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Die afrikanische Schlafkrankheit füht unweigerlich zum Tod wenn sie unerkannt und somit unbehandelt bleibt. Zur Therapie stehen nur sehr wenige Medikamente zur Verfügung, wovon die meisten bereits seit mehr als 50 Jahren im Einsatz sind. Unter der Therapie treten in ca. 5-10% der Fälle Enzephalopathien auf, die in vielen Fällen tödlich verlaufen. Bisher ist nicht sicher, wie der dahinterstehende Pathomechanismus verläuft. Zu dieser Frage wurden Untersuchungen des Glukosemetabolismus an Patienten im 2. Stadium der Schlafkrankheit durchgeführt. Es zeigte sich ein signifikanter Anstieg des durchschnittlichen Glukoseniveaus im Verlauf der Therapie. Des weiteren wurden unterschiedliche Verläufe von arzneimittel-induzierter Enzephalopathie klinisch beobachtet und beschrieben. / Human african Trypanosomiasis is a letal desease if left untreated. Most of the medicals used in late stage trypanosomiasis are older than 50 years. In 5-10% of the patients treatment induced encephalopathies occur which often lead to death. Therefore we investigated the glucosemetabolism in late stage patients treated with melarsoprol. a significant increasion of the blood glucose level was shown. Further clinical descriptions of treatment induced encephalopathies were made.

Die akute Form der afrikanischen Schlafkrankheit wird durch den Parasiten Trypanosoma brucei rhodesiense verursacht und führt unbehandelt zum exitus letalis. Da derzeit nur wenige, zum Teil hoch toxische Substanzen mit zunehmender Resistenzlage klinische Anwendung finden, ist die Entwicklung neuer Medikamente dringend erforderlich. Rhodesain ist eine essenzielle Cysteinprotease des Erregers und wird als potentielles Zielmolekül für die intelligente Wirkstoffentwicklung gehandelt. Inhibitoren, welche dieses Molekül im niedrigen mikromolaren Bereich inhibieren, konnten bereits vom Institut für Pharmazie der hiesigen Universität synthetisiert werden. Um die Inhibitoren hinsichtlich ihrer Selektivität, Affinität und Toxizität zu optimieren, ist deren röntgenstrukturbiologische Analyse im Komplex mit dem Zielmolekül Rhodesain notwendig. Rhodesain wurde in den Hefezellen Pichia pastoris, welche mit dem Vektor pPICZalphaB_RhodesainDeltaCmut transfiziert wurden, exprimiert und mittels Hydrophober-Wechselwirkungs- sowie Größenausschlußschromatographie gereinigt. Nadelförmige Kristalle konnten mit einer Reservoirlösung aus 1.6 M Ammoniumsulfat, 10% 1,4-Dioxan und 0.1 M MES pH6.9 sowie bei einer Temperatur von 20°C erhalten werden. Die Kristalle wurden mit dem Inhibitor UM112C getränkt und an der Europäischen Anlage für Synchrotronstrahlung ESRF (Grenoble) vermessen. Das Diffraktionsbild bei einer Wellenlänge von 0.97625 Å ergab ein für Proteine typisches Beugungsmuster mit einer Streuung bis 3.04 Å. Zur weiteren Analyse und Optimierung der Kristalle wurde das Projekt von Dipl.-Biol. Uwe Dietz im Rahmen seiner Dissertation und des Sonderforschungsbereichs SFB-630 übernommen. / The acute form of African Sleeping Sickness is caused by the parasite Trypanosoma brucei rhodesiense and leads to death if untreated. Since currently only few, in part highly toxic substances with an increasing rate of resistance are available, the development of new drugs is urgently needed. Rhodesain is the major cysteine protease of Trypanosoma brucei rhodesiense and therefore a potential target molecule for structure based drug design. Inhibitors that inhibit rhodesain in the micromolar range were already synthesized in the Insitute of Pharmacy at the University of Wuerzburg. Structural analysis of the rhodesain-inhibitor-complex will provide the basis for the development of an optimized inhibitor regarding affinity, selectivity and toxicity. Rhodesain was expressed in the yeast strain Pichia pastoris that was transfected with the vector pPICZalphaB_RhodesainDeltaCmut and purified using hydrophobic interaction and size exclusion chromatography. Spicular crystals were obtained in the presence of a reservoir solution containing 1.6 M ammonium sulfate, 10% Dioxane and 0.1 M MES pH 6.9 and at 20°C. Crystals were soaked with the inhibitor UM112C and analysed at the European Synchrotron Radiation Facility (Grenoble, France). The diffraction pattern at a wave length of 0.97625 Å shows a characteristic diffraction pattern of a macromolecule and the crystals diffracted to a resolution of 3.04 Å.

A dissertation submitted to the faculty of Science, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Master of Science. Johannesburg, 2014. / Human African Trypanosomiasis (HAT) is transmitted by Glossina species and remains a serious health problem in Africa. Many aspects of control of the disease have been implemented throughout the years but vector control of tsetse flies has proven to be the most efficient long-term solution. Vector control interventions have been implemented for many tsetse species but relatively little is known about the behaviour of the riverine species, Glossina fuscipes fuscipes. Increased knowledge of this species would improve vector control interventions. This study aimed at: i) understanding the behaviour of tsetse flies around visual devices and odour baits; ii) understanding the behaviour of the flies with regard to human activities; iii) understanding the interaction between the nutritional status of tsetse flies and their attraction to various trapping devices (biconical traps and electric nets); and iv) establishing an age determination curve for field-caught flies. Results showed that visual targets were better attractants then odour-based ones and electric nets performed better than biconical traps. The sticky traps caught 10x more flies (males) than the stationary biconical traps. Sticky traps caught more young flies than the biconical traps which caught more old flies. An age curve was established for flies ranging from 1 day to 60 days old and the fluorescence-based age determination technique, using pteridine levels, has been shown to work for this species. Understanding the behaviour of tsetse flies around trapping devices should lead to improved trapping efficiency. The data gathered will be of importance in assisting with designing and running the Lake Victoria region control operations planned by PATTEC and it will have application in G. f. fuscipes endemic regions in other parts of Africa.

Trypanosomatid parasites cause a wide range of so-called neglected diseases which affect over 27 million people every year. Current treatments are toxic and prone to resistance; therefore, it is imperative to identify novel protein targets and to develop more efficient treatments. Phosphofructokinase (PFK) is the third enzyme in glycolysis, and its reciprocal enzyme in gluconeogenesis is fructose-1,6-bisphosphatase (FBPase); in trypanosomatid parasites (Trypanosoma brucei [Tb], Trypanosoma cruzi [Tc] and Leishmania [Lm] species), both enzymes are recognised drug targets. This thesis describes biochemical and structural studies on these two allosteric enzymes that have been studied with two main purposes: 1) To understand their intrinsic behaviour. The allosteric mechanism of T. brucei PFK is described with the help of two novel crystal structures: TbPFK with the allosteric activator AMP, and mutant A288D located in the effector site. These studies have provided a better understanding of the effect of evolution on the allostery of PFK; and have introduced the first reproducible crystallisation of TbPFK via its A288D mutant. 2) To find novel inhibitors using in silico and high-throughput methods, and to investigate how the intrinsic behaviour relates to the mechanism of inhibition. Nanomolar selective inhibitors against TbPFK and TcPFK have been obtained and optimised to a novel family with low micromolar inhibitory activity against cultured parasites. Crystal structures with three of these inhibitors on TbPFK have helped us understand the structure-activity relationship. Moreover, novel crystal structures of TcPFK and LmFBPase, as well as reproducible crystallisation conditions for the latter enzyme and a mutant of TbPFK (A288D) will undoubtedly facilitate future drug discovery on these targets. Our long-term aim of finding novel drugs against sleeping sickness has been supported by the Wellcome Trust which has recently granted a Seeding Drug Discovery Award with the name “Optimisation of a trypanosome phosphofructokinase lead series to give candidates for treatment of the trypanosomatid based neglected disease Human African Trypanosomiasis”.

acase@tulane.edu / The United States is home to eleven species of triatomine insects that can carry and transmit the parasite that causes Chagas disease, Trypanosoma cruzi. Much of the natural history of these vectors remains unknown. This is especially true of the species native to southeastern Louisiana, Triatoma sanguisuga. To this end experiments were undertaken to understand the movements, blood meal sources, and associated vertebrates of T. sanguisuga. Sites of genetic variability were sequenced and compared to determine how related T. sanguisuga spatially aggregate. It is demonstrated that the cytochrome b gene can be used to identify sub-populations of these insects and monitor their spatial aggregation over a multi-year period. Genetic analysis suggests the presence of wide-spread sub-populations. The sources of vertebrate blood meals were determined from collected specimens to determine the most frequent bloodmeal sources. The most frequent bloodmeal sources were found to be from humans, cows, pigs, dogs, and raccoons - though some of these sequences are likely the result of laboratory contamination. Finally, habitat modeling of T. sanguisuga provides a framework for assessing the shared habitats of T. sanguisuga and its vertebrate food sources. By using publicly available reports of T. sanguisuga, a model representing suitable habitat for the vector was created. By comparing this distribution to similar distribution models of associated animals, it was determined that the most statistically significant overlap of suitable habitats occurs between T. sanguisuga and two small rodent species, Oryzomys palustris and Neotoma floridana. The present research adds significantly to the limited knowledge of the natural history of this vector species. The work provides a foundation on which to build evidence-based risk assessments of autochthonous human Chagas disease in southeastern Louisiana and beyond. / 1 / Samuel B Jameson