Enzyme in anderen Eukaryoten.

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.

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Trypanosoma cruzi is endemic in the U.S., infecting humans, dogs, and wildlife. This study identified a new geographic distribution for triatomine species within Texas based on 2,449 records obtained from published data and new field studies. Triatomine vectors of T. cruzi were reported from 97 counties covering all ecological zones. Triatoma gerstaeckeri was the most commonly collected species followed by T. sanguisuga. New field collections resulted in 233 specimens from 37 counties and a 52% T. cruzi infection rate. A second trypanosome, Blastocrithida triatomae was found in two specimens from different locations. A habitat suitability model for T. gerstaeckeri was developed using GIS and remote sensing applications. Forest and rangeland were the predominant land cover classes found within T. gerstaeckeri habitat, where as water and agriculture proved to have little influence on habitat suitability. Genetic variation of seven triatomine species from Texas was evaluated using cytochrome b DNA sequences from 61 new specimens. This is the first study of the taxonomic status of T. gerstaeckeri, T. indictiva, and T. neotomae using molecular markers. Intraspecific variation for T. sanguisuga and T. gerstaeckeri suggests significant gene flow across their ranges within Texas. Genetic variation of T. cruzi isolates from Texas was evaluated using SSU rRNA gene sequences. Included were 63 new sequences from five triatomine species, canine, baboon, and human isolates. Sequences partitioned into two groups in agreement with previous studies on U.S. isolates. Genetic variation of T. cruzi did not occur according to host, geographic location, or collection site. The extent of Chagas disease in domestic canines of Texas is described by geographic distribution, signalment, and clinical presentation and histopathology. Based on data from 553 cases, the geographic distribution in Texas is widespread (46 counties) and closely matches the distribution of the Triatomine vectors. Chagas disease was diagnosed in 33 breeds, primarily sporting/working dogs. This study represents the most comprehensive characterization of components of the Chagas disease transmission cycle in the U.S. to date. These findings should raise awareness among physicians, veterinarians, and public health practitioners regarding T. cruzi, its vectors, canine infection, and human risk for Chagas disease in Texas.