@phdthesis{HebronMwalwisi2018,
  author    = {Hebron Mwalwisi, Yonah},
  title     = {Assessment of Counterfeit and Substandard Antimalarial Medicines using High Performance Thin Layer Chromatography and High Performance Liquid Chromatography},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-145821},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Although the prevalence of substandard and counterfeit pharmaceutical products is a global problem, it is more critical in resource-constrained countries. The national medicines regulatory authorities (MNRA) in these countries have limited resources to cater for regular quality surveillance programmes aimed at ensuring that medicines in circulation are of acceptable quality. Among the reasons explained to hinder the implementation of these strategies is that compendial monographs are too complicated and require expensive infrastructures in terms of environment, equipment and consumables. In this study it was therefore aimed at developing simple, precise, and robust HPLC and HPTLC methods utilizing inexpensive, readily available chemicals (methanol and simple buffers) that can determine the APIs, other API than declared one, and which are capable of impurity profiling. As an outcome of this study, three isocratic and robust HPLC and two HPTLC methods for sulfadoxine, sulfalene, pyrimethamine, primaquine, artesunate, as well as amodiaquine have been developed and validated. All HPLC methods are operated using an isocratic elution mode which means they can be implemented even with a single pump HPLC system and standard C18 columns. The densitometric sulfadoxine/sulfalene and pyrimethamine method utilizes standard TLC plates as well as inexpensive, readily available and safe chemicals (toluene, methanol, and ethyl acetate), while that for artesunate and amodiaquine requires HPTLC plates as well as triethylamine and acetonitrile due to challenges associated with the analysis of amodiaquine and poorly the detectable artesunate. These HPTLC methods can be implemented as alternative to those requiring HPLC equipment e.g. in countries that already have acquired densitometer equipment. It is understood that HPTLC methods are less sensitive, precise and accurate when compared to HPLC methods, but this hindrance can easily be addressed by sending representative samples to third party quality control laboratories where the analytical results are verified using compendial HPLC methods on a regular basis. It is therefore anticipated that the implementation of these methods will not only address the problem of limited resources required for medicines quality control but also increase the number of monitored targeted antimalarial products as well as the number of resource- constrained countries participating in quality monitoring campaigns. Moreover, the experiences and skills acquired within this work will be applied to other API groups, e. g. antibiotics, afterwards.},
  subject      = {Instrumentelle Analytik},
  language  = {en}
}
@phdthesis{Schaefer2014,
  author    = {Schaefer, Frauke},
  title     = {Diagnosis and therapy of malaria under the conditions of a developing country - the example of Burkina Faso},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-102863},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Malaria is a challenging infection with increasing and wide-spread treatment failure risk due to resistance. With a estimated death toll of 1-3 Million per year, most cases of Malaria affect children under the age of five years in Sub-Saharan Africa. In this thesis, I analyse the current status of malaria control (focussing on diagnosis and therapy) in Burkina Faso to show how this disease burdens public health in endemic countries and to identify possible approaches to improvement. MB is discussed as a therapeutic option under these circumstances. Burkina Faso is used as a representative example for a country in Sub-Saharan Africa with high endemicity for malaria and is here portrayed, its health system characterised and discussed under socioeconomic aspects. More than half of this country's population live in absolute poverty. The burden that malaria, especially treatment cost, poses on these people cannot be under-estimated. A retrospective study of case files from the university pediatric hospital in Burkina Faso's capital, Ouagadougou, shows that the case load is huge, and especially the specific diagnosis of severe malaria is difficult to apply in the hospital's daily routine. Treatment policy as proposed by WHO is not satisfactorily implemented neither in home treatment nor in health services, as data for pretreatment clearly show. In the face of growing resistance in malaria parasites, pharmacological combination therapies are important. Artemisinins currently are the last resort of malaria therapy. As I show with homology models, even this golden bullet is not beyond resistance development. Inconsidered mass use has rendered other drugs virtually useless before. Artemisinins should thus be protected similar to reserve antibiotics against multi-resistant bacteria. There is accumulating evidence that MB is an effective drug against malaria. Here the biological effects of both MB alone and in combination therapy is explored via modeling and experimental data. Several different lines of MB attack on Plasmodium redox defense were identified by analysis of the network effects. Next, CQ resistance based on Pfmdr1 and PfCRT transporters as well as SP resistance were modeled in silico. Further modeling shows that MB has a favorable synergism on antimalarial network effects with these commonly used antimalarial drugs, given their correct application. Also from the economic point of view MB shows great potential: in terms of production price, it can be compared to CQ, which could help to diminuish the costs of malaria treatment to affordable ranges for those most affected and struk by poverty. Malaria control is feasible, but suboptimal diagnosis and treatment are often hindering the achievment of this goal. In order to achieve malaria control, more effort has to be made to implement better adjusted and available primary treatment strategies for uncomplicated malaria that are highly standardised. Unfortunately, campaigns against malaria are chronically underfinanced. In order to maximize the effect of available funds, a cheap treatment option is most important, especially as pharmaceuticals represent the biggest single matter of expense in the fight against malaria.},
  subject      = {Malaria},
  language  = {en}
}
@phdthesis{Kuehn2013,
  author    = {K{\"u}hn, Andrea},
  title     = {The molecular interplay of proteins expressed in the sexual stages and the induction of gamete formation in the malaria parasite Plasmodium falciparum},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-98028},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2013},
  abstract  = {Transmission of the malaria parasite from man to the mosquito requires the formation of sexual parasite stages, the gametocytes. The gametocytes are the only parasite stage that is able to survive in the mosquito midgut and to undergo further development - gamete formation and fertilization. Numerous sexual stage-specific proteins have been discovered, some of which play crucial roles for parasite transmission. However, the functions of many sexual stage proteins remain elusive. Amongst the sexual stage-specific proteins are the proteins of the PfCCp proteins family, which exhibit numerous adhesion domains in their protein structures. For four members of the protein family, PfCCp1 to PfCCp4 gene-disruptant parasite lines had been already studied. Amongst these, PfCCp2 and PfCCp3 showed an important role for development of the parasites in the mosquito. In the present work the study of gene-disrupted parasites of the PfCCp Protein family was completed. PfCCp5-KO and PfFNPA-KO parasite lines were characterized to a great extent and many properties were similar to those of other PfCCp proteins. The co-dependent expression previously reported to be a phenomenon of PfCCp proteins was also observed in these two mutants, although to lesser extent. When either PfCCp5 or PfFNPA were absent, all other proteins were detected in reduced abundance only. Co-dependent expression manifests exclusively on the protein level. Transcript levels were not altered as RT-PCR showed. Amongst PfCCp proteins numerous proteinproteins interactions are taking place. The previously described multimeric protein complexes also include further sexual stage-specific proteins like Pfs230, Pfs48/45 and Pfs25. Recently, a new component of PfCCp-based multimeric protein complexes had been identified. The protein was named PfWLP1 (WD repeat protein-like protein 1) due to its possession of several WD40 repeats. In the present study expression of this uncharacterized protein was investigated via indirect IFA. It was expressed in asexual blood stages and gametocytes. Upon gamete formation and fertilization its expression ceased. Another sexual stage protein studied in this work was PfactinII. It was shown to be exclusively expressed in sexual stages. In gametocytes it co-localizes with Pfs230 and correct localization of PfactinII depends on presence of Pfs230. Transcript analysis by means of RT-PCR revealed the expression of several components of the IMC in gametocytes. Furthermore, five or six myosin genes encoded in the P. falciparum genome were detected in gametocytes. Gametocyte egress was studied on the ultrastructural level via transmission electron microscopy and an inside-out type of egress was observed. Firstly, the membrane of the parasitophorous vacuole (PVM) was lysed and only thereafter the membrane of the red blood cell (RBCM) ruptured. Furthermore, a new inductor of gametogenesis was identified: The K+/H+ ionophore nigericin induced gametocytes activation in the absence of xanthurenic acid (XA), which is responsible for gamtetocyte activation in the mosquito midgut. Selective permeabilization of RBCM and PVM by the mild detergent saponin, showed that in the absence of these membranes male gametocytes were still able to perceive both XA and the drop in temperature. Thus, the receptors for both factors signaling the parasite transmission to the mosquito, seem to be of parasitic origin. LC/MS/MS analysis confirmed the ability of RBCs to take up XA. With malaria eradication on the agenda of malaria research targeting the sexual stages becomes a crucial part of intervention strategies. The sexual stages are especially attractive target as they represent a population bottleneck. The here reported findings on P. falciparum gametocytes provide several potential candidate proteins for developing tools to interrupt transmission from man to mosquito. Such tools might include Transmission blocking vaccines and drugs.},
  subject      = {Malaria},
  language  = {en}
}
@phdthesis{Ngwa2013,
  author    = {Ngwa, Che Julius},
  title     = {The mosquito midgut-specific stages of the malaria parasite as targets for transmission blocking interventions},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-83594},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2013},
  abstract  = {Die Tropenkrankheit Malaria, wird durch eine Infektion mit einzelligen Parasiten der Gattung Plasmodium verursacht und durch den Stich der weiblichen Anopheles-M{\"u}cke von Mensch zu Mensch verbreitet. Dabei kann eine erfolgreiche {\"U}bertragung des Parasiten auf den Menschen nur dann stattfinden, wenn der Parasit seine sexuelle Entwicklungsphase im Mitteldarm der M{\"u}cke erfolgreich durchl{\"a}uft. Ziel dieser Arbeit war es daher, die Wechselwirkungen des Malariaparasiten im Mitteldarm der M{\"u}cke in Hinblick auf die Identifizierung m{\"o}glicher neuer transmissionsblockierender Strategien zu untersuchen. Der Zweck von transmissionsblockierende Strategien ist es, der Verbreitung der Malaria durch die M{\"u}cke entgegenzuwirken, indem die Entwicklung des Parasiten in der M{\"u}cke unterbunden und dadurch der Lebenszyklus des Parasiten unterbrochen wird. Der Schwerpunkt der vorliegenden Arbeit lag auf insgesamt drei Aspekten. Der erste Aspekt der Arbeit befasste sich mit der Wechselwirkung zwischen dem Para-siten und der mikrobiellen Darmflora der M{\"u}cke. Dabei sollte der m{\"o}gliche Einfluss des Parasiten auf die Darmflora untersucht werden und weiterf{\"u}hrend die potentielle Verwendung von Darmbakterien als Vehikel f{\"u}r die Herstellung paratransgener M{\"u}cken erforscht werden. Vergleichende16S-rRNA- und DGGE-Analysen an der Darmflora des asiatischen Malariavektors Anopheles stephensi zeigten eine deutliche Reduktion der mikrobiellen Diversit{\"a}t w{\"a}hrend der Entwicklung vom Ei zur adulten M{\"u}cke. Zudem konnte das gram-negative Bakterium Elizabethkingia meningoseptica, das sich stadien- und generations{\"u}bergreifend verbreitet, als dominante Darmspezies bei im Labor aufgezogenen weiblichen und m{\"a}nnlichen An. stephensi festgestellt werden. Die Dominanz von E. meningoseptica wurde zudem nicht durch die Aufnahme von infiziertem Blut oder einer ver{\"a}nderten Nahrung beeinflusst. F{\"u}r die Studien wurde sowohl der humanpathogene Parasit P. falciparum als auch der Nagermalariaerreger P. berghei verwendet. Weiterf{\"u}hrende Versuche zeigten, dass Extrakte von E. meningoseptica antibakterielle, antifungale und antiplasmodiale Aktivit{\"a}ten aufwiesen, die ein m{\"o}glicher Grund f{\"u}r die Dominanz dieser Spezies im Mitteldarm des Vektors waren. Isolate von E. meningoseptica sind im Labor kultivierbar; dadurch stellt das Bakterium einen potentiellen Kandidaten zur Generierung von paratransgenen Anopheles-M{\"u}cken dar. Ein zweites Ziel dieser Arbeit war es, m{\"o}gliche Unterschiede in der Genexpression von P. falciparum darzustellen, die in den ersten 30 Minuten nach dessen {\"U}bertragung auf die M{\"u}cke erfolgen. Dies hatte zum einen zum Zweck, die durch den Wirtswechsel hervorgerufenen Genregulationen besser zu verstehen, und bot zum anderen die M{\"o}glichkeit, neue Proteine zu identifizieren, die als potentielle transmissionsblockierende Ziele genutzt werden k{\"o}nnen. Mittels supression substractive hybridization (SSH) konnten insgesamt 126 Gene identifiziert werden, deren Expression sich w{\"a}hrend der Gametogenese ver{\"a}ndert. Die identifizierten Gene konnten einer Vielzahl von putativen Funktionen wie zum Beispiel in der Signaltransduktion (17,5\%), im Zellzyklus (14,3\%) oder im Zytoskelett (8,7\%) zugeordnet werden. Des Weiteren wurden 7,9\% der Gene eine Funktion in der Proteastase und 6,4\% in metabolischen Prozessen zugeordnet. 12,7\% der Gene kodierten f{\"u}r zelloberfl{\"a}chenassoziierte Proteine. 11,9\% der Gene hatten anderen Funktionen, w{\"a}hrend 20\% der Gene keine putative Funktion zugeordnet werden konnte. Etwa 40\% der identifizierten Genprodukte waren bisher nicht in Proteomstudien nachgewiesen worden. In weiterf{\"u}hrenden Analysen wurden 34 Gene aus jeder ontologischen Gruppe ausgew{\"a}hlt und deren Expressionsver{\"a}nderung per quantitativer real time RT-PCR im Detail untersucht. F{\"u}r 29 Gene konnte dabei eine Transkriptexpression in Gametozyten nachgewiesen werden. Zudem wiesen 20 Gene eine erh{\"o}hte Expression in Gametozyten im Vergleich asexuellen Stadien auf. Insgesamt zeigten 8 Gene besonders hohe Transkriptlevel in aktivierten Gametozyten, was auf eine Funktion dieser Proteine w{\"a}hrend der {\"U}bertragung des Parasiten auf die M{\"u}cke hindeutet und diese somit potentielle Angriffspunkte f{\"u}r transmissionsblockierende Strategien darstellen k{\"o}nnten. Im letzten Teil dieser Arbeit stand die Untersuchung verschiedener antimikrobieller Substanzen in Bezug auf ihre transmissionsblockierenden Eigenschaften im Vordergrund. Die Substanzen waren entweder direkt aus der H{\"a}molymphe verschiedener Insekten isoliert oder rekombinant in transgenem Tabak exprimiert worden. Dabei wurden die rekombinanten Peptide so ausgew{\"a}hlt, dass sie entweder gegen die Mitteldarmstadien des Parasiten wirken oder m{\"u}ckenspezifische Rezeptoren blockieren, die der Parasit f{\"u}r seine weitere Entwicklung ben{\"o}tigt. Dabei konnte gezeigt werden, dass das antimikrobielle Molek{\"u}l Harmonin, ein Abwehrmolek{\"u}l aus der H{\"a}molymphe des asiatischen Marienk{\"a}fers Harmonia axyridis, antiplasmodiale als auch transmissions-blockierende Eigenschaften besitzt. Harmonin stellt daher eine potentielle Leitstruktur f{\"u}r die Entwicklung neuer Malariawirkstoffe dar},
  subject      = {Malariam{\"u}cke},
  language  = {en}
}
@article{KuehnPradel2010,
  author    = {Kuehn, Andrea and Pradel, Gabriele},
  title     = {The Coming-Out of Malaria Gametocytes [Review Article]},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-68196},
  year      = {2010},
  abstract  = {The tropical disease malaria, which results in more than one million deaths annually, is caused by protozoan parasites of the genus Plasmodium and transmitted by blood-feeding Anopheline mosquitoes. Parasite transition from the human host to the mosquito vector is mediated by gametocytes, sexual stages that are formed in human erythrocytes, which therefore play a crucial part in the spread of the tropical disease. The uptake by the blood-feeding mosquito triggers important molecular and cellular changes in the gametocytes, thus mediating the rapid adjustment of the parasite from the warm-blooded host to the insect host and subsequently initiating reproduction. The contact with midgut factors triggers gametocyte activation and results in their egress from the enveloping erythrocyte, which then leads to gamete formation and fertilization. This review summarizes recent findings on the role of gametocytes during transmission to themosquito and particularly focuses on the molecular mechanisms underlying gametocyte activation and emergence from the host erythrocyte during gametogenesis.},
  subject      = {Malaria},
  language  = {en}
}
@phdthesis{Simon2012,
  author    = {Simon, Nina Monica},
  title     = {Molecular interactions of the malaria parasite Plasmodium falciparum during the sexual reproduction in the mosquito midgut},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72403},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {The sexual phase of Plasmodium falciparum begins with the differentiation of intraerythrocytic sexual stages, termed gametocytes, in the human host. Mature gametocytes circulate in the peripheral blood and are taken up by the mosquito during the blood meal. These stages are essential for the spread of the malaria disease and form gametes in the mosquito midgut within minutes. A highly conserved family of six secreted proteins has been identified in Plasmodium falciparum. They comprise multiple adhesive domains and are termed PfCCp1 through PfCCp5, and PfFNPA. It was revealed in this work that PfCCp multi-domain adhesion proteins form protein complexes in gametocytes and on the surface of newly emerged macrogametes by adhesion domain-mediated binding. Co-Immunoprecipitation assays with activated gametocyte lysates show interactions between PfCCp proteins and indicate surface association via Pfs230 and Pfs25. Pfs230 is connected with the plasma membrane of the parasite by its interaction partner Pfs48/45. This protein is linked to the plasma membrane by a GPI anchor and presumably retains the multi-protein complex on the surface of newly emerged macrogametes in the mosquito midgut. A WD40 domain containing protein was identified to be part of this protein complex. It might serve as platform for the assembly of the multi protein complex or mediate the interplay among proteins, as suggested from known functions of the WD40 domain repeats. During egress from the host erythrocyte, the emerging gametes become vulnerable to factors of the human complement, which is taken up with the blood meal. In this thesis it was found that the complement system is active for about one hour post feeding. Macrogametes defend against complement-mediated lysis by co-opting the human complement regulators Factor H and FHL-1 from the blood-meal. These serum proteins bind via its SCR domains 5-7 to the surface of macrogametes. Once bound, they trigger complement inactivation of the alternative pathway, which prevents induction of complement lysis on the surface of the malaria parasite. Antibodies against Factor H are able to impair the sexual development in vitro and are able to block transmission to the mosquito. Interaction studies on endogenous proteins and immobilized recombinant proteins revealed the PfGAP50 protein as binding partner of Factor H and FHL-1. This protein was hitherto described as a glideosome-associated protein in invasive parasite stages, but has not yet been characterized in gametes. First localization studies indicate a relocation of PfGAP50 from the inner membrane complex to the surface of macrogametes. Malaria still persists as one of the deadliest infectious diseases worldwide. Investigations on the essential transmissive stages, gametocytes and gametes of Plasmodium falciparum, stood in the background of research for a long time. This work deciphered details on protein interactions on the surface of the malaria parasite and provides first information about coactions between the parasite and the human complement in the mosquito midgut.},
  subject      = {Plasmodium falciparum},
  language  = {en}
}
@phdthesis{Aminake2012,
  author    = {Aminake, Makoah Nigel},
  title     = {Towards malaria combination therapy: Characterization of hybrid molecules for HIV/malaria combination therapy and of thiostrepton as a proteasome-targeting antibiotic with a dual mode of action},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-71841},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {Malaria and HIV are among the most important global health problems of our time and together are responsible for approximately 3 million deaths annually. These two diseases overlap in many regions of the world including sub-Saharan Africa, Southeast Asia and South America, leading to a higher risk of co-infection. In this study, we generated and characterized hybrid molecules to target P. falciparum and HIV simultaneously for a potential HIV/malaria combination therapy. Hybrid molecules were synthesized by covalent fusion between azidothymidine (AZT) and dihydroartemisinin (DHA), tetraoxane or chloroquine (CQ); and a small library was generated and tested for antiviral and antimalarial activity. Our data suggest that dihyate is the most potent molecule in vitro, with antiplasmodial activity comparable to that of DHA (IC50 = 26 nM, SI > 3000), a moderate activity against HIV (IC50 = 2.9 µM; SI > 35) and safe to HeLa cells at concentrations used in the assay (CC50 > 100 µM). Pharmacokinetic studies further revealed that dihyate is metabolically unstable and is cleaved following an O-dealkylation once in contact with cytochrome P450 enzymes. The later further explains the uneffectiveness of dihyate against the CQ-sensitive P. berghei N strain in mice when administered by oral route at 20 mg/kg. Here, we report on a first approach to develop antimalarial/anti-HIV hybrid molecules and future optimization efforts will aim at producing second generation hybrid molecules to improve activity against HIV as well as compound bioavailability. With the emergence of resistant parasites against all the counterpart drugs of artemisinin derivatives used in artemisinin based combination therapies (ACTs), the introduction of antibiotics in the treatment of malaria has renewed interest on the identification of antibiotics with potent antimalarial properties. In this study we also investigated the antiplasmodial potential of thiostrepton and derivatives, synthesized using combinations of tail truncation, oxidation, and addition of lipophilic thiols to the terminal dehydroamino acid. We showed that derivatives SS231 and SS234 exhibit a better antiplasmodial activity (IC50 = 1 µM SI > 59 and SI > 77 respectively) than thiostrepton (IC50 = 8.95 µM, SI = 1.7). The antiplasmodial activity of these derivatives was observed at concentrations which are not hemolytic and non-toxic to human cell lines. Thiostrepton and derivatives appeared to exhibit transmission blocking properties when administered at their IC50 or IC90 concentrations and our data also showed that they attenuate proteasome activity of Plasmodium, which resulted in an accumulation of ubiquitinated proteins after incubation with their IC80 concentrations. Our results indicate that the parasite's proteasome could be an attractive target for therapeutic intervention. In this regard, thiostrepton derivatives are promising candidates by dually acting on two independent targets, the proteasome and the apicoplast, with the capacity to eliminate both intraerythrocytic asexual and transmission stages of the parasite. To further support our findings, we evaluated the activity of a new class of antimalarial and proteasome inhibitors namely peptidyl sulfonyl fluorides on gametocyte maturation and analogues AJ34 and AJ38 were able to completely suppress gametocytogenesis at IC50 concentrations (0.23 µM and 0.17 µM respectively) suggesting a strong transmission blocking potential. The proteasome, a major proteolytic complex, responsible for the degradation and re-cycling of non-functional proteins has been studied only indirectly in P. falciparum. In addition, an apparent proteasome-like protein with similarity to bacterial ClpQ/hslV threonine-peptidases was predicted in the parasite. Antibodies were generated against the proteasome subunits alpha type 5 (α5-SU), beta type 5 (β5-SU) and pfhslV in mice and we showed that the proteasome is expressed in both sexual and asexual blood stages of P. falciparum, where they localize in the nucleus and in the cytoplasm. However, expression of PfhslV was only observed in trophozoites and shizonts. The trafficking of the studied proteasome subunits was further investigated by generating parasites expressing GFP tagged proteins. The expression of α5-SU-GFP in transgenic parasite appeared to localize abundantly in the cytoplasm of all blood stages, and no additional information was obtained from this parasite line. In conclusion, our data highlight two new tools towards combination therapy. Hybrid molecules represent promising tools for the cure of co-infected individuals, while very potent antibiotics with a wide scope of activities could be useful in ACTs by eliminating resistant parasites and limiting transmission of both, resistances and disease.},
  subject      = {Malaria},
  language  = {en}
}
@phdthesis{Agarwal2010,
  author    = {Agarwal, Shruti},
  title     = {Functional characterization of four CDK-like kinases and one Calmodulin-dependent kinase of the human malaria parasite, Plasmodium falciparum},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-48522},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2010},
  abstract  = {Malaria still persists as one of the deadliest infectious disease in addition to AIDS and tuberculosis. lt is a leading cause of high mortality and morbidity rates in the developing world despite of groundbreaking research on global eradication of the disease initiated by WHO, about half a century ago. Lack of a commercially available vaccine and rapid spread of drug resistance have hampered the attempts of extinguishing malaria, which still leads to an annual death toll of about one million people. Resistance to anti-malarial compounds thus renders search for new target proteins imperative. The kinome of the human malaria parasite Plasmodium falciparum comprises representatives of most eukaryotic protein kinase groups, including kinases which regulate proliferation and differentiation processes. Several reports till date have suggested involvement of parasite kinases in the human host and as well as in the mosquito vector. Kinases essential for life cycle stages of the parasite represent promising targets for anti-malarial compounds thus, provoking characterization of additional malarial kinases. Despite extensive research on most plasmodial enzymes, very little information is available regarding the four identified members of the cyclin dependent kinase like kinase (CLK) family. Thus, the present thesis dealt with the functional characterization of four members of the PfCLK kinase family of the parasite denoted as PfCLK-1/Lammer, PfCLK-2, PfCLK-3 and PfCLK-4 with a special focus on the first two kinases. Additionally, one Ca2+/Calmodulin dependent putative kinase-related protein, PfPKRP, presumed to be involved in sexual stage development of the parasite, was investigated for its expression in the life cycle of the parasite. In other eukaryotes, CLK kinases regulate mRNA splicing through phosphorylation of Serine/Arginine-rich proteins. Transcription analysis revealed abundance of PfCLK kinase genes throughout the asexual blood stages and in gametocytes. By reverse genetics approach it was demonstrated that all four kinases are essential for completion of the asexual replication cycle of P. falciparum. PfCLK 1/Lammer possesses two nuclear localization signals and PfCLK-2 possesses one of these signals upstream of the C-terminal catalytic domains. Protein level expression and sub-cellular localization of the two kinases was determined by generation of antiserum directed against the kinase domains of the respective kinase. Indirect immunofluorescence, Western blot and electron microscopy data confirm that the kinases are primarily localized in the parasite nucleus, and in vitro assays show that both enzymes are associated with phosphorylation activity. Finally, mass spectrometric analysis of co immunoprecipitated proteins shows interactions of the two PfCLK kinases with proteins, which have putative nuclease, phosphatase or helicase functions. PfPKRP on the other hand is predominantly expressed during gametocyte differentiation as identified from transcriptional analysis. Antiserum directed against the catalytic domain of PfPKRP detected the protein expression profile in both asexual and gametocyte parasite lysates. Via immunofluorescence assay, the kinase was localized in the parasite cytoplasm in a punctuated manner, mostly in the gametocyte stages. Reverse genetics resulted in the generation of PfPKRP gene-disruptant parasites, thus demonstrating that unlike CLK kinases, PfPKRP is dispensable for asexual parasite survival and hence might have crucial role in sexual development of the parasite. On one hand, characterization of PfCLK kinases exemplified the kinases involved in parasite replication cycle. Successful gene-disruption and protein expression of PfPKRP kinase on the other hand, demonstrated a role of the kinase in sexual stage development of the parasite. Both kinase families therefore, represent potential candidates for anti-plasmodial compounds.},
  subject      = {Plasmodium falciparum},
  language  = {en}
}