Ratos que não expressam a proteína prion celular (PrPc) são mais sensíveis a crises epilépticas induzidas por diferentes protocolos. O hipocampo desses animais apresenta um brotamento supragranular de fibras musgosas semelhante ao observado em pacientes com epilepsia de lobo temporal relacionada a esclerose hipocampal (ELT-EH). Esses achados sugerem que a PrPc pode estar envolvida na epileptogênese da ELT-EH. Nós estudamos nessa tese a localização imunoistoquímica da PrPc no hipocampo de animais submetidos ao modelo de epilepsia de lobo temporal por pilocarpina (MELTP)em diferentes tempos de status epilepticus em ratos. Nesse trabalho induzimos estado de mal epileptico (EE) com o uso de pilocarpina em três diferentes grupos de ratos Wistar adultos. Os animais foram sacrificados 18 horas, 5 dias e 2 meses após a indução do EE. Os resultados foram comparados com cérebros controles de ratos que receberam injeções de solução salina. As lâminas foram processadas para coloração por hematoxilinaeosina, imunohistoquímica e neo-Timm. Observamos um aumento da expressão de PrPc nas regiões CA1 e CA3 do hipocampo 18 horas depois da injeção de pilocarpina. Essa expressão aumentada persistiu na região CA1 no quinto dia após a injeção. Não observamos diferenças significativas na expressão de PrPc durante a fase aguda do MELTP nas regiões CA2 e granular do hipocampo. No grupo crônico (2 meses) a PrPc foi observada na mesma localização em que se observou brotamento de fibras musgosas. Concluímos com esse trabalho que a expressão da PrPc é diferente nas diversas fases do modelo de epilepsia induzido por pilocarpina. A expressão transitória da proteína prion durante a fase aguda do modelo pode refletir mudanças de expressão visando tornar as células mais resistentes ao dano induzido pelas crises convulsivas. Alternativamente, essa expressão aumentada pode estar relacionadas à apotose ou então às fases iniciais da neuroplasticidade. A expressão de PrPc na mesma região dos brotamentos de fibras musgosas na fase crônica pode estar relacionada à neuroplasticidade, epileptogênese, neurotransmissão ou, ainda, estar implicada na proteção celular contra crises convulsivas recorrentes. Devido aos diversos achados relacionados a PrPc, sugerimos que o modelo de epilepsia do lobo temporal induzido pela pilocarpina possa ser um interessante modelo para o estudo do papel fisiológico da PrPc. / Mice lacking cellular prion protein (PrPc) are more sensitive to seizures induced by four different pharmacological protocols. The hippocampal formation of these animals exhibits supragranular mossy fiber sprouting which resembles that observed in patients with mesial temporal lobe epilepsy related to hippocampal sclerosis (MTLEHS). These findings suggest that the PrPc may be involved in epileptogenesis in MTLE-HS. Here we investigated the immunohistochemical localization of the PrPc in the hippocampus of animals submitted to the pilocarpine model of temporal lobe epilepsy (PMTLE). Status epilepticus (SE) was induced with pilocarpine in three different groups of adult Wistar rats. The animals were sacrificed 18 hours, 5 days, and 2 months after SE induction and the results were compared to the respective saline-injected control animals. Slices were processed for hematoxylin-eosin, PrPc immunohistochemistry and neo-Timm .PrPc was increased in the CA1 and in CA3 regions of the hippocampus 18 hours after pilocarpine injection. PrPc continued to be increased in the CA1 region of the hippocampus five days after pilocarpine injection. In the CA2 and granular regions of the hippocampus we did not observe significant differences in PrPc expression during the acute phase of PMTLE. In the chronic group, PrPc was expressed co-localized with mossy fiber sprouting. Cellular prion protein is differentially expressed at different phases of the pilocarpine model of epilepsy. Transient expression of PrPc during the acute phase of the pilocarpine model may reflect changes which may render cells more resistant to seizure-induced damage and may be related to apoptosis or may to the initial phases of neuroplasticity. During the chronic period, PrPc is co-expressed in the same regions of mossy fiber sprouting. In chronic animals, PrPc might be related to neuroplasticity, epileptogenic processes, neurotransmission, or alternatively may be implicated in cellular protection against recurrent seizures.

Growth and deposition of amyloid fibrils, polymers of proteins with a cross beta-sheet structure, are associated with a significant number of human pathologies including Alzheimer’s disease, Parkinson’s disease, prion diseases, type II diabetes, and senile systematic or dialysis-related amyloidoses. The broader objective of my research is to identify the basic mechanisms regulating nucleation and growth of amyloid fibrils. There is increasing evidence that amyloid formation may proceed along at least two distinct assembly pathways for the formation of rigid fibrils. One pathway involves the nucleated polymerization of the characteristic rigid fibrils from partially denatured monomers and the other proceeds via the growth of globular oligomers and their associated curvilinear fibrils (also known as protofibrils) which, in ways yet to be determined, transform into late-stage rigid fibrils. These oligomeric intermediates of fibril assembly, in particular, have been implicated as the predominant aggregate species causing cellular toxicity in amyloid diseases. Yet, amyloid oligomers and curvilinear fibrils are considered transient, metastable aggregates. This raises the question whether and how such transient aggregate species can be responsible for most of the cell/tissue toxicity? In this dissertation, I report on my investigation of several basic questions related to the mechanisms of amyloid formation. Using the model amyloid hen egg-white lysozyme, I participated in research to characterize the distinct kinetics of amyloid formation along distinct assembly pathways, to determine the morphological features of the various aggregate species emerging along either pathway, and to investigate the structural evolution of the monomers from their native state to the amyloid cross- sheet structure (chapter 3). Chapters 4-6 represent the core of my dissertation work. There I investigated whether amyloid aggregates from three different amyloid proteins, formed under denaturing condition, could undergo prion-like proliferation upon return to physiological solution conditions. I was also intimately involved in a project on the conditions inducing amyloid spherulites formation by polyglutamic acid and the mechanisms resulting in the formation of this often-overlooked amyloid aggregate structure (chapter 7). In the appendix I provide a short summary of the various experimental techniques I have used in the above experiments.

One of the key molecular events in the transmissible spongiform encephalopathies or prion diseases is the conformational conversion of the cellular prion protein PrPC into the misfolded and pathogenic isoform, PrPSc. Prion diseases are fatal neurodegenerative conditions affecting humans and other animal species, which present with diverse clinical and neuropathological phenotypes. In humans, prion diseases can occur as sporadic, familial or acquired forms. Sporadic Creutzfeldt–Jakob disease (sCJD) accounts for the majority of cases. The current classification system of human prion diseases recognizes several distinct clinico-pathological entities including sCJD, variant Creutzfeldt-Jakob disease (vCJD), Gerstmann–Straussler–Scheinker syndrome, fatal familial insomnia and variably protease-sensitive proteinopathy. Prion protein gene (PRNP) mutations and polymorphisms, and PrPSc types have a profound effect on these clinico-pathological phenotypes. Prion diseases of sheep and goats, cattle, and cervids are all actual animal health problems and present potential risks to human health. Thus far the only known zoonotic prion disease is bovine spongiform encephalopathy, which has resulted in vCJD in humans. The recognition of new forms of prion diseases in animal and humans has generated increased awareness of the animal and public health risks associated with prion disease. However the mechanisms involved in prion replication, transmission, and neurodegeneration remain poorly understood. This thesis uses in vitro PrP conversion assays (protein misfolding cyclic amplification and real time quaking-induced conversion) to model different aspects of human prion replication: Molecular susceptibility, genetic compatibility, spontaneous formation and the effect of molecules that might enhance or prevent conversion were each investigated in order to obtain a better understanding of the molecular mechanism of the prion replication. I have addressed the hypothesis that the major determinant factors in prion disease pathogenesis (PRNP genetics, PrPSc types and species barriers) are intrinsic to the prion protein conversion process and their effects can be faithfully recapitulated by in vitro conversion assays. The results shows that in vitro conversion assays used in this thesis can model the combined effects of different PrP type and genotypes, can replicate aspects of cross-species transmission potential and provide information about molecular barrier to zoonotic transmission, can model de novo PrPSc formation, and can assess the potential impact of chaperones on conversion of the human prion protein. In summary, this work provides evidence that the origin, propagation and transmission of prions can be meaningfully investigated in cell-free systems.

Iron is essential for virtually all organisms, yet it can be highly toxic if not properly regulated. Only the Lyme disease pathogen Borrelia burgdorferi has evolved to not require iron (Aguirre et al., 2013).Recent findings have characterised elements of the iron metabolism network, but understanding of systemic iron regulation remains poor. To improve understanding and provide a tool for in silico experimentation, a computational model of human iron metabolism has been constructed. COPASI was utilised to construct a model that included detailed modelling of iron metabolism in liver and intestinal cells. Inter-cellular interactions and dietary iron absorption were included to create a systemic computational model. Parameterisation was performed using a wide variety of literature data. Validation of the model was performed using published experimental and clinical findings, and the model was found to recreate quantitatively and accurately many results. Analysis of sensitivities in the model showed that, despite enterocytes being the only route of iron uptake, almost all control over the system is provided by reactions in the liver. Metabolic control analysis identified key regulatory factors and potential therapeutic targets. A virtual haemochromatosis patient was created and compared to a simulation of a healthy human. The redistribution of control in haemochromatosis was analysed in order to improve our understanding of the condition and identify promising therapeutic targets. Cellular prion protein (PrP) is an enigmatic protein, implicated in disease when misfolded, but its physiological role remains a mystery. PrP was recently found to have ferric-reductase capacity. Potential sites of ferric reduction were simulated and the findings compared to PrP knockout mice experiments. I propose that the physiological role of PrP is in the chemical reduction of endocytosed ferric iron to its ferrous form following transferrin receptor-mediated uptake.

No description available.

Les maladies neurodégénératives amyloïdes sont caractérisées par la dégénération et l'agrégation de protéines spécifiques. Ces processus d'agrégations restent mal compris par les spécialistes, et pour la plupart, hypothétiques seulement. Dans cette thèse, faîte en collaboration avec des biophysiciens, nous analysons ces méchanismes d'agrégations en nous basant sur des données expérimentales. Pour cela, la modélisation est une étape incontournable. Nous présentons deux modèles que nous confrontons aux expériences. Le premier modèle, connu de la littérature, est celui de Becker-Döring. Un système infini d'équations différentielles ordinaires. Ce premier modèle nous permet de reproduire se manière satisfaisante les premières étapes des expériences. Le second modèle que nous introduisons, se base sur une hypothèse réactionnelle additionnelle, formulée à partir des simulations du premier modèle, et qui consiste en la formation de fibres différentes. Ce deuxième modèle nous permet de mieux reproduire les expériences. / The amyloid neurodegenerative diseases are characterized by the degeneration and the aggregation of specific proteins. These aggregation processes remain misunderstood by specialists and, mostly, only hypothetical. In this thesis, and in collaboration with biophysicists, we analyze the mechanisms of aggregation, relying on experimental data. Modeling is then a must. We present two models which we compare with the experiments. The first model, well-known from the literature is the Becker-Döring system. An infinite system of ordinary differential equations. This first model allows us to reproduce satisfactorily the early stages of the experiments. The second model we introduce is based on an additional hypothesis which is about the formation of different fibers. This second model allows us to reproduce the experiments.

The thesis is focused on bus systems and their control by GUI. Basic energy management principles and the mostly known bus systems are analysed in the first – theoretical part of the paper. The theoretical part is then focused on software tool ETS 4, which is used for activation of system installation KNX. Software tool SmartServer, which is used for communication between bus system KNX and web interface, is described at the end of the first theoretical part. Two laboratory boards assembled of KNX and DALI parts were constructed in the second – practical part. Three laboratory tasks, which are prepared so they are related to theoretical description in previous part, were submitted in next step. Aim of the first task is to introduce basics of the board and PriOn interface. The second task is focused on DALI components and temperature control with PriOn regulating device. Creating of basic visualization and controlling of the board with SmartServer is described in the last task.

Transmissive spongiform encephalopathies, also called prion disorders, are fatal neurodegenerative diseases affecting mammals. In patients, the pathological prion protein (PrPTSE ) accumulates in CNS and causes death. Prions possess high binding affinity to surfaces. Moreover, they are highly resistant to conventional sterilization procedures which rise the risk of nosocomial transmission from patients in subclinical stage of prion disease through medical tools. In the thesis, we evaluate the efficiency of photodynamic inactivation (PDI) for prion decontamination. The PDI is induced by photoactivation of phthalocyanine (Pc) derivates AlPcOH(SO3)2, SiPc(OH)2(SO3)1-3 or ZnPc(SO3)1-3. Pc exposed to light generate reactive oxygen species, mainly singlet oxygen (O2(1 ∆g)). Production of O2(1 ∆g) in aqueous solution was confirmed by iodide method, quenching by NaN3 and oxidative degradation of uric acid. The photoactivation of Pc in infectious brain homogenate led to elimination of PrPres signal (= proteinase K-resistant PrPTSE fragment) below the detection limit of western blot by using nanomolar AlPcOH(SO3)2 concentration. The complete elimination of PrPres signal was accompanied with total protein concentration decrease by a maximum of 20% in brain homogenate No signs of protein fragmentation or...

Ein Ziel dieser Arbeit war die Entwicklung eines differentialdiagnostischen Tests (FLI-Test), der die Abgrenzung einer BSE- von einer Scrapieinfektion durch die direkte Untersuchung des Hirnstammmaterials ermöglicht. Bei einem Teil der dabei untersuchten deutschen klassi-schen Scrapiefälle wurde diese Charakterisierung zusätzlich im bis dahin zur Differenzierung verwendeten klassischen Mausbioassay durchgeführt, um die Ergebnisse aus dem FLI-Test zu verifizieren und um die vorhandenen Scrapieisolate weitergehend zu charakterisieren. Im zweiten Teil dieser Arbeit wurden die biochemischen Eigenschaften atypischer deutscher Scrapieisolate analysiert und ihre Infektiosität anhand von Übertragungsversuchen auf drei Wildtypmauslinien und eine transgene Mauslinie beurteilt. Darüber hinaus wurden diese Isolate dem klassischen BSE-Isolat gegenüber gestellt.

Leila Kupfer Einflüsse der Aminosäuresequenz und erregerspezifischer Eigenschaften auf die Konvertierbarkeit chimärer Prion-Proteine in vitro Institut für Tierhygiene und Öffentliches Veterinärwesen, Veterinärmedizinische Fakultät, Universität Leipzig und Institut für Neue und Neuartige Tierseuchenerreger des Friedrich-Loeffler-Institutes, Bundesforschungsinstitut für Tiergesundheit, Insel Riems Eingereicht im Februar 2007 127 Seiten, 40 Abbildungen, 4 Tabellen, 230 Literaturangaben, 13 Seiten Anhang Schlüsselwörter: zellfreie Konversion, chimäres Prion-Protein, Aminosäuresequenz, stammspezifische Eigenschaften Die genauen molekularen Mechanismen bei der Entstehung der transmissiblen spongiformen Enzephalopathien (TSE) sind immer noch nicht eindeutig geklärt. Es wird vermutet, dass das auslösende Ereignis, die irreversible Umfaltung oder Konversion eines körpereigenen Membranproteins, des zellulären Prion-Proteins PrPC in seine krankheitsassoziierte, Proteinase K (PK) resistente Isoform PrPSc, eine ‚autokatalytische’ Konversionsreaktion initiiert. In verschiedenen Studien wurde gezeigt, dass dabei die Aminosäuresequenzen des PrPC und PrPSc die Effizienz dieser Konversionsreaktion sowohl bei Übertragungen innerhalb derselben Spezies als auch über Speziesgrenzen hinweg beeinflussen. Am Friedrich-Loeffler-Institut wurden in vergangenen Arbeiten zwei transgene Inzucht-Mauslinien etabliert, die auf der Basis von amino- und carboxyterminalen murinen Anteilen ein chimäres PrPC mit zentralen ovinen (Tgmushp XIX) oder bovinen (Tgmubo XIII) Sequenzen exprimieren. Erstaunlicherweise erwiesen sich die Tgmubo XIII-Mäuse als nahezu resistent gegen verschiedene TSE-Erreger aus Schaf, Rind und Maus, während die Tiere der Linie Tgmushp XIX ausgesprochen gut infizierbar waren. Da sich die PrP-Sequenz dieser beiden Mauslinien um lediglich vier Aminosäurereste unterscheidet, war es Ziel dieser Studie zu ermitteln, welcher Aminosäure-Austausch die verminderte Infizierbarkeit der Tgmubo XIII bedingt. Um kostenspielige und zeitaufwenige Tierversuche zu vermeiden, wurde hierzu ein zellfreier Konversionsansatz gewählt. Dieser birgt den Vorteil, dass die molekularen Einflüsse auf die Konversion in einem stark vereinfachten System innerhalb weniger Tage festgestellt werden können. Insgesamt wurden 19 verschiedene PrPC-Mutanten kloniert, in E. coli rekombinant exprimiert, affinitätschromatographisch aufgereinigt und im zellfreien Konversionsassay mit Zusammenfassung 85 ebenfalls hochaufgereinigtem PrPSc aus Gehirnextrakten final BSE- oder Scrapie-infizierter (drei unterschiedliche Erregerstämme: Me7, 22A, 87V) Mäuse für bis zu drei Tage inkubiert. Die stattgefundene Konversion der Mutanten wurde anschließend mittels PK-Verdau festgestellt. Die im Tierversuch ermittelten Ergebnisse für Tgmubo XIII und Tgmushp XIX konnten so in vitro für PrP-mubo und PrP-mushp bestätigt werden, d.h. nur letzteres wurde in ein PK-resistentes PrPres-Fragment konvertiert. Ausgehend von PrP-mushp wurden die vier unterschiedlichen Positionen gegen die jeweils entsprechende Aminosäure aus PrP-mubo mutiert. Dabei zeigte der Austausch von Asparagin gegen Serin auf Position 142 der chimären Sequenz den stärksten Effekt: PrP-mushpN142S ließ sich nur durch Koinkubation mit BSE-, aber nicht mehr durch Scrapie-PrPSc konvertieren. Substitutionen durch Alanin oder Glutamin führten zu einer weiteren Minderung der Konversionsrate. Auch beim Austausch mehrerer Aminosäuren in Kombination mit Serin an Position 142 wurde der hemmende Effekt dieser Substitution deutlich. Einen ähnlichen, wenn auch nicht ganz so starken Einfluss hatten Aminosäureaustausche auf Position 185. Wurde dort anstelle von Glutamin Glutamat eingefügt, konnte das so entstandene Konstrukt PrP-mushpQ185E nicht mehr durch Me7 aber noch durch 22A, 87V und BSE konvertiert werden. Wurde Glutamin durch Alanin oder Asparagin ersetzt, verminderte sich die Konversionsrate für 22A, 87V und BSE. Diese Effekte traten nur bei chimärem PrPC auf. Der singuläre Austausch der entsprechenden Aminosäuren 146 und 189 der ovinen Sequenz gegen die des bovinen PrPC zeigten keine derartige Hemmung der Konvertierbarkeit. Genauso wenig hatte die Deletion des Aminoterminus der chimären Prion-Proteine einen hemmenden Effekt. Lediglich nach Inkubation mit mauspassagierter BSE stellte sich das PrPres-Fragment des PrP-mushpΔ94 als Doppelbande im Immunoblot dar, deren Bedeutung jedoch unbekannt ist. Durch den zellfreien Konversionsassay konnten die im Tierversuch ermittelten Ergebnisse bezüglich der Empfänglichkeit der transgenen, chimäres Prion-Protein exprimierenden Mauslinien bestätigt werden. Die Erkenntnis, dass zwei Aminosäuren der chimären Sequenz für die Inkonvertibilität verantwortlich sind, zeigt, dass ein direkter Zusammenhang zwischen der Primärstruktur des Prion-Proteins und dessen Überführbarkeit in seine pathologische Isoform besteht. Zusätzlich konnten stammspezifische Effekte auf die zellfreie Konversion ermittelt werden, die zum einen ebenfalls von der Aminosäuresequenz des rekombinanten PrPC, zum anderen aber auch von den Eigenschaften des jeweiligen eingesetzten Stammes abhängen. Die in der vorgestellten Arbeit ermittelten Ergebnisse untermauern die Prionhypothese, wonach einzelne Aminosäuren des Prion-Proteins die Erregervermehrung maßgeblich beeinflussen können. Allerdings werfen die beobachteten stammspezifischen Effekte auch bisher ungelöste Fragen zu den dabei zugrunde liegenden Mechanismen auf