>Magister Scientiae - MSc / Knowledge of haematological 'normdata', of experimental animals, and the biological variables that affect it is essential in order to recognise variations from the normal. In addition, the haemopoietic system may be regarded in principle as good material for studies of the cellular events associated with ageing. These considerations, together with the well documented effects of age on various physiological processes, prompted an investigation into the effects of donor age on several blood parameters. Review of the literature revealed that age-related changes in blood parameters have been reported for several species, but the documentation thereof is incomplete, inconsistent and inconclusive in many respects. Blood samples from male Wistar rats of nine different biological ages, ranging from birth to 96 weeks of age, were analysed for haematological and biochemical parameters. These included the blood cell counts, erythrocytic indices, haemoglobin concentration, haematocrit, erythrocytic 2,3-diphosphoglycerate and adenosine triphosphate levels, and erythrocytic glucose 6-phosphate dehydrogenase and pyruvate kinase activities. Data was obtained which demonstrates that all blood parameters measured underwent significant, although not al~ays regular, age-related changes. These changes were found to be more marked during the first month of life than at any other period. Evidence is also presented to show that the depressed haemoglobin concentration during the early postnatal life may not imply a condition of 'physiologic anaemia' as was previously thought. Since the blood profile exhibits only slight changes from about 24 weeks of age, it does not seem that the haemopoietic system of the old rat deteriorates significantly as to constitute a limiting factor for the animal's life. However, the importance of taking an animal's age into account when blood parameters constitute experimental results is emphasised. The second phase of this study involved a detailed investigation of the effect of the animal's age on erythrocytes in particular. These cells have limited life-spans, and are often used as models in studies of cellular ageing. Special emphasis was therefore placed on comparing the relative effects of host and cellular ageing on the properties of these cells. Erythrocytes from rats between one and 48 weeks of age were separated into two populations by a modification of the conventional density gradient centrifugation technique. The two populations were assumed to differ in mean cell age and were analysed for erythrocytic indices, phosphate ester concentrations and the activities of glucose 6-phosphate dehydrogenase and pyruvate kinase. Evidence is presented to show that ageing rat erythrocytes exhibit a decrease in volume, phosphate ester content and enzyme activities while the cellular haemoglobin concentration increases. Differences in the mean cell age however, does not seem to account for the donor-age-related effects observed in the whole blood parameters. Rather, the significant differences found in the characteristics of similarly aged red cells, between variously aged donors, demonstrate that the biological age of the organism influences the red cells and probably the ageing thereof in vivo. The contribution of the changing status of the erythrocyte's environment of progressively older animals, to alterations which take place in the ageing red cell is discussed.

This thesis will be organized into two chapters discussing the placental expression of two proteins, pyruvate kinase M2 (PKM2) and heat shock protein 27 (HSP 27), in human placentas. Understanding the mechanisms of placental metabolism in healthy and diseased placentas helps us understand how placenta disorders occur and how we can treat these disorders. The goal is to investigate these proteins to gain an understanding of their roles in placental disorders and help decrease maternal and fetal mortality rates. Chapter one covers the background of pyruvate kinase M2 (PKM2) in cancer and embryonic tissues, and the expression of PKM2 in the human placenta. Cancer PKM2 has been studied extensively, but little is know about the role of placental PKM2. Expression of PKM2 is confirmed in normal human placenta samples and described in preeclamptic and intrauterine growth restriction (IUGR) affected human placentas. Proteins associated with elevated PKM2 in cancer are also associated with elevated PKM2 in human placentas. Comparing normal and diseased placenta samples helps understand the similarities between cancer PKM2 and placental PKM2. Understanding the mechanisms of placental metabolism and PKM2 expression in the human placenta will clarify how the placenta is affected by preeclampsia and IUGR and the role placental PKM2 plays in each of these diseases. Chapter two will cover a paper that I wrote on the expression of phosphorylated heat shock protein 27 (HSP27) in the human placenta. Heat shock proteins are involved in the stress response and help inhibit apoptosis. The object of the study was to look for correlations between p-HSP27 and apoptosis in human and ovine placenta samples. P-HSP27 was quantified in human placenta samples and in placenta sampled collected from ovine models. Pregnant control and hyperthermic sheep models were used to quantify expression of p-HSP27 across gestation. This study showed similarities between human IUGR and our ovine IUGR model, suggesting a link between decreased p-HSP27 and increased apoptosis in IUGR.

This thesis explores the pathogenesis of calciphylaxis, a rare and potentially fatal complication of chronic kidney disease (CKD) characterized by calcification and thrombosis of small- to medium-sized arteries. A range of bench techniques, including cell culture, genetic analysis, and immunofluorescence, were utilized in combination with human samples from patients with calciphylaxis and healthy controls. The results revealed a pathway that may modulate the thrombotic phenotype in these patients and, in turn, may serve as a targetable therapeutic axis. This work provides a foundation for further research and clinical advances in the field of calciphylaxis. Moreover, this study has the potential to inform the development of therapeutic interventions that could greatly improve the outcomes of CKD patients suffering from calciphylaxis. / 2026-03-05T00:00:00Z

A psoríase é uma doença inflamatória crônica com uma elevada incidência, que afeta a pele. A patogênese da psoríase caracteriza-se pela participação de inúmeras células, incluindo os queratinócitos que são as principais células efetoras da citocina IL-17, críticas para a doença, que produzidas pelas células T. Evidências crescentes sugerem o importante papel da piruvato quinase M2 (PKM2) na regulação da resposta inflamatória, mas o mecanismo subjacente permanece obscuro. Nesse sentido, no presente estudo investigamos o papel da PKM2 no desenvolvimento da psoríase. Observamos o aumento de PKM2 em biópsia humana, em modelo de psoríase induzida por imiquimode e em modelos espontâneos K14-IL-17Aind e DC-IL-17Aind. Em adição, esse aumento observado na enzima foi predominante nos queratinócitos e isso foi associado a marcadores de ativação de queratinócitos. Utilizando o inibidor de PKM2, Shikonin (SKN), como abordagem farmacológica, observamos que o tratamento com esse composto foi capaz de reverter a psoríase experimental e a reduzir marcadores associados a doença como: K17, LCN2, TNF-?, KC, S100A8, S100A9, IL-6 e IL-17A. Associado a isso, observamos a redução na frequência de células T (?? e ??) produtoras de IL-17 e do número de neutrófilos na pele em modelo de imiquimode após inibição da PKM2. O SKN, também, reduziu o número de neutrófilos no modelo DC-IL-17Aind. Em nosso próximo passo, observamos que queratinócitos HACAT estimulados com IL-17A apresentou um aumento da expressão de PKM2 e que a sua inibição foi associada a redução da ativação de queratinócitos e de mediadores inflamatórios como a IL-8. Além disso, a deleção da PKM2, utilizando a tecnologia CRISPR/Cas9, reduziu a expressão do receptor de IL-17. Por fim, o desenvolvimento da psoríase por imiquimode foi atenuada em animais deficientes para PKM2 em queratinócitos (K14-PKM2fl/+), no qual foi observado a redução de neutrófilos na pele e, além disso, evidenciamos a redução da expressão de IL-17A nesses animais. O conjunto de resultados apresentados nesse trabalho demonstram que a PKM2 apresenta um papel crítico no desenvolvimento da psoríase e que a ativação do receptor de IL-17 promove um aumento da PKM2 em queratinócitos e esta contribui para ativação de mediadores que é responsável diretamente para o desenvolvimento da psoríase. Esses resultados, ainda, sugerem a PKM2 como um biomarcador para diagnóstico da psoríase e consequentemente, um potencial alvo terapêutico para tratamento dessa doença e outras doenças inflamatórias. / Psoriasis is a chronic inflammatory skin disease with high incidence in the global population. The pathogenesis of psoriasis is characterized by involvement of many cells, including keratinocytes that are targets for IL-17-producing T cells. Evidences suggests a critical role of pyruvate kinase M2 (PKM2) in inflammatory response, but the underlying mechanism remains unclear. In this context, here we investigated the role of PKM2 in the development of psoriasis. We observed overexpression of PKM2 in psoriatic human skin, imiquimod-induced psoriasis and spontaneous K14-IL-17Aind and DC-IL-17Aind models. In addition, the overexpression of this enzyme was observed in keratinocytes associated with keratinocytes activation markers. Using the PKM2 inhibitor, Shikonin (SKN), as a pharmacological approach, we observed that the treatment with this compound was able to reduce experimental psoriasis and disease-associated markers such as K17, LCN2, TNF-?, KC, S100A8, S100A9, IL-6 and IL-17A. Moreover, we observed reduction of frequency of IL-17-producing T cells (?? and ??) and the number of neutrophils in the skin after imiquimod application plus inhibition of PKM2. SKN, also, reduced the number of neutrophils in the DC-IL-17Aind model. In our next step, we observed overexpression of PKM2 in human keratinocytes HACAT stimulated with IL-17A and that its inhibition was associated with less keratinocytes activation and inflammatory mediators such as IL-8. In addition, deletion of PKM2, using CRISPR/Cas9 technology, reduced IL-17 receptor expression. Finally, the development of imiquimod-induced psoriasis was attenuated in PKM2-deficient mice in keratinocytes (K14-PKM2f/+), with reduction in the number of neutrophils in the skin. In addition, we evidenced the reduction of IL-17A expression these animals. Taken together, these results demonstrate that PKM2 plays a critical role in the development of psoriasis and that IL-17 receptor activation promotes an increase of PKM2 in keratinocytes and this contributes to the release of mediators that is directly responsible for development of psoriasis. These results, suggest PKM2 as a biomarker for the diagnosis of psoriasis and consequently a potential therapeutic target for the treatment of this disease and other inflammatory diseases.

This thesis will be organized into two chapters discussing the role of hypoxia in the human placenta. The goal of this thesis is to characterize pyruvate kinase M2, mammalian target of rapamycin, mitochondrial function, and cell invasion in hypoxic conditions in the trophoblast. Understanding the mechanisms of placental metabolism can lead to further treatments for placental diseases. Chapter one covers the background of intrauterine growth restriction, hypoxia, placental metabolism, and pyruvate kinase M2 (PKM2). Little is currently understood about the role of the mitochondria in placental diseases. Expression of PKM2, trophoblast cell invasion, and mitochondrial function is shown to be inhibited by hypoxia. PKM2 inhibition decreases trophoblast cell invasion and nuclear expression of PKM2, but increases mitochondrial function. Studying how hypoxia affects the placenta during placental diseases can help clarify the mechanisms by which these diseases occur. Chapter two further characterizes the background of intrauterine growth restriction and hypoxia. It also covers the background of mammalian target of rapamycin. The objective of this chapter was to assess activated mTOR in the trophoblast in hypoxia. Decreased placental and fetal weights, as well as trophoblast cell invasion were observed in hypoxia. A decrease in the activation of mTOR was also found in the hypoxic placenta. This study could provide insight into the physiological relevance of the pathways and could be targeted to help alleviate placental diseases.

No description available.

Referat: Background: Human African Trypanosomiasis (HAT) also called sleeping sickness is an infectious disease of humans caused by an extracellular protozoan parasite. The disease, if left untreated, results in 100% mortality. However, the available drugs are full of severe drawbacks and fail to escape the fast development of trypanosoma resistance. Due to the probable similarities in cell metabolism among tumor and trypanosoma cells, some of the current registered drugs against HAT were derived from cancer chemotherapeutic research. Here too, for the first time, we have demonstrated that the simple ester, ethyl pyruvate, comprises such properties. On the other hand initial studies have confirmed the efficacy of protease inhibitors in treatment of Trypanosoma cruzi, Plasmodium falciparum and Leishmania major. However, studies on efficacy and specific proteases inhibition using HIV-1 protease inhibitors on T. brucei cells remain untouched. Methodology/Principal findings: The current study covers efficacy and corresponding target evaluation of ethyl pyruvate and HIV-1 protease inhibitors (ritonavir and saquinavir) on T. brucei cell lines using a combination of biochemical techniques including cell proliferation assays, enzyme kinetics, zymography, phase contrast microscopic video imaging and ex vivo drug toxicity tests. We have shown that ethyl pyruvate effectively kills trypanosomes most probably by net ATP depletion through inhibition of pyruvate kinase (Ki=3.0±0.29 mM). The potential of this compound as an anti-trypanosomal drug is also strengthened by its fast acting property, killing cells within three hours post exposure. This was demonstrated using video imaging of live cells as well as concentration and time dependency experiments. Most importantly, this drug produced minimal side effects in human erythrocytes and is known to easily cross the blood-brain-barrier (BBB) which makes it a promising candidate for effective treatment of the two clinical stages of sleeping sickness. Trypanosome drug resistance tests indicate irreversible killing of cells and a low chance of drug resistance development under applied experimental conditions. In addition to ethyl pyruvate our experimental study on HIV-1 protease inhibitors showed that both ritonavir (RTV) (IC50=12.23 µM) and saquinavir (SQV) (IC50=11.49 µM) effectively inhibited T. brucei cells proliferation. The major proteases identified in these cells were the cysteine- (~29kDa Mr) and metallo- (~66kDa Mr) proteases. Their proteolytic activity was, however, not hampered by either of these two protease inhibitors. Conclusion/Significance: Our results present ethyl pyruvate as a safe and fast acting drug. Hence, because of its predefined property to easily cross the BBB, it can probably be a new candidate agent to treat the heamolymphatic as well as neurological stages of sleeping sickness. Similarly, HIV-1 protease inhibitors, SQV and RTV, exhibited their antitrypanosomal potential but require further anlysis to identify their specific targets.

Referat: Background: Human African Trypanosomiasis (HAT) also called sleeping sickness is an infectious disease of humans caused by an extracellular protozoan parasite. The disease, if left untreated, results in 100% mortality. However, the available drugs are full of severe drawbacks and fail to escape the fast development of trypanosoma resistance. Due to the probable similarities in cell metabolism among tumor and trypanosoma cells, some of the current registered drugs against HAT were derived from cancer chemotherapeutic research. Here too, for the first time, we have demonstrated that the simple ester, ethyl pyruvate, comprises such properties. On the other hand initial studies have confirmed the efficacy of protease inhibitors in treatment of Trypanosoma cruzi, Plasmodium falciparum and Leishmania major. However, studies on efficacy and specific proteases inhibition using HIV-1 protease inhibitors on T. brucei cells remain untouched. Methodology/Principal findings: The current study covers efficacy and corresponding target evaluation of ethyl pyruvate and HIV-1 protease inhibitors (ritonavir and saquinavir) on T. brucei cell lines using a combination of biochemical techniques including cell proliferation assays, enzyme kinetics, zymography, phase contrast microscopic video imaging and ex vivo drug toxicity tests. We have shown that ethyl pyruvate effectively kills trypanosomes most probably by net ATP depletion through inhibition of pyruvate kinase (Ki=3.0±0.29 mM). The potential of this compound as an anti-trypanosomal drug is also strengthened by its fast acting property, killing cells within three hours post exposure. This was demonstrated using video imaging of live cells as well as concentration and time dependency experiments. Most importantly, this drug produced minimal side effects in human erythrocytes and is known to easily cross the blood-brain-barrier (BBB) which makes it a promising candidate for effective treatment of the two clinical stages of sleeping sickness. Trypanosome drug resistance tests indicate irreversible killing of cells and a low chance of drug resistance development under applied experimental conditions. In addition to ethyl pyruvate our experimental study on HIV-1 protease inhibitors showed that both ritonavir (RTV) (IC50=12.23 µM) and saquinavir (SQV) (IC50=11.49 µM) effectively inhibited T. brucei cells proliferation. The major proteases identified in these cells were the cysteine- (~29kDa Mr) and metallo- (~66kDa Mr) proteases. Their proteolytic activity was, however, not hampered by either of these two protease inhibitors. Conclusion/Significance: Our results present ethyl pyruvate as a safe and fast acting drug. Hence, because of its predefined property to easily cross the BBB, it can probably be a new candidate agent to treat the heamolymphatic as well as neurological stages of sleeping sickness. Similarly, HIV-1 protease inhibitors, SQV and RTV, exhibited their antitrypanosomal potential but require further anlysis to identify their specific targets.:Bibliographic description ii Acronyms iii 1. Introduction 1 1.1. Disease background 1 1.2. Epidemiological distribution and disease transmission dynamics 1 1.3. Biology and life cycle of the trypanosomatidea 3 1.4. Public health significance 4 1.5. Clinical stages and disease progression 5 1.6. Current challenges of disease control 6 1.7. Current drugs and their clinical applications 9 1.8. Targets for drug discovery 12 1.8.1. Energy metabolism 12 1.8.2. Proteolysis 17 1.9. Ethyl pyruvate 18 1.10. HIV-1 Protease Inhibitors 21 2. Aim of the study 22 3. Materials and Methods 24 4. Results 31 5. Discussion 45 6. Conclusion 53 7. Supporting information 54 8. Summary 56 9. References 62 Erklärung über die eigenständige Abfassung der Arbeit 77 Curriculum vitae 78 Publications and Presentations 81 Acknowledgement 83

Indiana University-Purdue University Indianapolis (IUPUI) / The expression of pyruvate dehydrogenase kinase (PDK) 2 and 4 are increased in the fasted state to inactivate the pyruvate dehydrogenase complex (PDC) by phosphorylation to conserve substrates for glucose production. To assess the importance of PDK2 and PDK4 in regulation of the PDC to maintain glucose homeostasis, PDK2 knockout (KO), PDK4 KO, and PDK2/PDK4 double knockout (DKO) mice were generated. PDK2 deficiency caused higher PDC activity and lower blood glucose levels in the fed state while PDK4 deficiency caused similar effects in the fasting state. DKO intensified these effects in both states. PDK2 deficiency had no effect on glucose tolerance, PDK4 deficiency produced a modest effect, but DKO caused a marked improvement, lowered insulin levels, and increased insulin sensitivity. However, the DKO mice were more sensitive than wild-type mice to long term fasting, succumbing to hypoglycemia, ketoacidosis, and hypothermia. Stable isotope flux analysis indicated that hypoglycemia was due to a reduced rate of gluconeogenesis. We hypothesized that hyperglycemia would be prevented in DKO mice fed a high saturated fat diet for 30 weeks. As expected, DKO mice fed a high fat diet had improved glucose tolerance, decreased adiposity, and were euglycemic due to reduction in the rate of gluconeogenesis. Like chow fed DKO mice, high fat fed DKO mice were unusually sensitive to fasting because of ketoacidosis and hypothermia. PDK deficiency resulted in greater PDC activity which limited the availability of pyruvate for oxaloacetate synthesis. Low oxaloacetate resulted in overproduction of ketone bodies by the liver and inhibition of ketone body and fatty acid oxidation by peripheral tissues, culminating in ketoacidosis and hypothermia. Furthermore, when fed a ketogenic diet consisting of low carbohydrate and high fat, DKO mice also exhibited hypothermia, ketoacidosis, and hypoglycemia. The findings establish that PDK2 is more important in the fed state, PDK4 is more important in the fasted state, survival during long term fasting depends upon regulation of the PDC by both PDK2 and PDK4, and that the PDKs are important for the regulation of glucose and ketone body metabolism.