Universiẗat, Diss., 2003--Giessen.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 134-157).

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Each year, between 300 and 500 million people develop malaria. Close to 3 million people die as a result. Due to resistance there are now few drugs left which are effective against malaria; one is a Chinese herbal remedy called qinghaosu (artemisinin). A section of this thesis represents an attempt to understand further the mode of action of this antimalarial agent. Several mechanisms involving iron have been proposed and the main categories reviewed (chapter 3). Techniques including ESR spectrometry, fluorescence and absorbance measurements were used to further probe the mode of action of qinghaosu. The findings of these studies provide support for a mechanism proposed by Wu et al., involving cleavage of the peroxy bridge in qinghaosu with the ferrous ion as opposed to antimalarial activity being due to the generation of high valent iron-oxo species. The ether derivative of qinghaosu, arteether has been studied using 2D NMR in order to provide further insight into the unusual structure of this compound (chapter 4). A comparison of the sp2 lactone functionality in qinghaosu with the sp3 ether group in arteether was performed. Evidence is presented for different conformations and chemical shift values. The immune system protects the human body from invasion by foreign substances or cells. A key part of this immune response comes from cellular components known as macrophages. The killing process of macrophages appears to involve nitric oxide. However, this natural defence mechanism is not very efficient as the body is susceptible to invasion by microbes. The term microbe includes both parasites and bacteria. The toxicity of nitric oxide (NO) and NO donor compounds has been investigated using the bacterium E. coli (chapter 5). In addition, studies have been performed using parasites. Malarial parasites (Plasmodia) are difficult to culture, therefore Leishmania parasites which cause the tropical disease leishmaniasis were used as a model (chapter 6). Evidence is presented for the toxic nature of NO donor compounds towards both parasites and bacteria. However, qinghaosu and its derivatives did not prove effective against Leishmania mexicana parasites in contrast with their reported antimalarial activity. The conclusions of this study suggest that qinghaosu possesses a toxicity mechanism selective for malarial parasites.

Orientadores: Sergio Roberto Peres Line, Paulo Sergio Flores Campos / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-09T19:33:47Z (GMT). No. of bitstreams: 1 Campos_MariaIsabelaGuimaraes_D.pdf: 3191665 bytes, checksum: ffbd544df321690468c5974728a9d0a8 (MD5) Previous issue date: 2007 / Resumo: O objetivo deste estudo foi investigar a ocorrência dos polimorfismos IL-1A (-889C/T), IL-1B (-511C/T), IL-1B (+3953C/T), IL-1RN VNTR (intron 2), IL-6 (-174G/C) e TNF-A (-308G/A) em 92 pacientes com sinais imaginológicos de alterações degenerativas da ATM e em 102 indivíduos controles. O DNA dos voluntários foi extraído a partir de células epiteliais da mucosa bucal e submetido às técnicas de PCR (reação em cadeia da polimerase) e RFLP (polimorfismo no comprimento do fragmento de restrição) para a detecção dos possíveis alelos. Os produtos do PCR/RFLP foram analisados em géis de poliacrilamida a 10%, corados por prata. Os polimorfismos foram analisados individualmente e sob a forma de haplótipos. O programa Arlequin (ver. 3.01) foi utilizado para calcular o desequilíbrio de ligação entre os loci polimórficos da IL-1 e determinar seus possíveis haplótipos. Adicionalmente, um questionário foi aplicado aos voluntários para investigar a presença de fatores locais e sistêmicos associados a DTMs. Os dados do questionário, bem como, as freqüências alélicas, genotípicas e haplotípicas foram comparadas entre os dois grupos com auxílio do teste qui-quadrado (p<0,05). O método de Mantel-Haenszel foi utilizado para o cálculo de odds ratio (OR), com intervalo de confiança (IC) a 95% para estimar o risco de cada fator avaliado na ocorrência da doença. As análises estatísticas foram realizadas com auxílio do programa Epi Info (ver. 6.04). Estresse e/ou ansiedade (p=0,001), uso freqüente de ansiolíticos ou antidepressivos (p=0,000), hábitos parafuncionais (p=0,000), histórico de trauma facial (p=0,005), queixas constantes de dores em outras articulações (p=0,000), presença de alterações sistêmicas (p=0,000) e histórico de familiares com DTM (p=0,000) se mostraram significativamente mais freqüentes nos pacientes do grupo teste. Considerando o polimorfismo IL-1B (+3953C/T), o alelo C e genótipo C/C mostraram maior ocorrência no grupo teste do que no controle (p=0,000), aumentando em 3,24 e 6,35 vezes o risco de desenvolvimento de DTM, respectivamente. A presença do genótipo C/T do polimorfismo IL-1A (-889C/T) aumentou em aproximadamente 1,87 vezes o risco em desenvolver DTM. Os loci polimórficos IL-1B (+3953C/T), IL-1A (-889C/T) e IL-1RN se mostraram em desequilíbrio de ligação e foram analisados simultaneamente, demonstrando que indivíduos portadores do respectivo haplótipo TC1 apresentaram-se 0,11 vezes mais protegidos em desenvolver DTM. Considerando o polimorfismo IL-6 (-174G/C), indivíduos portadores do alelo C e do genótipo C/G apresentaram um risco 1,60 e 1,98 vezes maior em desenvolver DTM, respectivamente. O presente estudo mostra que polimorfismos nos genes da IL-1 e IL-6 estão associados ao desenvolvimento de DTMs na população estudada e corrobora evidências anteriores de que fatores psicológicos, locais e sistêmicos devem atuar em conjunto no desenvolvimento de DTMs / Abstract: The aim of this study was to investigate the occurrence of IL-1A (-889C/T), IL-1B (-511C/T), IL-1B (+3953C/T), IL-1RN VNTR (intron 2), IL-6 (-174G/C) and TNF-A (-308G/A) polymorphisms in 92 patients with degenerative changes of temporomandibular joint. Control group comprised 102 healthy individuals. Genomic DNA was obtained from oral mucosa cells and polymorphisms were identified by PCR (polymerase chain reaction) and RFLP (restriction fragment length polymorphism). Restriction products were visualized by electrophoresis on vertical 10% polyacrylamide gels, followed by silver staining. The Arlequin software (ver.3.01) was used to determine haplotype frequencies and linkage disequilibrium among the IL-1 polymorphic sites. The polymorphisms were analyzed individually and in haplotypes. In addition, a questionnaire was answered by volunteers in order to assess the presence of local and systemic factors associated to TMDs. Data of the questionnaires, frequencies of alleles, genotypes and haplotypes were compared between the two groups using the chi-squared test (p<0.05). The odds ratio (OR) was calculated with 95% confidence intervals (CI) to estimate the relative risk of each factor on the disease occurrence. The statistical analyses were performed using Epi Info software (ver. 6.04). Stress and/or anxiety (p=0.001), frequent usage of anxiolytic or antidepressant drugs (p=0.000), oral parafunctions (p=0.000), history of facial trauma (p=0.005), presence of systemic diseases (p=0.000), complaints of pain in other joints (p=0.000) and history of relatives with TMD (p=0.000) were significantly more frequent in patients of test group. For the IL-1B (+3953C/T) polymorphism, allele C and genotype C/C were significantly frequent in patients of test group (p=0,000), showing 3,24- and 6,35-folds increased risk for TMD development, respectively. Individuals with genotype C/T of IL-1A (-889C/T) polymorphism showed a 1,87-folds increased risk for TMD development. The polymorphisms IL-1B (+3953C/T), IL-1A (-889C/T) and IL-1RN were in linkage disequilibrium and were analyzed in haplotypes. The respective haplotype TC1 showed a protective effect increased in 0,11-folds. Individuals with allele C and genotype C/G of IL-IL-6 (-174G/C) polymorphism showed a 1,60- and 1,98-folds increased risk for TMD development, respectively. The present study shows that IL-1 and IL-6 polymorphisms are associated with development of TMDs in the studied population and corroborates previous evidences that psychological, local and systemic factors should act in combination to promote development of TMDs / Doutorado / Histologia e Embriologia / Doutor em Biologia Buco-Dental

The mechanism of action of the hemopoietic growth factor, murine interleukin-3 (mIL-3), was investigated using an mIL-3-dependent multipotential hemopoietic cell line, B6SUtA. These cells were first used to identify the mIL-3 surface receptor as a monomeric 67 kDa protein with a pI of approximately 6.2. Further studies suggested the presence of an additional mIL-3 binding protein with an apparent molecular mass of 140 kDa. Then, in an attempt to gain some insights into the mechanism of action of mIL-3, molecules other than mIL-3 were tested to determine their effects on cell proliferation. Murine granulocyte -macrophage colony-stimulating factor (mGM-CSF) was found to be as potent as mIL-3 in stimulating B6SUtA cells. In addition, sodium orthovanadate, an inhibitor of phosphotyrosine phosphatase, and 12-0-tetradecanoyl-phorbol-13-acetate (TPA), a known activator of protein kinase C, both stimulated DNA synthesis in these cells, suggesting that protein phosphorylation might be involved in the mechanism of action of mIL-3 and mGM-CSF. To assess this possibility, intact B6SUtA cells exposed for brief periods to mIL-3, mGM-CSF or TPA were analyzed for changes in phosphorylation patterns using metabolic [superscript]32p-labeling. Both mIL-3 and mGM-CSF induced the serine-specific phosphorylation of a 68 kDa cytosolic protein, while all three agents stimulated the serine-specific phosphorylation of a 67 kDa membrane protein. Furthermore, using antibodies to phosphotyrosine, it appeared that mIL-3 stimulated tyrosine phosphorylation of 67 kDa and 140 kDa membrane proteins, as well as of 40, 55 and 90 kDa cytosolic proteins. The 90 kDa protein was also tyrosine phosphorylated in response to mGM-CSF, suggesting that this phosphorylation results from a common step in mIL-3 and mGM-CSF-stimulated signaling pathways. These phosphotyrosine containing proteins were not detected in TPA-treated cells. Moreover, evidence from a variety of studies is presented that the 140 kDa but not the 67 kDa mIL-3 receptor becomes phosphorylated on tyrosine residues when B6SUtA cells bind mIL-3. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Zinc is an essential biological trace element required for proper immune functioning. Zinc deficient individuals have been reported to suffer compromised immune responses and increased levels of inflammatory cytokines. Inflammation is integral to the pathology of many disease states, ranging from pathogen dependent infectious disease to non-infectious disease such as cancer, heart disease, diabetes and stroke. One of the main mediators of inflammation is the pro-inflammatory cytokine interleukin-1beta (IL-1beta). Production of IL-1beta occurs via a two step process; firstly the transcription of an inactive pro-form is initiated, followed by protease activation leading to the cleavage of IL-1beta to a mature form. Here it is shown that in vitro zinc depletion of macrophages, using the zinc chelators TPEN and DTPA, leads to pro-IL-1beta cleavage and furthermore to increased release of active IL-1beta. This would suggest that zinc depletion induces activation of proteases that cleave IL-1beta. Caspase-1, ASC, PP2A, cathepsin B and cathepsin G are all shown to regulate zinc depletion-induced IL-1beta release in macrophages. The cell death proteins XIAP and caspase-8 have also been identified to be regulated by zinc depletion in macrophages and there is literature to suggest that these proteins may contribute to IL-1beta processing and release. By identifying a role for zinc depletion in IL-1beta processing we move closer to identifying potential therapeutic targets for zinc deficiency induced inflammatory disease. Zinc also has regulatory roles in the expression of IL-1beta. Here a systems biology approach is utilised to create an explicit representation of the pathways involved in IL-1beta expression. In many in vivo and in vitro models, transcription of pro-Interleukin-1beta is induced by the gram negative cell wall component lipopolysaccharide (LPS). A systematically curated network map of IL-1 transcription has been created. The map encompasses interactions at the macrophage cell membrane, where LPS binds Toll-like receptor 4 (TLR4); the resulting cytoplasmic signalling cascades, including MAPK and NF-kappaB; and finally the specific transcription factor interactions in the nucleus. By creating this model we aim to enable the production of dynamic models of IL-1 transcription.

The pathogenesis of osteoarthritis (OA) is complex, but likely involves destruction of articular cartilage by endogenous enzymes (Dingle 1979). Factors controlling this are not well understood. Cetabolin, a 21,000 molecular weight peptide structurally end functionally related to interleukin-1, stimulates living but not killed chondrocytes in vitro to degrade their matrix (Fell and Jubb 1977, Saklatvala et al. 1983), suggesting it is not itself a degradative enzyme but functions as a control factor. The work in this thesis investigated the possible role of cetabolin in the pethogenesis of OA by measuring catabolin production by cultures of synovium excised from the canine anterior cruciate ligament transection model of OA. Normal cenine synovium in culture was shown to produce a factor which can stimulate the release of glycoseminoglycens from living cenine articular cartilage in culture. The total emount of cetebolin produced by cultures of synovium from experimentally induced OA synovium is statistically significantly greater (p<0.05) than that produced by normal synovium. When calculated per gram of synovium, there was no statistically significant difference. This suggests that a possible role for cetebolin in the pathogenesis of OA might be related to the degree of synovial hypertrophy. / Medicine, Faculty of / Graduate

Most studies of the induction of cytokine synthesis in monocytes have employed an exogenous triggering agent such as lipopolysaccharide. However, in nonseptic inflammatory responses (e.g. rheumatoid arthritis) monocyte activation occurs as a result of T cell‐generated signals. In previous reports, we and others have demonstrated that contact‐dependent T cell‐generated signals are capable of contributing to macrophage activation. We have shown that plasma membranes from anti‐CD3 activated purified peripheral CD4+ T cells (TmA) but not from resting CD4+ cells (TmR) induce monocytes to synthesize interleukin (IL)‐1 in the absence of co‐stimulatory cytokines. Studies to determine the expression kinetics of the molecule(s) unique to activated CD4+ T cells which interact with monocytes to induce IL‐1 revealed that optimal expression occurred at 6 h post activation. This matched the previously reported kinetics of expression of CD40 ligand (CD40L) on activated peripheral T cells, implicating the CD40‐CD40L interaction as a candidate for the initiator of the IL‐1 signaling event. The ability of TmA to induce IL‐1 synthesis in resting monocytes could be markedly reduced by addition of a monoclonal anti‐CD40L antibody, 5c8. In addition, a monoclonal anti‐CD40 IgM (BL‐C4) proved dramatic in its ability to induce resting monocytes to synthesize IL‐1. In summary, these results demonstrate that the CD40‐CD40L interaction provides a critical component of CD4+ T cell contact‐dependent activation of monocyte IL‐1 synthesis.

IL-15 is a pleiotropic cytokine which influences a variety of immune and inflammatory responses. Our lab has previously demonstrated that IL-15 and IL-15Rα promote atherosclerosis. However, the mechanisms by which IL-15 affect atherosclerosis development were not fully defined. In this study, we reported that overexpression of the IL-15 gene resulted in an increase of granzyme B level in the atherosclerotic plaque of ApoE deficient mice. Furthermore, we observed that leukocytes-specific genetic deletion of IL-15Rα reduced the granzyme B level within atherosclerotic lesions from LDLr deficient mice. Collectively, our data shows one of the mechanistic pathways by which IL-15 promotes atherosclerosis development. Moreover, we tested the role of IL-15 in carotid artery disease. It has been reported previously that immunization of low density lipoprotein receptor deficient mice against IL-15, by inoculating them with bacteria harboring an IL-15 expression plasmid, led to reduced development of diet induced atherosclerosis in carotid arteries whose diameter was restricted to induce a hemodynamic stress. Others, however, reported that injection of wild type mice with an antibody against IL-15 triggered increased neointima formation in carotid arteries that were partially ligated. In our study, we found no differences in the amount of collar/diet induced atherosclerosis in control apoE KO mice and in IL-15/apoE dKO mice.Therefore,inactivation of IL-15 gene expression does not appear to affect the rapid onset of atherosclerosis in carotid arteries of ApoE KO mice induced by a combination of high fat diet and hemodynamic stress. / Thesis / Master of Science (MSc)