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11	Probing reaction conditions and cofactors of conformational prion protein changes underlying the autocatalytic self-propagation of different prion strains Boerner, Susann 15 July 2014 (has links) Prionen sind das infektiöse Agens transmissibler spongiformer Enzephalopathien von Tieren und Menschen. Prionen bestehen hauptsächlich aus einer abnormal gefalteten und aggregierten Isoform des zellulären Prionproteins (PrP). Die Replikation von Prionen findet mutmaßlich durch keiminduzierte Polymerisation des Prionproteins statt. Es existieren verschiedene Prionstämme, die unterschiedliche Eigenschaften aufweisen, aber vom selben zellulären Prionprotein abstammen können. Neben PrP scheinen Kofaktormoleküle an der Prionreplikation beteiligt zu sein. Weiterhin wird angenommen, dass Kofaktoren bei der Definition von Stammeigenschaften beteiligt sind, sowie ein Einfluss auf die Infektiosität von Prionen besteht. In dieser Arbeit wurden die Auswirkungen verschiedener Kofaktoren auf die Replikation von vier Hamster-adaptierten Prionstämmen in vitro mittels der Methode der „Protein Misfolding Cyclic Amplification“ (PMCA) untersucht. Es wurden stammabhängige Unterschiede bezüglich der Anforderungen an die Replikationsbedingungen in der PMCA, sowie Kofaktor-Selektivitäten festgestellt. Der Einfluss von Kofaktoren wurde durch den Vergleich ausgewählter biologischer, biochemischer und biophysikalischer Eigenschaften von in vitro erzeugten PMCA Produkten (PrPres) mit denen nativer Prionkeime untersucht. Es zeigte sich, dass Kofaktoren Stammeigenschaften, wie die biologische Keimaktivität in primären Gliazellkulturen und biochemische Eigenschaften, wie die Migration in SDS-Gelen, beeinflussen können. Um festzustellen, ob unterschiedliche Kofaktorbedingungen während der PMCA messbare Veränderungen der Proteinkonformation hervorrufen, wurde PMCA generiertes PrPres mittels FT-IR Spektroskopie in einer Pilotstudie charakterisiert. Erste Befunde zeigten spektrale Unterschiede zwischen den Proteinkeimen und deren PMCA Produkten bei allen Stämmen, unabhängig von den Kofaktorbedingungen. / Prions are the causative agent of transmissible spongiform encephalopathies in animals and humans such as scrapie, bovine spongiform encephalopathy (BSE) and Creutzfeldt-Jakob disease (CJD). Prions are thought to be composed essentially of a misfolded and aberrantly aggregated isoform of the cellular prion protein (PrP) and to replicate by seeded PrP polymerization. Prions may exist in the form of distinct strains that differ in their phenotypic characteristics although they are derived from the same cellular prion protein. Cofactor molecules other than PrP may be involved in prion replication and may be a determinant of strain properties. Furthermore, cofactors may also be required for conveying infectivity. The present study examined the effects of different cofactor molecules on the replication efficacy of four hamster adapted prion agents using the method of serial protein misfolding cyclic amplification (PMCA) as in vitro assay for PrP misfolding and aggregation. The study revealed strain dependent differences of PMCA conditions and cofactors required for efficient in vitro replication. The impact of cofactors was assessed by comparative analyses of selected biological, biochemical and biophysical properties of PMCA products (PrPres) and native prion seeds. The biological seeding activity as monitored in a primary hamster glial cell assay, and biochemical properties such as electrophoretic migration in SDS-gels, were affected differently by different cofactors. In order to define the impact of putative cofactors on the molecular conversion of PrP in more detail, changes in the spatial structure associated with different cofactor molecule conditions during amplification of PrPres in PMCA was monitored by Fourier transform-infrared (FT-IR) spectroscopic analysis. Largely preliminary data revealed spectral differences between native prion seeds and progeny PMCA generated PrPres for all prion strains, but no variations due to different cofactor conditions. Prion Prionprotein Zellassay Kofaktoren Prion Prion protein Transmissible spongiform encephalopathy Cell assay Conversion cofactors 570 Biowissenschaften, Biologie 32 Biologie WD 5280 ddc:570
12	Investigating the porphyrias through analysis of biochemical pathways. Ruegg, Evonne Teresa Nicole January 2014 (has links) ABSTRACT The porphyrias are a diverse group of metabolic disorders arising from diminished activity of enzymes in the heme biosynthetic pathway. They can present with acute neurovisceral symptoms, cutaneous symptoms, or both. The complexity of these disorders is demonstrated by the fact that some acute porphyria patients with the underlying genetic defect(s) are latent and asymptomatic while others present with severe symptoms. This indicates that there is at least one other risk factor required in addition to the genetic defect for symptom manifestation. A systematic review of the heme biosynthetic pathway highlighted the involvement of a number of micronutrient cofactors. An exhaustive review of the medical literature uncovered numerous reports of micronutrient deficiencies in the porphyrias as well as successful case reports of treatments with micronutrients. Many micronutrient deficiencies present with symptoms similar to those in porphyria, in particular vitamin B6. It is hypothesized that a vitamin B6 deficiency and related micronutrient deficiencies may play a major role in the pathogenesis of the acute porphyrias. In order to further investigate the porphyrias, a computational model of the heme biosynthetic pathway was developed based on kinetic parameters derived from a careful analysis of the literature. This model demonstrated aspects of normal heme biosynthesis and illustrated some of the disordered biochemistry of acute intermittent porphyria (AIP). The testing of this model highlighted the modifications necessary to develop a more comprehensive model with the potential to investigated hypotheses of the disordered biochemistry of the porphyrias as well as the discovery of new methods of treatment and symptom control. It is concluded that vitamin B6 deficiency might be the risk factor necessary in conjunction with the genetic defect to trigger porphyria symptoms. Acute attack Acute intermittent porphyria Aminolevulinate Aminolevulinate dehydratase Aminolevulinate synthase B vitamins Biomodeling Cofactors Computational modeling Congenital erythopoietic porphyria Coproporphyrinogen oxidase Erythropoietic protoporphyria Ferrochelatase GABA Glucose therapy Glycine Heme Heme biosynthesis Heme deficiency Heme therapy Hepatoerythropoietic porphyria Hepatorenal Tyrosemia Hereditary coproporphyria Iron Iron deficiency anemia Kinetics Lead poisoning Liver transplant Micronutrients PLP Porphobilinogen Porphobilinogen deaminase Porphyria Porphyria cutanea tarda Porphyrins Prophylaxis Protoporphyrinogen oxidase Pyridoxal Pyridoxal phosphate Pyridoxine Serotonin Succinyl-CoA TCA cycle Tryptophan Tryptophan pyrrolase Uroporphyrinogen decarboxylase Uroporphyrinogen synthase Variegate porphyria Vitamin B6 Vitamin therapy Vitamins X-linked protoporphyria X-linked sideroblastic anemia

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Probing reaction conditions and cofactors of conformational prion protein changes underlying the autocatalytic self-propagation of different prion strains

Investigating the porphyrias through analysis of biochemical pathways.