Estimates from radiocarbon-dated packrat middens indicate that the high elevation woodland communities of the Madrean Sky Islands were continuous as recently as 8,000 to 12,000 years ago. A number of population studies on a diverse collection of taxa have investigated the extent to which the Madrean Sky Island system has limited gene flow among mountain ranges. The results of several of these studies indicate that population divergences may be more ancient than the Holocene. Yarrow’s spiny lizards, Sceloporus jarrovii, were sampled from eight sites representing seven mountain ranges. The populations of S. jarrovii are host to the malarial parasite, Plasmodium chiricahuae. DNA sequences from the lizards and their malarial parasites were used to reconstruct the evolutionary relationships and estimate the ages of the populations for both host and parasite. The findings of these analyses indicate that the sky island populations of S. jarrovii have been isolated for hundreds of thousands of years and did not experience gene flow during the last woodland expansion. In contrast, the results indicate that the malarial infection occurred more recently, possibly during the Holocene woodland expansion. In addition, the prevalence of the malarial infection was compared to multiple attributes of the lizards. This analysis revealed a negative relationship between the genetic diversity of the lizard populations and the prevalence of infection. Furthermore, lizard populations with lower prevalence of infection have a lower frequency of multiclonal infections.

Malaria is a debilitating and deadly disease that afflicts over 200 million people and kills over 600 thousand each year. Due to quickly evolving drug resistance and lack of an affordable vaccine, novel interventions are needed to fight the Plasmodium parasites that cause malaria. Targeting Plasmodium inside their mosquito hosts is one approach that could complement other preventative and medicinal interventions by reducing the ability of the mosquitoes to transmit the disease to humans. The research presented here uses paratransgenesis, the genetic modification of symbiotic bacteria within the mosquito midgut, to provide antimalarial protein to the mosquito and to interfere with the life cycle of Plasmodium within the insect host. <br>This research has produced three new antimalarial paratransgenic tools. The first tool is a set of new antimalarial effector proteins that were constructed by converting anti-Plasmodium mouse antibodies into single-chain variable fragment (scFv) versions for expression by bacteria. These antibodies bind to Plasmodium surface proteins and interfere with critical steps in the parasite life cycle. The second tool is a modified bacterial species, Pantoea agglomerans , which was engineered to secrete diverse antimalarial proteins via the hemolysin secretion pathway. Modified P. agglomerans were fed to mosquitoes and were capable of inhibiting the invasion of Plasmodium within the midgut. The third tool is another modified bacterial species, Asaia sp. SF2.1. Native Type II secretion signals were discovered that enable the creation of paratransgenic strains of these bacteria. Modified strains of Asaia sp. SF2.1 were also demonstrated to interfere with the invasion of Plasmodium within the mosquito. <br>These tools have laid the groundwork for the future use of paratransgenic bacteria to combat malaria in the wild. Asaia sp. SF2.1 bacteria, in particular, are capable of spreading throughout mosquito populations, so they provide their own drive mechanism to establish themselves within the mosquito vectors of malaria. While further modifications will be required to make these bacteria ready for field use, the findings of this research provide proof of concept that the bacteria are suitable for eventual use in malaria transmission-blocking interventions. / Bayer School of Natural and Environmental Sciences; / Biological Sciences / PhD; / Dissertation;

Intermittent fevers caused by Plasmodium parasites have been known for millennia, and have caused untold human suffering. Today, millions of people are afflicted by malaria each year, and hundreds of thousands die. Historically, the most successful synthetic antimalarial drug was chloroquine, as it was safe, inexpensive, and highly efficacious. However, plasmodial resistance to chloroquine now greatly limits its utility. Previously in our laboratories it has been shown that attachment of a "reversal agent moiety" to the side chain of chloroquine can result in the restoration of activity against chloroquine-resistant strains of P. falciparum malaria. In the first part of the work presented here, a study has been made of the importance of the quinoline ring substitution pattern to the activity of such reversed chloroquines. The compounds presented here include those bearing a substituent in the 2-, 5, 6-, 7-, and/or 8- position, and include those with chloro, bromo, iodo, fluoro, nitro, trifluoromethyl, methyl, and methoxy substituents. For reversed chloroquines, 2-, 5-, and 8- substituents have been found to decrease in vitro antiplasmodial activity against P. falciparum relative to 7-chloro substitution, whereas 6- and 7- substituted compounds with various substituents have in many cases similar activity to that of 7-chloro substituted compounds. Little difference has been observed between 6- and 7- substitution, or between chlorine and a methyl group in position 6. In most cases these effects on activity are directionally similar to those observed for chloroquine analogs without an attached reversal agent, but the magnitude of the effect is generally smaller, suggesting that the activities of reversed chloroquines are less affected by modifications to the quinoline ring system than is true for chloroquine analogs without an attached reversal agent. The second portion of this work presents an asymmetrical bis-quinoline (PL241) that is highly active against P. falciparum malaria, with an IC50 of less than 0.1 nM for all strains tested. Mechanistic studies have been performed in which the substitution patterns of the two quinoline rings of PL241 are modified in ways that indicate that either ring system is equally capable of participating in the antimalarial activity of these compounds. The excellent in vitro antiplasmodial activity of PL241 makes this a compound of great interest for further development as a potential antimalarial drug. In the third part of this work, a survey has been made of antimalarial treatments recommended in the European medical literature from the time of Pliny the Elder (active in the first century A.D.) through the advent of modern malaria chemotherapy in the early twentieth century. In the fifteen primary sources utilized in this study, 251 distinct substances - primarily plants - were identified as having likely been used in the treatment of malaria. Of the 38 substances that were described in three or more sources, at least fifteen have been examined by other workers for antiplasmodial activity; in many cases, they were found to have antiplasmodial activity in vitro or in vivo. However, the majority of the phytotherapies for malaria identified in this project have not yet been tested against Plasmodium species, and may provide valuable leads in the search for new compounds active against drug-resistant malaria.

Malaria is a disease of global importance, causing hundreds of thousand of deaths a year. The majority or deaths are caused by Plasmodium falciparum, a parasite transmitted by the mosquito Anopheles. Its pathogenicity largely results from an ability to transform infected erythrocytes by creating knob-like structures that result in endothelial adhesion. Two major components of these knob structures have been identified as P. falciparum erythrocyte membrane protein 1 (PfEMP1) and knob-associated histidine rich protein (KAHRP). The extracellular fragment of PfEMP1 is responsible for antigenic variability and cytoadherence while its intracellular domain (ATS) connects to the cytoskeleton via interactions with other plasmodium-encoded proteins. In addition, perforin-like proteins (PLPs) with a MACPF domain have been identified in the genome of Plasmodium. PLPs are highly conserved and are expressed in various life-cycle stages of the parasite. They are believed to form pores in membranes of the host cell but their structure is yet unknown. The aim of the work in this thesis was to obtain new information about the structure and role of malaria proteins, thus giving a better understanding of the disease and its possible treatment. Studies of numerous designed constructs of the ATS family were carried out using biophysical methods including high resolution NMR and CD. These revealed that ATS domains are mainly unstructured with a relatively small folded core, consisting of a bundle of α-helices. Surprisingly, no evidence could be found for ATS binding to KAHRP in solution conditions although previous pull-down data had indicated an interaction. Bioinformatics analysis and yeast-two-hybrid data suggested, however, that there is a conserved protein interaction epitope on the central flexible part of ATS. It was shown, using fluorescence anisotropy measurements, that this part of ATS associates with a parasite protein containing a PHIST (Plasmodium helical interspersed sub-telomeric) domain. Expression constructs of the PLP protein family were designed and manufactured, with the aim of enabling structural studies of this putative pore protein.

The virulence of Plasmodium falciparum is largely attributed to the ability of asexual blood-stage parasites to cytoadhere to the microvascular endothelium of the human host. This pathogenic behavior is mediated by the primary parasite virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1), an understanding of which is crucial to develop interventions to ameliorate the morbidity and mortality of P. falciparum malaria. The work presented in this thesis describes the application of a phenotype-to-genotype experimental approach to identify novel parasite proteins involved in the trafficking and display of PfEMP1. Guided by the overall hypothesis that the in vitro culture-adapted parasite line 3D7 harbors 1 or more genetic determinants of impaired PfEMP1 trafficking, surface PfEMP1 levels were first measured in 3D7, the presumably trafficking-competent parasite line HB3, and 16 unique progeny from an HB3 x 3D7 genetic cross (chapter 2). These phenotypes were then combined with genome-wide SNP data in QTL analysis to identify genetic polymorphisms potentially responsible for the impaired trafficking in 3D7 (chapter 3). A near-significant QTL containing a single protein-coding gene, the putative kinesin Pf3D7_1245600, was identified, characterized, and investigated in CRISPR-Cas9-driven allele-exchange parasite transfection experiments to establish a causal link between the gene and PfEMP1 trafficking (chapter 4). The parasite transfections were unsuccessful, but the potential role of Pf3D7_1245600 in PfEMP1 trafficking was indirectly assessed through the disruption of microtubules with colchicine (chapter 4), which significantly impacted the surface PfEMP1 levels of HB3 but not 3D7. The findings of this thesis suggest that kinesins and microtubules may play previously unconsidered roles in the regulation, production, or trafficking of PfEMP1.

El objetivo de esta investigación retrospectiva, fue determinar la asociación existente entre mutaciones puntuales en los genes dihidrofolato reductasa (Pfdhfr) y dihidropteroato sintasa (Pfdhps) de Plasmodium falciparum y la respuesta clínica en pacientes con diagnóstico de malaria no complicada causada por P. falciparum, admitidos en un estudio de eficacia in vivo de sulfadoxina-pirimetamina (SP) llevado a cabo en dos áreas de la región Loreto en 1999. Se tomaron muestras de sangre de 86 pacientes antes de administrarles SP, las que se analizaron usando PCR-anclado específico de alelo para estudiar a los codones S108N/T, N51I, C59R, I164L y C50R del gen Pfdhf y los codones A436G, A437G, K540E, A581G y A613S/T del gen Pfdhps, encontrándose que las infecciones causadas por parásitos con 3 mutaciones en Pfdhfr (108Asn/51Ile/164Leu) y 2 (581Gli/437Gli) ó 3 mutaciones en Pfdhps (581Gli/437Gli/540Glu) denominados el quíntuple mutante y el séxtuple mutante, respectivamente, se encontraban asociadas con la falla del tratamiento con SP. Además, se estableció que cuanto más alto era el número de mutaciones tanto en Pfdhfr como en Pfdhps, más alto era el riesgo de fallar al tratamiento con SP, según resultado del análisis de regresión logística empleado para asociar a estas dos variables. Este estudio contribuye en brindar evidencias científicas de la asociación existente entre las variables en la región Loreto contribuyendo a su validación y su futuro uso para estudios de vigilancia de fármaco resistencia a SP no solo en el Perú sino en general para los países de América del Sur que comparten territorio de la selva Amazónica. / -- The objective of this retrospective study was to determine the asociation between point mutations in dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) Plasmodium falciparum genes, and clinical outcome of patients with non complicated P.falciparum malaria diagnosis, admitted to a in vivo sulphadoxine-pirymethamine (SP) drug efficacy study conducted in 1999, in two areas of Loreto region. We used allelic specific-nested PCR to analize 86 blood samples collected before SP treatment, and study mutations at codons S108N/T, N51I, C59R, I164L and C50R in Pfdhfr and codons A436G, A437G, K540E, A581G y A613S/T in Pfdhps genes, and found that Infections caused by parasites harbouring 3 mutations in Pfdhfr (108Asn/51Ile/164Leu) and either 2 or 3 mutations in Pfdhps (581Gly/437Gly and 540Glu) called the quintuple and sextuple mutants, respectively, were associated to failure with SP treatment. Logistic regression analysis was used to look for association between the variables, helping to establish the higher the number of mutations in both Pfdhfr and Pfdhps genes, the higher the risk of treatment failures when using SP.The contribution of this study is to provide scientific evidences of the association between both variables in Loreto region supporting its validation and future application in surveillance studies for SP drug resistance, that can be conducted not only in Peru but also in South American countries that share the Amazon basin territory.

Long-distance migratory birds can encounter a wide range of parasites. Various populations of birds within one species use different migration routes and can also winter in different places. It can be supposed that birds which use different migration routes should be infected with different parasites. To study the relationship between the migration and the distribution of parasites we chose the worldwide species barn swallow (Hirundo rustica) and the avian malaria parasites. Swallows migrate long distances in different migrating routes. Some populations of swallows do not migrate, they are resident. This is the reason why swallow is a good model species for finding the answers to questions whether the populations using different migration routes are infected with different parasites or not and whether or not the diversity of parasites is wider in populations which migrate long distances in comparison with the resident populations. The malaria lineages of the genus Plasmodium and Haemoproteus were detected using nested PCR and sequencing. 1242 samples from 8 different localities from the USA, Europe and Asia were tested. We detected 24 different malaria lineages. Within the genus Plasmodium 4 of 16 lines were detected for the first time and in the genus Haemoproteus 3 of 6 lines were detected for the first...

Protozoan parasites of the genus Plasmodium are the causative agent of malaria. The most severe form of human malaria is caused by P. falciparum, responsible for approximately three quarters of a million deaths each year. One major problem in the treatment of malaria is resistance to existing chemotherapies. Consequently, there is an urgent need to identify and validate novel drug targets. A possible recently identified drug target is the PfNitA protein of P. falciparum which contains orthologues in other Plasmodium species but is absent from humans. The gene is annotated as a putative formate-nitrite transporter and orthologues are found in a range of prokaryotes as well as the lower eukaryotes algae and fungi. To determine the biological function of the protein, pfnita was expressed in Escherichia coli strains lacking the endogenous formate and nitrite transporters. In order to analyse the essentiality of the gene a reverse genetics approach was taken and the data discussed. Results indicate that the PfNitA protein is located in the plasma membrane and digestive vacuole of intraerythrocytic parasites suggesting a role in the uptake or excretion of metabolites. A second complexity with regard to treatment is the lack of a vaccine. A problem in crating a vaccine is antigenic variation. The PIR family of proteins contain a so-called hypervariable domain that has led to the suggestion that the family may play a role in antigenic variation. The objective of the work carried out in this thesis was to investigate the topology and structure of the PcCir2 protein of Plasmodium chabaudi, using E. coli as the expression host. The topology of Cir2 has been examined by means of reporter fusions and overexpression/purification studies undertaken towards crystallisation. As the PcCir2 amino acid sequence does not show significant homology to other proteins, structural data may provide insights into potential functional or binding domains.

In Säugetieren existieren im wesentlichen zwei Abwehrsysteme gegen oxidativen Streß, in welchen die Glutathionreduktase (GR) und Thioredoxinreduktase (TrxR) Schlüsselenzyme sind. Ein einzelnes Gen der Taufliege, genannt dmtrxr-1, kodiert sowohl für die durch alternatives Splicing entstehende cytoplasmatische und mitochondriale Form der DmTrxR-1. Zum Teil innerhalb des dmtrxr-1-Gens findet sich auf dem Komplementärstrang ein weiteres Gen, welches sniffer genannt wurde. In Kooperation wurde nachgewiesen, daß dieses Gen essentiell zur Verhinderung alterungsbedingter Neurodegeneration ist. Durch biochemische Charakterisierung konnte das rekombinant hergestellte Produkt dieses Gens in der vorliegenden Arbeit als Carbonylreduktase, ein zu den Kurzketten-Dehydrogenasen (short-chain dehydrogenases) gehörendes Enzym, identifiziert werden. Sniffer weist das für Carbonylreduktasen typische Substratspektrum mit Phenanthrenequinone als bestem Substrat auf und wird von Flavonoiden wie Quercetin und Rutin sowie Hydroxymercuribenzoat gehemmt. In verschiedenen Ansätzen konnten Kristalle des rekombinanten Proteins gewonnen werden, die inzwischen in Kooperation vermessen wurden und so zu einer Kristallstruktur mit einer Auflösung von 1,7 Angström führten. Durch diese Arbeiten konnte zum ersten Mal eine Verbindung zwischen einem charakterisierten Gen (snifffer), oxidativem Streß und neurodegenerativen Effekten auf molekularer Ebene nachgewiesen werden. Parasiten haben während ihres Lebenszyklus einen hohen Bedarf an Energie und sind abhängig von einer starken Syntheseleistung. Zur Bewältigung dieses Stresses benötigen sie hohe Aktivitäten an Adenylatkinase (AK; ATP + AMP  2 ADP) und GTP-AMP-Phosphotransferase (GAK; GTP + AMP  GDP + ADP). Beide Enzyme wurden in Blutstadien des Malariaparasiten Plasmodium falciparum identifiziert und die entsprechenden Gene der PfAK und PfGAK auf den Chromosomen 10 und 4 respektive lokalisiert. Klonierung und heterologe Expression in E. coli ergab enzymatisch aktive Proteine mit einer Größe von 28,9 (PfAK), bzw. 28,0 kDa (PfGAK). Das rekombinante Protein der PfAK entspricht in seinen biochemischen Charakteristika denen der authentischen PfAK. Dies gilt auch für eine mögliche Assoziation mit einem stabilisierenden Protein mit einem Molekulargewicht von ca. 70 kDa und der hohen Substratspezifität für das Monophosphat-Nukleotid AMP. Die Spezifität für das Triphosphat-Substrat ist weniger stringent. Das beste Triphosphat-Substrat ist ATP mit einem Vmax-Wert von 75 U/mg und einem kcat von 2800 min-1. Die Sequenz der PfAK enthält eine amphiphatische Helix, welche als notwendig für die Translokation zytosolischer Adenylatkinasen in den Intermembranraum der Mitochondrien beschrieben wurde. Die PfGAK bevorzugt GTP und AMP als Substrat (100 U/mg; kcat = 2800 min-1 bei 25°C) und zeigt als Besonderheit keine messbare Aktivität mit ATP. Im Gegensatz zu ihrem Ortholog im Menschen (AK3) enthält die Sequenz der PfGAK ein Zinkfinger-Motiv und bindet Eisenionen. Erste Immunfluoreszenz-Analysen lokalisieren die PfGAK in den Mitochondrien. PfAK und PfGAK werden von den Dinukleosid-Pentaphosphat-Verbindungen AP5A beziehungsweise GP5A gehemmt. Die Ki-Werte liegen mit ca. 0.2 µM ungefähr 250-fach niedriger als die KM-Werte der entsprechenden Nukleotidsubstrate. Zur Lösung der vor allem im Rahmen einer rationalen Medikamentenentwicklung notwendigen Kristallstruktur des Zielmoleküls konnten bereits Kristalle der PfGAK erhalten werden. / In mammalia, two major systems with glutathione reductase (GR) and thioredoxin reductase (TrxR) as key enzymes defend the organism against oxidative stress. The single copy gene dmtrxr-1 codes for both the cytoplasmic and mitochondrial form of DmTrxR-1, generated by alternative splicing. Another gene, located on the complementary strand partially within the dmtrxr-1 gene, could be identified and was named sniffer. This gene is essential for prevention of age-related neuro-degeneration, as could be shown in a cooperation with the group of Prof. Schneuwly. In this thesis, biochemical characterization of the recombinant protein identified sniffer as a carbonyl reductase, an enzyme belonging to the short-chain-dehydrogenases. Sniffer shows the typical substrate spectrum of carbonyl reductases with phenthrenequinone as best substrate and is inhibited by the flavonoids quercetin and rutin and also by hydroxymercurybenzoate (HMB). Protein crystals could be obtained under different conditions. In a cooperation with the group of Prof. Klebe, these already lead to a crystal structure with a resolution of 1.7 angstrom. The work on sniffer is the first that directly links a characterized gene (sniffer), oxidative stress and neurodegeneration on the molecular level. For coping with energetic and synthetic challenges, parasites require high activities of adenylate kinase (AK; ATP + AMP  2 ADP) and GTP:AMP phosphotransferase (GAK; GTP + AMP  2 ADP). These enzymes were identified in bloodstream stages of Plasmodium falciparum. The genes encoding PfAK and PfGAK are located on chromosomes 10 and 4, respectively. Molecular cloning and heterologous expression in E. coli yielded enzymatically active proteins of 28.9 (PfAK) and 28.0 kDa (PfGAK). Recombinant PfAK resembles authentic PfAK in its biochemical characteristics including the possible association with a stabilizing protein and the high specificity for AMP as the mononucleotide substrate. Specificity is less stringent for the triphosphate, with ATP as the best substrate (75 U/mg; kcat = 2160 min-1). PfAK contains the sequence of the amphiphatic helix that is known to mediate translocation of the cytosolic protein into the mitochondrial intermembrane space. PfGAK exhibits substrate preference for GTP and AMP (100 U/mg; kcat = 2800 min-1); notably, there is no detectable activity with ATP. In contrast to its human orthologue (AK3), PfGAK contains a zinc finger motif and binds ionic iron. The dinucleoside pentaphosphate compounds AP5A and GP5A inhibited PfAK and PfGAK, respectively, with Ki values of appr. 0.2 µM which is more than 250-fold lower than the KM values determined for the nucleotide substrates. The disubstrate inhibitors are useful for studying the enzymatic mechanism of PfAK and PfGAK as well as their function in adenine nucleotide homeostasis; in addition, the chimeric inhibitors represent interesting lead compounds for developing nucleosides to be used as antiparasitic agents. To elucidate the structure which is necessary for the use as a drug target, crystallization studies have been performed and the first crystals could be obtained.

Diese Arbeit untersucht zelluläre Netzwerke mit dem Ziel, die so gewonnenen Einsichten medizinisch beziehungsweise biotechnologisch zu nutzen. Hierzu müssen zunächst Proteindomänen und wichtige regulatorische RNA Elemente erkannt werden. Dies geschieht für regulatorische Elemente in Nukleinsäuren am Beispiel von Iron Responsive Elements (IREs) in Staphylococcus aureus, wobei sich solche Elemente in viel versprechender Nähe zu exprimierten Sequenzen finden lassen (T. Dandekar, F. Du, H. Bertram (2001) Nonlinear Analysis 47(1): 225-34). Noch bedeutsamer als Ziele zur Medikamentenentwicklung gegen Parasiten sind Domänenunterschiede in Struktur und Sequenz bei Proteinen (T. Dandekar, F. Du, H. Bertram (2001) Nonlinear Analysis 47(1): 225-34). Ihre Identifikation wird am Beispiel eines potentiellen Transportproteins in Plasmodium falciparum exemplarisch dargestellt. Anschließend wird das Zusammenwirken von regulatorischen Elementen und Domänen in Netzwerken betrachtet (einschließlich experimenteller Daten). Dies kann einerseits zu allgemeineren Schlussfolgerungen über das Netzwerkverhalten führen, andererseits für konkrete Anwendungen genutzt werden. Als Beispiel wählten wir hier Redoxnetzwerke und die Bekämpfung von Plasmodien als Verursacher der Malaria. Da das gesamte Redoxnetzwerk einer lebenden Zelle mit Methoden der pH Wert Messung nur unzureichend zu erfassen ist, werden als alternative Messmethode für dieses Netzwerk Mikrokristalle der Glutathionreduktase als Indikatorsystem nach digitaler Verstärkung experimentell genutzt (H. Bertram, M. A. Keese, C. Boulin, R. H. Schirmer, R. Pepperkok, T. Dandekar (2002) Chemical Nanotechnology Talks III - Nano for Life Sciences). Um komplexe Redoxnetzwerke auch bioinformatisch zu modulieren, werden Verfahren der metabolischen Fluxanalyse vorgestellt und verbessert, um insbesondere ihrer Verzahnung besser gerecht zu werden und solche Netzwerke mit möglichst wenig elementaren Flussmoden zutreffend beschreiben zu können. Die Reduktion der Anzahl von Elementarmoden bei sehr großen metabolischen Netzwerken einer Zelle gelingt hier mit Hilfe unterschiedlicher Methoden und führt zu einer vereinfachten Darstellungsmöglichkeit komplexer Stoffwechselwege von Metaboliten. Dabei dient bei jeder dieser Methoden die biochemisch sinnvolle Definition von externen Metaboliten als Grundlage (T. Dandekar, F. Moldenhauer, S. Bulik, H. Bertram, S. Schuster (2003) Biosystems 70(3): 255-70). Allgemeiner werden Verfahren der Proteindomänenklassifikation sowie neue Strategien gegen mikrobielle Erreger betrachtet. In Bezug auf automatisierte Einteilung von Proteinen in Domänen wird ein neues System von Taylor (2002b) mit bekannten Systemen verglichen, die in unterschiedlichem Umfang menschlichen Eingriffs bedürfen (H. Bertram, T. Dandekar (2002) Chemtracts 15: 735-9). Außerdem wurde neben einer Arbeit über die verschiedenen Methoden aus den Daten eines Genoms Informationen über das metabolische Netzwerk der Zelle zu erlangen (H. Bertram, T. Dandekar (2004) it 46(1): 5-11) auch eine Übersicht über die Schwerpunkte der Bioinformatik in Würzburg zusammengestellt (H. Bertram, S. Balthasar, T. Dandekar (2003) Bioforum 1-2: 26-7). Schließlich wird beschrieben, wie die Pathogenomik und Virulenz von Bakterien der bioinformatischen Analyse zugänglich gemacht werden können (H. Bertram, S. Balthasar, T. Dandekar (2003) Bioforum Eur. 3: 157-9). Im letzten Teil wird die metabolische Fluxanalyse zur Identifikation neuer Strategien zur Bekämpfung von Plasmodien dargestellt: Beim Vergleich der Stoffwechselwege mit Glutathion und Thioredoxin in Plasmodium falciparum, Anopheles und Mensch geht es darum, gezielte Störungen im Stoffwechsel des Malariaerregers auszulösen und dabei den Wirt zu schonen. Es ergeben sich einige interessante Ansatzpunkte, deren medizinische Nutzung experimentell angestrebt werden kann. / The objective of this thesis is to obtain information, which may be advantageous for biotechnical and medical purposes. In order to achieve this aim it is first necessary to identify protein domains and essential regulatory RNA elements. In case of regulatory RNA elements this is accomplished by investigating Iron Responsive Elements (IREs) in Staphylocuccus aureus as a model. In this case these elements are found in much promising vicinity to open reading frames coding for proteins (T. Dandekar, F. Du, H. Bertram (2001) Nonlinear Analysis 47(1): 225-34). Even more significant for the purpose of developing pharmaceuticals against parasites are differences of structure and sequence in protein domains (T. Dandekar, F. Du, H. Bertram (2001) Nonlinear Analysis 47(1): 225-34). Their identification is shown in a potential transport protein in Plasmodium falciparum. Subsequently the interaction of regulatory elements and domains in networks is considered (including experimental data). The resulting observations may lead to general conclusions concerning network reaction, as well as specific applications. Our example and field of interest are redox networks and Plasmodia causing malaria. It is not possible to cover the redox network state of a living cell using only pH measurements. Therefore small crystals of glutathione reductase are employed as a more suitable indicator, whose signal is digitally amplified (H. Bertram, M. A. Keese, C. Boulin, R. H. Schirmer, R. Pepperkok, T. Dandekar (2002) Chemical Nanotechnology Talks III - Nano for Life Sciences). In order to bioinformatically modulate complex redox networks techniques of metabolic flux analysis are presented. They are also improved particularly to advance the understanding of interdependences and to facilitate the correct comprehension of such networks with as few elementary flux modes as possible. In this thesis the reduction of the number of elementary modes of large and intertwined metabolic networks succeeds with various methods. This leads to a simpler model of complex metabolic functions. For each of the methods used in this process the biochemically justified definition of external and internal metabolites constitutes the basis (T. Dandekar, F. Moldenhauer, S. Bulik, H. Bertram, S. Schuster (2003) Biosystems 70(3): 255-70). In a more general sense methods of protein domain classification and new strategies for the control of microbial pathogens are considered. In reference to automated classification of protein domains a new system by Taylor (2002b) is compared with traditional systems, which require a varying degree of human intervention (H. Bertram, T. Dandekar (2002) Chemtracts 15: 735-9). In addition different methods of acquiring information on the cellular metabolic network from genomic data is discussed (H. Bertram, T. Dandekar (2004) it 46(1): 5-11). Furthermore a survey of the main fields of bioinformatic research in Würzburg is given (H. Bertram, S. Balthasar, T. Dandekar (2003) Bioforum 1-2: 26-7). Finally it is outlined how pathogenicity and virulence of bacteria may be made accessible to bioinformatic analysis (H. Bertram, S. Balthasar, T. Dandekar (2003) Bioforum Eur. 3: 157-9). In the conclusion metabolic flux analysis is used for the identification of new strategies in the battle against Plasmodia: The comparison of metabolic pathways with glutathione and thioredoxin in Plasmodium falciparum, Anopheles and man aims at raising planned dysfunctions in the metabolism of Plasmodium or Anopheles without harming the human host. Valuable suggestions for medical applications and pharmacological targets are obtained.