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Plasmodium falciparum and Babesia divergens are parasites of the same phylogeny apicomplexa and they invade and replicate in the human erythrocytes. Both the parasites differently modify the host cell membrane during and after infection, providing an excellent example of parallel but distinct adaptations of the parasites to survive in the RBC. During invasion of the host erythrocyte, the parasite targets specific entry sites on the RBCs membrane, which is also the initial step in the biogenesis of the parasitophorous vacuole (PV). The parasitophorous vacuolar membrane (PVM) surrounds the parasite and thus P. falciparum remains surrounded by the vacuolar membrane for most of the parasite’s development. On the other hand in B. divergens, vacuolar membrane fate is unclear, owing to the lack of suitable markers. The electron microscopic studies suggest the disintegration of the PVM in the later stages of parasite maturation. As the differentiated erythrocyte does not endocytose or phagocytose, events leading to the formation of the PVM, in particular the contribution of the erythrocyte membrane (RBCM) are unknown. In order to understand better the internalization of RBCM proteins by parasites, we wanted to determine whether the lipid rafts, which are concentrated patches of lipids and proteins on the erythrocyte membrane, play any such role as specific entry site´ in the biogenesis of PV and also determine whether both parasites respectively internalize the same type of lipids/proteins or not. Recent work has shown that there is no internalization of major RBCM proteins, but the proteins which were found internalized are comparatively present in low abundance in the RBCM and are associated with the lipid rafts. In this work I used B. divergens and also P. falciparum infected erythrocytes to do a comparative investigation of the host cell lipids/proteins, whether they are included or excluded in PV. As a result (i) we found that both parasites recruit and exclude the same set of proteins. (ii) we used cholera toxin B subunit which binds to the ganglioside GM1, to follow the fate of this lipid raft associated glycophingolipid during PVM formation and disintegration. (iii) we observed that GM1 remains in the vicinity of P. falciparum during maturation but disappears from the vicinity of B. divergens as the parasite matures, consistent with a disintegration of the PVM. In conclusion, it appears that there is similarity between the early events in the PVM formation in both the parasites. All the biochemical pathways are dependent on the balance of the transport mechanisms and counteractions like the import of extracellular metabolites, export of the intracellular metabolites and proper waste disposal of unused metabolites. Thus the second part of my work was focused on the increased permeability of the host cell membrane after infection and appearance of new permeability pathways (NPP) in the infected erythrocyte. The erythrocyte which is save heaven´ for the parasite have limited resources and metabolism, that the parasite can use. The intracellular parasite is far more active inside the erythrocyte than outside cell. After invasion the small metabolically active merozoite stage of each genus (Plasmodium and Babesia) grows rapidly into other developmental stages (ring form and trophozoite form) inside the erythrocyte and thereby synthesizing DNA, RNA, proteins and lipids in large amounts. In order to fulfil all the essential requirements, the growing parasite induces changes in the membrane permeability of the erythrocyte and takes up solutes from the extracellular media via NPP. One among the other important amino acids required by the parasite is L-glutamate. The main aim of my work was to observe and characterize the transport of L- glutamate into B. divergens infected erythrocytes, and compare it with P. falciparum infected erythrocytes. As it is known that P. falciparum infected erythrocytes have both low- and high-affinity (EAAT3) glutamate transporter activated. The transport system present in B. divergens infected erythrocytes is unclear, therefore it is interesting to know the details of transport mechanism and see if it is parasite specific mechanism. Like P. falciparum, after infection of erythrocytes, B. divergens also induces the uptake of several solutes which are not taken up by non-infected erythrocytes. We found that B. divergens infected erythrocytes show uptake of glutamate via a low-affinity transport system. The other important characteristics of the glutamate transport observed was Na+-independency, non-saturability and non-stereo selectivity. There was also enhanced uptake of glutamate in the presence of choline. In conclusion, it appears that both parasites induce different mechanisms or transport systems for glutamate uptake and also the activation of the transport system is parasite specific., Plasmodium falciparum und Babesia divergens sind Parasiten der selben phylogenetischen Apicomplexa, die Erythrozyten befallen und sich replizieren. Beide Parasiten modifizieren die Zellmembran während und nach der Infektion und geben so ein hervorragendes Beispiel für parallele aber unterschiedliche Adaption von Parasiten um im RBC zu überleben. In dieser Arbeit werden Aufnahme und Transport verschiedener Lösungen durch die veränderte Zellmembran untersucht.

Vesicles are extensively used as a model for understanding dynamics and deformation of red blood cells at the individual level but also regarding collective phenomena and rheology. Vesicles’ membranes withstand to bending but do not have a shear resistance, unlike red blood cells, but they still share several dynamical properties with red blood cells, like tank-treading and tumbling under linear shear flow, or parachute and slipper shapes under Poiseuille flow. The red blood cells are known to form train of cells in the microcirculation attributed to attractive hydrodynamic interactions. We investigate numerically several kind of problems such as: (i) the dynamics of isolated cells; (ii) the hydrodynamic coupling between the red blood cells (by using vesicles as a model) subject to a Poiseuille flow under different confinements; (iii) the aggregation of red blood cells and formation of rouleaux; and (iv) the contribution of macromolecules in the formation of clusters under flow condition. The obtained results give a new insight into the physics of deformable objects under confinement that are transposable to the flow of red blood cells in the microcirculation. Vesikel werden in der aktuellen Forschung extensiv als Modelsystem genutzt um die Dynamik und die Deformation von roten Blutzellen (RBCs) zu verstehen. Dies sowohl auf dem individuellen Level wie auch für kollektive Phänomene und die Rheologie. Die Membran von Vesikel ist nicht dehnbar jedoch verformbar und eine Scherung erfolgt widerstandslos. Rote Blutzellen haben vergleichbare Eigenschaften, auch wenn eine Scherung nicht widerstandslos erfolgt. So wird unter einem linearen Scherfluss das tank-treading (vergleichbar mit der Bewegung einer Panzerkette) oder eine Taumelbewegung beobachtet. Im Poiseuille-Fluss werden hingegen parachute (fallschirmförmig) oder slipper (schuhförmig) Konfigurationen angenommen. Es ist bekannt, dass rote Blutzellen in der Mikrozirkulation zu Aneinanderreihungen neigen. Dies geschieht aufgrund der anziehenden hydrodynamischen Wechselwirkung. Es wurden verschiedene Problemstellungen untersucht: (i) die Dynamik einzelner Zellen, (ii) die hydrodynamische Kopplung zwischen roten Blutzellen (als Vesikel im Modellsystem) in einem Poiseuille-Fluss bei verschiedenen geometrischen Einschränkungen, (iii) die Aggregation und die Bildung von Rollen bei roten Blutzellen und (iv) der Beitrag von Makromolekülen an der Clusterbildung unter Flussbedingungen. Die dargestellten Untersuchungen geben neuartige Einblicke in die Physik von verformbaren Objekten in eingeschränkten Geometrien welche bedeutend sind für das Verständnis der Bewegung von roten Blutzellen in der Mikrozirkulation.

Plasmodium falciparum and Babesia divergens are parasites of the same phylogeny apicomplexa and they invade and replicate in the human erythrocytes. Both the parasites differently modify the host cell membrane during and after infection, providing an excellent example of parallel but distinct adaptations of the parasites to survive in the RBC. During invasion of the host erythrocyte, the parasite targets specific entry sites on the RBCs membrane, which is also the initial step in the biogenesis of the parasitophorous vacuole (PV). The parasitophorous vacuolar membrane (PVM) surrounds the parasite and thus P. falciparum remains surrounded by the vacuolar membrane for most of the parasite’s development. On the other hand in B. divergens, vacuolar membrane fate is unclear, owing to the lack of suitable markers. The electron microscopic studies suggest the disintegration of the PVM in the later stages of parasite maturation. As the differentiated erythrocyte does not endocytose or phagocytose, events leading to the formation of the PVM, in particular the contribution of the erythrocyte membrane (RBCM) are unknown. In order to understand better the internalization of RBCM proteins by parasites, we wanted to determine whether the lipid rafts, which are concentrated patches of lipids and proteins on the erythrocyte membrane, play any such role as specific entry site´ in the biogenesis of PV and also determine whether both parasites respectively internalize the same type of lipids/proteins or not. Recent work has shown that there is no internalization of major RBCM proteins, but the proteins which were found internalized are comparatively present in low abundance in the RBCM and are associated with the lipid rafts. In this work I used B. divergens and also P. falciparum infected erythrocytes to do a comparative investigation of the host cell lipids/proteins, whether they are included or excluded in PV. As a result (i) we found that both parasites recruit and exclude the same set of proteins. (ii) we used cholera toxin B subunit which binds to the ganglioside GM1, to follow the fate of this lipid raft associated glycophingolipid during PVM formation and disintegration. (iii) we observed that GM1 remains in the vicinity of P. falciparum during maturation but disappears from the vicinity of B. divergens as the parasite matures, consistent with a disintegration of the PVM. In conclusion, it appears that there is similarity between the early events in the PVM formation in both the parasites. All the biochemical pathways are dependent on the balance of the transport mechanisms and counteractions like the import of extracellular metabolites, export of the intracellular metabolites and proper waste disposal of unused metabolites. Thus the second part of my work was focused on the increased permeability of the host cell membrane after infection and appearance of new permeability pathways (NPP) in the infected erythrocyte. The erythrocyte which is save heaven´ for the parasite have limited resources and metabolism, that the parasite can use. The intracellular parasite is far more active inside the erythrocyte than outside cell. After invasion the small metabolically active merozoite stage of each genus (Plasmodium and Babesia) grows rapidly into other developmental stages (ring form and trophozoite form) inside the erythrocyte and thereby synthesizing DNA, RNA, proteins and lipids in large amounts. In order to fulfil all the essential requirements, the growing parasite induces changes in the membrane permeability of the erythrocyte and takes up solutes from the extracellular media via NPP. One among the other important amino acids required by the parasite is L-glutamate. The main aim of my work was to observe and characterize the transport of L- glutamate into B. divergens infected erythrocytes, and compare it with P. falciparum infected erythrocytes. As it is known that P. falciparum infected erythrocytes have both low- and high-affinity (EAAT3) glutamate transporter activated. The transport system present in B. divergens infected erythrocytes is unclear, therefore it is interesting to know the details of transport mechanism and see if it is parasite specific mechanism. Like P. falciparum, after infection of erythrocytes, B. divergens also induces the uptake of several solutes which are not taken up by non-infected erythrocytes. We found that B. divergens infected erythrocytes show uptake of glutamate via a low-affinity transport system. The other important characteristics of the glutamate transport observed was Na+-independency, non-saturability and non-stereo selectivity. There was also enhanced uptake of glutamate in the presence of choline. In conclusion, it appears that both parasites induce different mechanisms or transport systems for glutamate uptake and also the activation of the transport system is parasite specific.

In this work, the rheological properties of human blood are investigated by two different approaches. The flow properties of plasma, the liquid component of blood, is analyzed under three different conditions: shear flow, elongational flow and contraction flow. Up to now, the plasma was considered as a Newtonian fluid, while the non-Newtonian properties of blood were only attributed to the red blood cells. The performed experiments reveal a viscoelastic behavior of the plasma which has to be considered in future studies. In addition to the plasma, also diluted polymer solutions are analyzed in order to find a good model solution for plasma. The second part concerns the red blood cells. Their adhesion to linear aggregates is held responsible for the well-known shear thinning behavior of blood but the reason for the cluster formation is still not clear. The interaction energy between two red blood cells and the distribution of different sized clusters flowing through narrow channels are measured under the influence of the two macromolecules dextran and fibrinogen. As the aggregates are actually broken at high shear rates, the current understanding is that they would not play a role for the properties of blood flow. However, an increased amount of clusters at physiological fibrinogen concentrations can be shown, even at shear rates which are common in the microvascular system, which clarifies that the aggregation cannot be neglected in the description of blood flow through the capillary network. In dieser Arbeit werden die rheologischen Eigenschaften von menschlichem Blut mit zwei verschiedenen Zugängen untersucht. Im ersten Teil werden die Fließeigenschaften von Plasma unter drei Bedingungen analysiert: Scherfluss, Dehnungsfluss und Kontraktionsfluss. Bis jetzt galt Plasma als Newtonsche Flüssigkeit, während die nicht Newtonschen Eigenschaften von Blut ausschließlich den roten Blutzellen zugeordnet wurden. Die durchgeführten Experimente zeigen ein viskoelastisches Verhalten von Plasma, welches in zukünftigen Studien zu berücksichtigen ist. Der zweite Teil beschäftigt sich mit den roten Blutzellen. Ihre Adhäsion zu linearen Aggregaten wird für das wohlbekannte scherverdünnende Verhalten von Blut verantwortlich gemacht, aber der Grund für die Entstehung der Cluster ist noch immer unklar. Die Interaktionsenergie zwischen zwei Blutzellen und die Verteilung verschieden großer Cluster, die durch schmale Kanäle fließen, werden unter dem Einfluss der beiden Makromoleküle Dextran und Fibrinogen gemessen. Da die Aggregate bei größeren Scherraten eigentlich aufgebrochen werden, war man bisher der Meinung, sie würden keine Rolle für den Blutfluss spielen, aber ein erhöhter Anteil von Clustern bei physiologischen Fibrinogenkonzentrationen, sogar bei Scherraten die im mikrovaskulären System üblich sind, stellt klar, dass die Aggregation bei der Beschreibung des Blutflusses im kapillaren Netzwerk nicht vernachlässigt werden kann.

submitted by Philip Klepeisz Zsfassung in dt. Sprache Wien, Univ. für Bodenkultur, Diss., 2013 OeBB

Ill., graph. Darst.

Das Ziel der vorliegenden Arbeit war, die Etablierung komplexer Autoantikörper- und miRNA-Expressionsprofile in peripherem Blut. Mit Hilfe der Analyse von über 1800 Autoantigenen wurde die Genauigkeit für verschiedene vergleichende Klassifikationen bestimmt. Lungentumorpatienten konnten von gesunden Probanden mit einer Genauigkeit von 98% getrennt werden. Patienten mit niedrig-gradigen Lungentumoren wurden mit einer Genauigkeit von 93,41% von Gesunden getrennt. Eine Trennung von Patienten mit NSCLC bzw. SCLC von Gesunden erzielte Genauigkeiten über 95%. Die Trennung der Lungentumorpatienten von Patienten mit COPD erreichte eine Genauigkeit von 76,75%. COPD-Patienten wurden von Gesunden mit 97,60% Genauigkeit getrennt. Entsprechende Unterscheidungen bei diesen Erkrankungen waren mit anderen Biomarkern bisher nicht mit vergleichbaren Ergebnissen möglich. Durch die Expressionsanalyse von über 800 miRNAs konnten Lungentumorpatienten mit einer Genauigkeit von 95,4% von Gesunden unterschieden werden. Insgesamt wurden in Blutzellen von Lungentumorpatienten 27 signifikant deregulierte miRNAs identifiziert. Mit vorliegender Studie konnten erstmals solide Tumoren anhand eines miRNA-Expressionsprofils in Blutzellen klassifiziert werden. In vorliegender Arbeit konnten komplexe Autoantikörper- und miRNA-Expressionsprofile in peripherem Blut von Lungentumorpatienten etabliert werden. Mit diesen Profilen wurden vergleichende Klassifikationen mit hoher Genauigkeit erreicht. The presented study aimed at the establishment of complex autoantibody and miRNA expression profiles in peripheral blood. With the analysis of more than 1800 autoantigens, the accuracy of several classification tasks was determined.Lung cancer patients were differentiated from healthy subjects with an accuracy of 98%. Patients with low-grade lung cancer were differentiated from healthy subjects with an accuracy of 93.41%. The differentiation of patients with NSCLC or SCLC from healthy subjects reached an accuracy of more than 95%. The differentiation of lung cancer patients from patients with COPD reached an accuracy of 76.75%. COPD patients were differentiated from healthy subjects with an accuracy of 97.60%. Until now, corresponding classifications with these diseases were not possible with comparable results with other biomarkers. With the expression analysis of more than 800 miRNAs lung cancer patients were differentiated from healthy subjects with an accuracy of 95.4%. A total of 27 significantly deregulated miRNAs were identified. With the presented study, it was possible for the first time to classify solid cancer based on the miRNA expression analysis in blood cells. In the presented work, we were able to establish complex autoantibody and miRNA expression profiles in peripheral blood of lung cancer patients. Using these profiles we were able to perform several classifications with high accuracy.

submitted by: Andrea Mayer Graz, Univ., Diss., 2009 OeBB

Background Glyoxalases (Glo1 and Glo2) are involved in the glycolytic pathway by detoxifying the reactive methylglyoxal (MGO) into D-lactate in a two-step reaction using glutathione (GSH) as cofactor. Inhibitors of glyoxalases are considered as anti-inflammatory and anti-carcinogenic agents. The recent finding that various polyphenols modulate Glo1 activity has prompted us to assess curcumin's potency as an Glo1 inhibitor. Methodology/Principal Findings Cultures of whole blood cells and tumor cell lines (PC-3, JIM-1, MDA-MD 231 and 1321N1) were set up to investigate the effect of selected polyphenols, including curcumin, on the LPS-induced cytokine production (cytometric bead-based array), cell proliferation (WST-1 assay), cytosolic Glo1 and Glo2 enzymatic activity, apoptosis/necrosis (annexin V-FITC/propidium iodide staining; flow cytometric analysis) as well as GSH and ATP content. Results of enzyme kinetics revealed that curcumin, compared to the polyphenols quercetin, myricetin, kaempferol, luteolin and rutin, elicited a stronger competitive inhibitory effect on Glo1 (Ki = 5.1±1.4 µM). Applying a whole blood assay, IC50 values of pro-inflammatory cytokine release (TNF-a, IL-6, IL-8, IL-1v) were found to be positively correlated with the Ki-values of the aforementioned polyphenols. Moreover, whereas curcumin was found to hamper the growth of breast cancer (JIMT-1, MDA-MB-231), prostate cancer PC-3 and brain astrocytoma 1321N1 cells, no effect on growth or vitality of human primary hepatocytes was elucidated. Curcumin decreased D-lactate release by tumor cells, another clue for inhibition of intracellular Glo1. Conclusions/Significance The results described herein provide new insights into curcumin's biological activities as they indicate that inhibition of Glo1 by curcumin may result in non-tolerable levels of MGO and GSH, which, in turn, modulate various metabolic cellular pathways including depletion of cellular ATP and GSH content. This may account for curcumin's potency as an anti-inflammatory and anti-tumor agent. The findings support the use of curcumin as a potential therapeutic agent.

Dehydroepiandrosteron wirkt hauptsächlich indirekt über nachfolgende Umwandlung in Androgene und Östrogene sowie deren Zwischenprodukte in den peripheren Zielzellen, respektive Immunzellen. In vitro Versuche erbrachten den Nachweis von gesteigerter Interleukin-2 Sekretion durch T-Lymphozyten nach Gabe von DHEA, wohingegen es zu einer Inhibierung der Interleukin-6 Sekretion kam. Dementgegen wird im Altersprozess ein Abfall von DHEA und Interleukin-2 beobachtet bei gleichzeitigem Anstieg von Interleukin-6. Diese Arbeit beschäftigt sich mit der Frage, in wieweit humane periphere mononukleäre Blutzellen (PBMCs) über Expression und funktionale Aktivität der im DHEA-Downstream-Metabolismus essentiellen Enzyme verfügen und ob es alters- bzw. geschlechtsspezifische Unterschiede gibt. Die Studie wurde an 8 gesunden jüngeren Frauen sowie 8 gesunden jüngeren und 8 gesunden älteren Männern durchgeführt. Zur Bestimmung der mRNA-Expression der im DHEA-Downstreammetabolismus notwendigen Steroidenzyme in humanen PBMCs wurden qualitative, semiquantitative und quantitative RT-PCR Analysen sowie PBMC-Enzymfunktionsassays durchgeführt. Hierbei wurden PBMCs mit radioaktiv markiertem 4-14C-Testosteron, 4-14C-Androstendion bzw. 4-14C-DHEA inkubiert, extrahiert und mittels Dünnschichtchromatographie separiert. Die anschließende quantitative Auswertung der Konversionsprodukte erfolgte durch Phosphorimager-Analyse. Zusammenfassend lässt sich aussagen, dass humane periphere mononukleäre Blutzellen (PBMCs) ausgehend von DHEA über alle notwendigen Steroidenzyme für einen effizienten Downstreammetabolismus zu aktiven Androgenen verfügen. Dies konnte sowohl durch den qualitativen und quantitativen Nachweis von mRNA der betreffenden Enzyme als auch durch die nachgewiesene Aktivität in PBMC-Enzymfunktionsassays bestätigt werden. Dabei zeigten sich signifikante alters- und geschlechtsspezifische Veränderungen im Androgenmetabolismus. Insbesondere konnte eine erhöhte 17beta-HSD5-Aktivität und -Expression in der älteren männlichen und weiblichen Probandengruppe im Vergleich zur jüngeren männlichen nachgewiesen werden, was sich in signifikant höheren Konversionsraten von DHEA zu Androstenediol und Androstenedion zu Testosteron widerspiegelte. Ebenso konnte eine signifikant erhöhte 5alpha-Reduktase-Aktivität in der älteren männlichen Probandengruppe im Vergleich zur jüngeren männlichen aufgezeigt werden. Dementgegen waren die Konversionsraten von DHEA zu Androstenedion via 3beta-Aktivität unter den einzelnen Probandengruppen ähnlich. Die vermehrte Konversion von DHEA zum immunmodulatorisch wirksamen Metabolit Androstendiol sowie von Androstendion zu Testosteron entspricht einer vermehrten Androgenaktivierung. Dieses Hochregulieren könnte einen kompensatorischen Mechanismus der peripheren Zielzelle darstellen, dem sinkenden DHEA-Serumspiegel während des Alterungsprozesses entgegenzuwirken und kann einen endokrinen Hinweis auf eine „Immunseneszenz“ geben., CONTEXT: Dehydroepiandrosterone (DHEA) mainly exerts indirect action via downstream conversion toward sex steroids within peripheral target cells including immune cells. In vitro DHEA has been shown to enhance IL-2 release from T lymphocytes, whereas it inhibits IL-6 secretion. Conversely, aging is associated with a decline in both DHEA and IL-2, whereas IL-6 increases. OBJECTIVE: The objective of the study was to investigate age and sex-related differences in expression and functional activity of steroidogenic enzymes involved in downstream conversion of DHEA in peripheral blood mononuclear cells (PBMCs). DESIGN: This study was cross-sectional. PARTICIPANTS/SETTING: Healthy young men (n = 8) and women (n = 8) and healthy middle-aged men (n = 8) were studied in an academic setting. MEASURES: mRNA expression of steroidogenic enzymes in PBMCs was measured by qualitative and quantitative RT-PCR analysis and enzyme activity assays after incubation of PBMCs with radiolabeled DHEA, 4-androstene-3,17-dione (androstenedione), and testosterone. RESULTS: RT-PCR analysis showed expression of all enzymes required for DHEA conversion toward active androgens and to the immune-stimulatory metabolite androstenediol. Steroid conversion patterns indicated a particularly increased activity of 17beta-hydroxysteroid dehydrogenase type 5 (17beta-HSD5) in the older men and young women demonstrated by significantly higher conversion rates of DHEA to androstenediol and of androstenedione to testosterone (all P < 0.05). By contrast, conversion of DHEA to androstenedione via 3beta-HSD occurred at a similar rate. Quantitative RT-PCR analysis revealed increased expression of 17beta-HSD 5 mRNA in PBMCs from the older men. CONCLUSIONS: Our results provide evidence for significant changes in sex steroid metabolism by human PBMCs with aging, which may represent an endocrine link to immune senescence.

In dieser Arbeit wurde der Einfluß von Doxycyclin auf die Cytokin- und Lipidmediatorsynthese sowie das Proliferationsverhalten humaner Leukozyten untersucht. Die PBMC und PMN wurden durch einen diskontinuierlichen doppelten Ficoll-Gradienten getrennt. Die Zellen wurden in Anwesenheit von Doxycyclin unter gleichzeitiger Stimulation mit TSST-1, ConA, SEB und LPS für 24 h bei 37 [$\textdegree$]C unter Zellkulturbedingungen inkubiert. Die freigesetzten Cytokine wurden mittels ELISA. Die Analyse der Freisezung von Leukotrienen erfolgte durch eine HPLC. Die Zellproliferationsmessung erfolgte kolorimetrisch. Bei den aktivierten Zellen kam es zu einer konzentrationsabhängigen Inhibition der Cytokin-und Lipidmediatorsynthese. Die Proliferation der PBMC sowie die Cytokinproduktion der PMN wurde durch Doxycyclin kaum beeinflusst. Diese Arbeit zeigt, dass Doxycyclin in vitro die Freisetzung von Cytokinen und Lipidmediatoren inhibieren kann.