[pt] Derivados do núcleo 2,1,3-benzotiadiazola (BTD) são altamente emissivos e têm reconhecida aplicação em diversas áreas da tecnologia da luz (células solares sensibilizadas por corantes, sondas fluorescentes, dispositivos emissores tipo OLEDs, etc). Derivados de BTD substituídos com grupos estireno têm sido descritos como ótimas sondas fluorescentes para estudo de conformações de proteínas. Além disso, o grupo de pesquisa do LaSOQF tem produzido novos derivados de BTD luminescentes e buscado diferentes aplicações para os mesmos. Assim sendo, o objetivo desse trabalho foi desenvolver/otimizar a síntese de novos derivados de BTD estiril-substituídos. Nesse planejamento almejou a obtenção de compostos com intensa luminescência a partir da substituição de um grupo estiril por grupos 4-metóxifenil, 4-metoxifenóxi ou 2-bifenil, buscando acentuar o caráter de transferência de carga intramolecular (ICT) e, também, características de emissão aumentada induzida por agregação (AIEE). Para a obtenção dos compostos desejados, os derivados bromados de BTD foram submetidos a reações de Heck com o estireno. Dessa forma, as moléculas alvo foram obtidas com rendimentos entre 36 por cento e 87 por certo. Em seguida, a caracterização fotofísica dos fluoróforos foi realizada e foram obtidos máximos de absorção entre 404 e 450 nm e máximos de emissão entre 503 e 578 nm, indicando a alta estabilidade do estado excitado (delta lambda igual 90-121 nm). Os valores determinados de [fi]FL (0,31-0,69) confirmaram a alta emissividade dos compostos. Ademais, o composto 4-(4-metoxifenóxi)-7-estirilbenzo[c][1,2,5]tiadiazola apresentou características de AIEE. As novas moléculas desenvolvidas estão sendo aplicadas como sondas fluorescentes para a determinação de água em soluções etanólicas comerciais. / [en] The 2,1,3-benzothiadiazole (BTD) core derivatives are highly emissive and with recognized application in several areas of light technology (dye-sensitized solar cells, fluorescent probes, emitters devices such as OLEDs, etc.). BTD derivatives substituted with styrene group have been described as excellent fluorescent probes for protein conformations study. In addition, the LaSOQF research group has been producing new luminescent BTD derivatives and proposed different applications for them. Therefore, the objective of this work was to develop/optimize the synthesis of novel styryl-BTD derivatives. This design aimed at obtaining more luminescent compounds from the replacement of one of the styryl groups by 4-methoxyphenyl, 4-methoxyphenoxy or 2-biphenyl, seeking for intramolecular charge transfer (ICT) and also aggregated-induced enhanced emission (AIEE) characteristics. To obtain the desired compounds, brominated BTD derivatives were submitted to Heck reactions with styrene. Thus, the target fluorophores were obtained with yields ranging between 36 per cent and 87 per cent. Then, photophysical characterization of the fluorophores was carried out and absorption maxima between 404 and 450 nm and emission maxima between 503 and 578 nm were obtained, indicating the high stability of the excited state (delta lambda equal 90-121 nm). The determined values of [fi]FL (0.31-0.69) confirmed the high emissivity of the compounds. In addition, the compound 4-(4-methoxyphenoxy)-7-styrylbenzo[c] [1,2,5]thiadiazole showed of AIEE properties. The novel molecules developed are being applied as fluorescent probes for determination of water in commercial ethanolic solutions.

En övergång till bilar utrustade med avancerade automatiska säkerhetssystem (ADAS) och även utvecklingen mot självkörande fordon innebär ökad trafik på den lokala databussen. Det finns således ett behov av att både minska den faktiska mängden data som överförs, samtidigt som värdet på datat ökas. Data aggregation tillämpas i dagsläget inom områden såsom trådlösasensornätverk och mindre mobila robotar (WMR’s) och skulle kunna vara en del av en lösning. Denna rapport avser undersöka aggregation av sensordata i ett tidskänsligt system. För ett användarfall gällande halka under konvojkörning testas en aggregationsstrategi genom implementation på en fysisk demonstrator. Demonstratorn består av ett autonomt fordon i mindre skala som befinner sig i en konvoj med ett annat identiskt fordon. Resultaten pekar mot att ett viktat medelvärde, som i realtid anpassar sin viktning baserat på specifika sensorers koherens, med fördel kan användas för att estimera fordonshastighet baserat på individuella hjuls sensordata. Därefter kan en slip ratio beräknas, vilket avgör om fordonet befinner sig i ett tillstånd av halka eller ej. Begränsningar för den undersökta strategin inkluderar antalet icke-halkande hjul som behövs för tillförlitliga resultat. Simulerade resultat antyder att extra hastighetsreferenser behövs för tillförlitliga resultat. Relaterat till användarfallet konvojkörning föreslås att andra fordon används som hastighetsreferens. Detta skulle innebära en ökad precision för estimeringen av fordonshastigheten samt utgöra en intressant sammanslagning av områdena samarbetande cyberfysiska system (CO-CPS) och dataaggregation. / With an impending shift to more advanced safety systems and driver assistance (ADAS) in the vehicles we drive, and also increased autonomousity, comes increased amounts of data on the internal vehicle data bus. There is a need to lessen the amount of data and at the same time increase its value. Data aggregation, often applied in the field of environmental sensing or small mobile robots (WMR’s), could be a partial solution. This thesis choses to investigate an aggregation strategy applied to a use case regarding slip detection in a vehicle convoy. The approach was implemented in a physical demonstrator in the shape of a small autonomousvehicle convoy to produce quantitative data. The results imply that a weighted adaptive average can be used for vehicle velocity estimation based on the input of four individual wheel velocities. There after a slip ratio can be calculated which is used to decide if slip exists or not. Limitations of the proposed approach is however the number of velocity references that is needed since the results currently apply to one-wheel slipon a four-wheel vehicle. A proposed future direction related to the use case of convoy driving could be to include platooning vehicles as extra velocity references for the vehicles in the convoy, thus increasing the accuracy of the slip detection and merging the areas of CO-CPS and data aggregation.

Superaggregates are clusters formed by diverse aggregation mechanisms at different scales. They can be found in fluidized nanoparticles and soot formation. An aggregate, with a single aggregation mechanism, can be described by the fractal dimension, df , which is the measure of the distribution and configuration of primary particles into the aggregates. Similarly, a su-peraggregate can be analyzed by the different fractal dimensions that are found at each scale. In a fractal structure aggregate, a self-similarity can be identified at different scales and it has a power law relation between the mass and aggregate size, which can be related to properties like density or light scattering. The fractal dimension, df , can be influenced by aggregation mechanism, particles concentration, temperature, residence time, among other variables. More-over, this parameter can help on the estimation of aggregates’ properties which can help on the design of new processes, analyze health issues and characterize new materials.A multi-dimensional soot aggregate was simulated with the following approach. The first aggregation stage was modeled with a Diffusion Limited cluster-cluster aggregation (DLCA) mechanism, where primary clusters with a fractal dimension, df1, close to 1.44 were obtained. Then, the second aggregation stage was specified by Ballistic Aggregation (BA) mechanism, where the primary clusters generated in the first stage were used to form a superaggregate. All the models were validated with reported data on different experiments and computer models. Using the Ballistic Aggregation (BA) model with primary particles as the building blocks, the fractal dimension, df2, was close to 2.0, which is the expected value reported by literature. However, a decrease on this parameter is appreciated using primary clusters, from a DLCA model, as the building blocks because there is a less compact distribution of primary particles in the superaggregate’s structure.On the second aggregation stage, the fractal dimension, df2, increases when the superaggre-gate size increases, showing an asymptotic behavior to 2.0, which will be developed at higher scales. Partial reorganization was implemented in the Ballistic Aggregation (BA) mechanism where two contact points between primary clusters were achieved for stabilization purposes. This implementation showed a faster increase on the fractal dimension, df2, than without par-tial reorganization. This behavior is the result of a more packed distribution of primary clusters in a short range scales, but it does not affect the scaling behavior of multi-dimensional fractal structures. Moreover, the same results were obtained with different scenarios where the building block sizes were in the range from 200 to 300 and 700 to 800 primary particles.The obtained results demonstrate the importance of fractal dimension, df , for aggregate characterization. This parameter is powerful, universal and accurate since the identification of the different aggregation stages in the superaggregate can increase the accuracy of the estimation of properties, which is crucial in physics and process modeling.

Huntington’s Disease (HD) ist eine neurodegenerative Erkrankung, die sich durch motorische, kognitive sowie psychiatrische Beeinträchtigungen auszeichnet. Die Verlängerung eines Polyglutamin (polyQ)-Abschnittes im Protein Huntingtin (Htt) über 37 Qs hinaus bedingt die Aggregation des mutierten Proteins (mHtt) und dessen Ablagerung in neuronalen Einschlüssen. Als potenzieller Modulator der polyQ-abhängigen mHtt Aggregation und Toxizität wurde der Q-reiche Transkriptionstransaktivator SS18L1 in silico identifiziert. Rekombinantes Volllängen-SS18L1 sowie die beiden verkürzten Fragmente SS18L1_NM und SS18L1_C weisen in wässriger Lösung einen hohen Anteil an Random-Coil-Strukturen auf und bilden Oligomere. Alle SS18L1-Proteine verzögern dosisabhängig die spontane Aggregation eines Htt Exon 1 Fragmentes mit 49 Glutaminen (Ex1Q49). Dabei wird die Entstehung SDS-stabiler Ex1Q49 Aggregate durch die Stabilisierung von Ex1Q49 Mono- und Oligomeren gebremst. In HEK293 Zellen verringern rekombinante SS18L1-Proteine sowohl die Anzahl der SDS-unlöslichen Ex1Q49-Aggregate als auch die mHtt-vermittelte Zytotoxizität. Auch hierbei scheint eine Stabilisierung früher Aggregatspezies, wahrscheinlich durch die Interaktion der SS18L1-Proteine mit dem jeweiligen mHtt Fragment, eine wesentliche Rolle zu spielen. Entsprechende Interaktionen konnten mittels LUMIER-Studien und konfokaler Mikroskopie nachgewiesen werden. Humanes, exogenes SS18L1 unterdrückt die polyQ-bedingte Aggregation in einem C. elegans-Modell für HD und in transgenen R6/2 HD-Mäusen sind die Mengen an endogenem SS18L1 im Vergleich zu Wildtyp-Mäusen verändert. Beides weist darauf hin, dass SS18L1 auch in vivo von Relevanz sein könnte. Dafür spricht zudem, dass murines SS18L1 in Gehirnen von R6/2-Mäusen mit neuronalen mHtt-Aggregaten co-lokalisiert. Die Ergebnisse dieser Arbeit könnten einen Ausgangspunkt für die Entwicklung neuer Therapieansätze und die weitere Erforschung der HD Pathologie darstellen. / Huntington’s Disease (HD) is a neurodegenerative disease, which is characterised by motor, cognitive and psychiatric disturbances. The abnormal extension of an N-terminal polyQ tract in the protein huntingtin (Htt) results in aggregation of the mutant protein (mHtt) and the deposition of neuronal inclusions. The Q-rich transcriptional transactivator SS18L1 was identified in silico as a potential modulator of polyQ-mediated mHtt aggregation and toxicity. Recombinant full-length SS18L1 and the truncated fragments SS18L1_NM and SS18L1_C have a high random-coil content and form oligomeric structures in aqueous solutions. In addition, all three proteins delay the spontaneous aggregation of an Htt exon 1 fragment with a stretch of 49 glutamines (Ex1Q49). The formation of SDS-resistant Ex1Q49 aggregates is postponed in a concentration-dependent manner as monomers and oligomers, appearing early in the amyloid formation cascade, are stabilised. In mammalian cells recombinant SS18L1 proteins reduce both the number of SDS-stable Ex1Q49 aggregates and mHtt-induced cytotoxicity. These effects are likely due to the stabilisation of early aggregation intermediates, which could result from interactions of the SS18L1 proteins with the respective mutant Htt exon 1 fragment. Such interactions have been demonstrated employing a LUMIER assay and confocal microscopy. Exogenous human SS18L1 suppresses polyQ-mediated aggregation in a C. elegans model of HD and levels of endogenous SS18L1 are altered in transgenic R6/2 HD mice compared to wild type mice. As a consequence, SS18L1 might be of relevance in vivo. This is also supported by the finding that murine SS18L1 interacts with mHtt inclusions in R6/2 mice. The results of this study could provide a basis for the development of a therapeutical strategy or for the further elucidation of HD pathology.

Neurodegenerative Erkrankungen (NDs) wie Alzheimer (AD), Parkinson (PD), und amyotrophe lateral Sklerose (ALS) sind Hirnerkrankungen, die durch unlösliche Proteinaggregate in Neuronen oder im Extrazellularraum charakterisiert sind. In dieser Arbeit habe ich für verschiede bekannte und vorhergesagte neurodegenerative Krankheitsproteine (NDPs) Proteininteraktionsnetzwerke erstellt, um mögliche gemeinsame Krankheitsmechanismen genauer zu studieren. Mit Hilfe eines automatisierten Hefe-Zwei-Hybrid-Systems (Y2H) konnte ich 18.663 Protein-Protein-Interaktionen (PPIs) für 449 wildtyp und 22 mutierte Proteine identifizieren. Eine genaue funktionelle Analyse der Interaktionspartner von korrespondierenden wildtyp und mutierten Proteinen ergab deutliche Unterschiede zum einen im Fall von allen untersuchten Proteinen und insbesondere im Fall vom ALS Krankheitsprotein TDP-43. Die identifizierten PPIs wurden außerdem verwendet um krankheitsspezifische Netzwerke zu erstellen und um Proteine zu identifizieren, die mit mehreren NDPs verbunden sind. Ich habe auf diese Weise vier Proteine (APP, IQSEC1, ZNF179 und ZMAT2) gefunden, die mit bekannten NDPs with Huntingtin, TDP-43, Parkin und Ataxin-1 interagieren und so fünf verschiedene NDs miteinander verbinden. Die Reduktion der mRNA Expression von IQSEC1, ZNF179 oder ZMAT2 mit Hilfe von siRNA führte zu einer Verstärkung von pathogenen Mechanismen wie der Aggregation von mutiertem Huntingtin und TDP-43 sowie der Hyperphosphorylierung des Proteins Tau. Außerdem habe ich 22 Proteine entdeckt, die die Aggregation von TDP-43 deutlich verändern und außerdem Mitglieder in sieben vorhergesagten Proteinkomplexen sind. Die Proteinkomplexe habe ich durch Kombination von Interaktionsdaten und Daten eines siRNA Screenings vorhergesagt. Zusätzlich habe ich herausgefunden, dass die Proteine eines vorhergesagten Komplexes, nämlich HDAC1, pRB, HP1, BRG1 und c-MYC, die Aggregation von TDP-43 durch Veränderung von dessen Genexpression beeinflussen. / Neurodegenerative diseases (NDs) such as Alzheimer’s disease (AD), Parkinson’s disease (PD) or amyotrophic lateral sclerosis (ALS) are progressive brain disorders characterized by the accumulation of insoluble protein aggregates in neuronal cells or the extracellular space of patient brains. To elucidate potential common pathological mechanisms in different NDs, I created comprehensive interaction networks for various known and predicted neurodegenerative disease proteins (NDPs). I identified 18,663 protein-protein interactions (PPIs) for 449 bioinformatically selected wild-type target proteins and 22 mutant variants of 11 known NDPs by using an automated yeast two-hybrid (Y2H) system. The functional analysis of the interaction partners of corresponding wild-type and mutant NDPs revealed strong differences in the case of all 11 NDPs and especially for the ALS protein TDP-43. The identified PPIs were used to generate networks for individual NDs such as AD or PD and to identify proteins that are connected to multiple NDPs. For example, I found that five neurodegenerative diseases are connected by four proteins (APP, ZMAT2, ZNF179 and IQSEC1) that link known NDPs such as huntingtin, TDP-43, parkin, ataxin-1 and SOD1. Analysis of publicly available gene expression data suggested that the mRNA expression of the four proteins is abnormally altered in brains of ND patients. Moreover, the knock-down of IQSEC1, ZNF179 or ZMAT2 aggravates pathogenic disease mechanisms such as aggregation of mutant huntingtin or TDP-43 as well as hyperphosphorylation of tau. Additionally, I identified 22 modifiers of TDP-43 aggregation, which are members in 7 protein complexes. These complexes were predicted based on combined data from PPI as well as siRNA screenings. Finally, I found that the proteins HDAC1, pRB, HP1, BRG1 and c-MYC, which form one of the predicted complexes, influence TDP-43 aggregation by altering its mRNA expression.

Rapide Fortschritte in der Netzwerk- und Speichertechnologie haben dazu geführt, dass Informationen über viele verschiedene Quellen wie z.B. Personal Computer oder Datenbanken verstreut sind. Weil diese Informationen oft auch sehr heterogen sind, wurde gleichzeitig die Entwicklung effektiver Softwaretechniken zur Datensammlung und -integration vorangetrieben. Diese werden beispielsweise in Online-Katalogen von Bibliotheken oder in Internetsuchmaschinen eingesetzt und ermöglichen eine breitgefächerte Suche von Informationen unterschiedlichster Art und Herkunft. In sensiblen Anwendungsgebieten kann der Einsatz solcher Techniken aber zu einer Gefährdung der Privatsphäre der Datenhalter führen. Bei der Erforschung häufig auftretender Krankheiten beispielsweise sammeln und analysieren Wissenschaftler Patientendaten, um Muster mit hohem Erkrankungspotenzial zu erkennen. Dazu werden von den Forschern möglichst präzise und vollständige Daten benötigt. Der Patient hat dagegen großes Interesse am Schutz seiner persönlichen Daten. Dieser Interessenkonflikt zwischen Datenhaltern und Nutzern tritt auch in anderen Konstellationen wie beispielsweise in Internetdiensten auf, die die Eingabe von persönlichen Finanz- und Steuerdaten erfordern. Oft kann ein qualitativ höherwertiger Dienst angeboten werden, wenn persönliche Informationen preisgegeben werden. Über die hierzu notwendige Abwägung von Datenschutz und Dienstqualität sind sich nicht alle Datenhalter im Klaren und neigen zu Extremverhalten wie der Übermittlung aller persönlicher Daten oder gar keiner. Diese Dissertation erforscht den Grenzbereich zwischen den scheinbar konträren Interessen von Datenhaltern und Dienstnutzern. Dabei werden insbesondere die technischen Möglichkeiten zur Modellierung und Beschreibung dieses Bereiches betrachtet. Die erarbeiteten Techniken sollen den beteiligten Parteien ermöglichen, den bestehenden Konflikt unter Einbeziehung ihrer Präferenzen zur beiderseitigen Zufriedenheit zu lösen. Die Beiträge dieser Dissertation sind im Einzelnen: - Eine Klassifizierung von Dienstarchitekturen im Hinblick auf Datenschutzprobleme Verschiedene Dienstarchitekturen werden nach ihrer Datenschutzproblematik klassifiziert. Für jede Kategorie werden praktische Anwendungen erläutert. - Entwurf, Analyse und Implementierung einer verschlüsselungsbasierten Dienstarchitektur in einer nicht vertrauenswürdigen 2-Parteien-Umgebung Es werden Gründe für Vertrauen von Datenhaltern in Anbieter von netzbasierten Diensten dargestellt. Für Fälle, in denen dieses Vertrauen alleine nicht ausreicht, wird eine Datenschutz garantierende Dienstarchitektur abgeleitet, die auf einem modifizierten Verschlüsselungsalgorithmus basiert. Wichtige Datenbankoperationen und arithmetische Elemente werden auf die verschlüsselten Daten übertragen und in beispielhaften Diensten zum Einsatz gebracht. - Entwurf, Analyse und Implementierung einer aggregationsbasierten Dienstarchitektur in einer nicht vertrauenswürdigen 3-Parteien-Umgebung Am Beispiel eines den Datenschutz verletzenden Gesundheitsberichts wird gezeigt, wie Methoden des Operations Research dazu eingesetzt werden können, aus veröffentlichten Statistiken enge Intervalle für vertrauliche numerische Daten abzuleiten ("Intervallinferenz"). Zur Lösung des Interessenkonflikts zwischen Datenhaltern und Dienstnutzern wird die Verwendung eines sogenannten Datenschutzmediators vorgeschlagen. Dessen Kernkomponente ist die "Audit & Aggregate" Methodologie, die das Auftreten von Intervallinferenz aufdecken und verhindern kann. - Quantifizierung der Datenschutzabwägungen und Schlussfolgerungen für den elektronischen Handel Es werden verschiedene Ansätze zur Quantifizierung der Datenschutzabwägungen betrachtet und Schlussfolgerungen für den elektronischen Handel gezogen. Zusammengefasst versucht diese Arbeit, (a) die Wahrnehmung von Datenhaltern und Dienstnutzern für den bestehenden Interessenkonflikt zu erhöhen, (b) einen Rahmen zur Modellierung der Datenschutzabwägungen bereitzustellen und (c) Methoden zu entwickeln, die den Interessenkonflikt zur beiderseitigen Zufriedenheit beilegen können. / Recent developments in networking and storage technology have led to the dissemination of information over many different sources such as personal computers or corporate and public databases. As these information sources are often distributed and heterogeneous, effective tools for data collection and integration have been developed in parallel. These tools are employed e.g. in library search catalogues or in Internet search engines to facilitate information search over a wide range of different information sources. In more sensitive application areas however, the privacy of the data holders can be compromised. In medical disease research for example, scientists collect and analyze patient data for epidemiological characterizations and for the construction of predictive models. Whereas the medical researchers need patient data at the highest level of detail, patients are only willing to provide data when their privacy is guaranteed. This conflict of interest between the data holders and the users occurs in many different settings, for example in the use of web-based services that require confidential input data such as financial or tax data. The more accurate and rich the provided private information, the higher the quality of the provided service. Not all data holders are aware of this trade-off and for lack of knowledge tend to the extremes, i.e. provide no data or provide it all. This thesis explores the borderline between the competing interests of data holders and service users. In particular, we investigate the technical opportunities to model and describe this borderline. These techniques allow the two opposing parties to express their preferences and to settle the conflict with a solution that is satisfactory to both. The specific contributions of this thesis are the following: - Privacy classification of service architectures We present a privacy classification of different service architectures after the number of involved parties and the reactivity of the data provision. For each class, we provide examples of practical applications and explain their relevance by discussing preceding cases of real-world privacy violations. - Design, analysis and implementation of an encryption-based service architecture in an untrusted two-party environment We analyze the foundations of trust in web-based services and point out cases where trust in the service provider alone is not enough e.g. for legal requirements. For these cases, we derive a new privacy-preserving architecture that is based on an adapted homomorphic encryption algorithm. We map important database and arithmetic operations from plain data to encrypted data, and we present sample services that can be carried out within the framework. - Design, analysis and implementation of an aggregation-based service architecture in an untrusted three-party environment Using a privacy-compromising health report as a running example through the thesis, we show how mathematical programming can be used to derive tight intervals for confidential data fields from non-critical aggregated data. We propose a new class of privacy mediators that settle the conflict between data holders and service users. A core component is the "audit & aggregate" methodology that detects and limits this kind of disclosure called interval inference. - Quantification of the privacy trade-off and implications for electronic commerce and public policy We analyze several frameworks to quantify the trade-off between data holders and service users. We also discuss the implications of this trade-off for electronic commerce and public policy. To summarize, this thesis aims to (a) increase data holders'' and service users'' awareness of the privacy conflict, (b) to provide a framework to model the trade-off and (c) to develop methods that can settle the conflict to both parties'' satisfaction.

Das Amyloid-beta Peptid (Ab) ist der Hauptbestandteil der extrazellulären Plaques bei der Alzheimerschen Krankheit. Das Ziel der vorliegenden Arbeit ist es, die Mechanismen der Wechselwirkungen des Ab mit der Plasmamembran und der nachfolgenden zellulären Aufnahme aufzuklären. Die Aggregation, die zelluläre Aufnahme und die Zytotoxizität von Ab42 wurden durch Verwendung von fluoreszenzmarkierten Ab42 in einem Neuroblastomzellkulturmodell untersucht. Sowohl bei Inkubation mit Monomeren als auch mit Aggregaten wurde in den Zellen Ab42 detektiert. Dabei binden Ab42 Monomere und kleine Aggregate zunächst an die Zellmembran. Allerdings erfolgt keine direkte Aufnahme von Monomeren in die Zelle. Erst nach Ausbildung von Aggregaten mit geordneter Sekundärstruktur wurde Ab42 in den endozytotischen Vesikel detektiert. Voraussetzung für den an der Membran ablaufenden Aggregationsprozess ist, dass die Monomere oberhalb einer kritischen Konzentration anwesend sind, um eine Bildung von beta-Faltblatt-Strukturen (bF) und entsprechenden Aggregaten zu ermöglichen. Ab42 Aggregate, die sich durch eine bF auszeichneten, benötigten keine kritische Schwellenkonzentration für die endozytotische Aufnahme. Eng mit der Aufnahme von Ab42 Aggregaten war die Veränderung des zellulären Metabolismus verbunden. Um die Wechselwirkung zwischen Ab und der Membrannäher zu charakterisieren, wurden Modellmembransystemen einschl. riesigen Membranvesikeln genutzt. Dabei wurde beobachtet, dass sowohl Ab42 als auch Ab40 Einstülpungen in der Membran induzieren können. Kleine Aggregate beider Isoformen, die noch keine bF aufweisen, interagierten bevorzugt mit der ungeordneten Lipidphase und induzierten dabei eine negative Membrankrümmung. Diese Beobachtungen legen den Schluss nahe, dass möglicherweise das Ab selbst den endozytotischen Prozess unterstützt oder diesen sogar einleiten könnte. Dies könnte auch auf eine mögliche physiologische Funktion von Ab Aggregaten, die nicht toxisch sind, hindeuten. / The accumulation of Amyloid beta peptide 1-42 (Ab42) in extracellular plaques is one of the pathological hallmarks of Alzheimer’s disease. Several studies have suggested that a cellular reuptake of Ab42 may be a crucial step in its cytotoxicity, but mechanisms of Ab-membrane interaction and subsequent cellular uptake are not yet understood. The first aim of the present study is to answer the question whether aggregate formation is a prerequisite or a consequence of Ab-membrane interaction and of Ab endocytosis. We visualized aggregate formation of fluorescently labeled Ab42 by Förster resonance energy transfer and tracked its internalization by human neuroblastoma cells. Both monomeric and aggregated Ab42 entered the cells, however, monomer uptake faced a concentration threshold and occurred only at concentrations and time scales that allowed beta-sheet-rich (bS) aggregates to form. By uncoupling membrane binding from internalization, we found that Ab42 monomers as well as small aggregate species bound rapidly to the plasma membrane and formed bS aggregates. These structures were subsequently taken up and accumulated in endocytic vesicles. This process correlated with inhibition of cellular metabolism activities. Our data therefore imply that the formation of bS aggregates at the cell membrane is a prerequisite for Ab42 uptake and cytotoxicity. The second aim of the study is to investigate the Ab-membrane interaction in vitro by using giant unilamellar vesicles and giant plasma membrane vesicles as model membrane systems. We found that both Ab isoforms, Ab42 and Ab40, interacted with the liquid disordered phase of model membranes. Early aggregation intermediates, which did not yet bind to the amyloiddophilic dye Thioflavin T, induced negative membrane curvature. The ability of Ab to induce membrane deformation suggests that Ab may facilitate its own endocytosis. It also hints at a possible physiological function of non-toxic Ab aggregate species.

Master of Science / Biochemistry and Molecular Biophysics / Jianhan Chen / Proteins are fundamental building blocks of life in an organism, and to function properly, they must adopt an appropriate three-dimensional conformation or conformational ensemble. In protein aggregation diseases, proteins misfold to incorrect structures that allow them to join together and form aggregates. A wide variety of proteins are involved in these aggregation diseases and there are multiple theories of their disease mechanism. However, a common theme is that they aggregate into filamentous structures. Therapies that target the process by which the aggregating proteins assemble into these similar fibril-like structures may by effective at countering aggregation diseases. This requires models that can accurately describe the assembly process of the fibrils. An analytical theory was recently described where fibrils grow by the templating of peptides onto an existing amyloid core and the kinetics of the templating process is modeled as a random walk in the backbone hydrogen bonding space. In this thesis, I present my work integrating molecular simulation with this analytical model to investigate the dependence of fibril growth kinetics on peptide sequence and other molecular details. Using the Aβ16-22 peptide as a model system, we first calculate the rate matrix of transitions among all possible hydrogen bonding microscopic states using numerous short-time simulations. These rates were then used to construct a kinetic Monte Carlo model for simulations of long-timescale fibril growth. The results demonstrate the feasibility of using such a theory/simulation framework for bridging the significant gap between fibril growth and simulation timescales. At the same time, the study also reveals some limits of describing the fibril growth as a templating process in the backbone hydrogen bonding space alone. In particular, we found that dynamics in nonspecifically bound states must also be considered. Possible solutions to this deficiency are discussed at the end.

Master of Science / Department of Agronomy / Charles W. Rice / Biotic interactions among mycorrhizal fungi, nematodes, plants and other microbial communities can have significant effects on the dynamics of C and nutrient cycling. The specific objectives of this study were (1) to evaluate the effects of grazing and mycorrhizal symbiosis on the allocation and storage of C, especially for plant above-and belowground biomass, (2) evaluate the biotic rhizosphere interactions and their role in C cycling, (3) determine the soil microbial community structure as a result of the plant-mycorrhizal symbiosis, and (4) determine the effect of mycorrhizal fungal abundance on soil aggregation. The soil for the experiment was sampled from the Ap horizon of a fine-silty, mixed, superactive, mesic Cumulic Hapludolls located at Konza Prairie Biological Station, Manhattan KS. The experiment was a three-way factorial in a complete randomized block design with four replications. The three factors were mycorrhizae (M), nematodes (N), and phosphorus (P). In a greenhouse study, 96 microcosms (52×32×40cm) were planted to Andropogon gerardii Vit. so that a third of the microcosms could be destructively sampled at the end of each growing season for three years. Plant biomass was separated into aboveground, rhizomes, and roots. All components were dried and weighed at harvest. Mycorrhizal fungi and P increased plant aboveground biomass, while nematodes decreased plant aboveground biomass compared to non-inoculated controls. As expected, P increased plant root biomass, while mycorrhizae increased plant rhizome biomass. Nematodes decreased both above- and belowground biomass. Phospholipid and neutral lipid fatty acid (PLFA and NLFA) analysis were determined for both soil and roots. Water-stable aggregates were separated using a modified Yoder wet-sieving apparatus and analyzed for mass, total C and N, and the isotopic composition of C. There was a positive relationship between AM fungal abundance in the soil and the mass of the largest macroaggregates (>2000µm) after the 3rd year (r=0.67). The effect of roots on the macroaggregate (>2000µm) fraction was not apparent. Phosphorus significantly increased smaller macroaggregates (250-2000µm), along with significantly enhanced plant root biomass, which indirectly demonstrated the effect of roots on the formation of macroaggregates (250-2000µm). The addition of P induced more plant derived C into the aggregates than the non-P amended microcosms as suggested by the [superscript]13C content of the aggregates. Our results confirmed the importance of biotic and abiotic interactions among mycorrhizae, nematodes, and phosphorus on plant growth and the resulting effect on the soil C cycle and soil aggregation.

Knee osteoarthritis (OA) is the commonest form of lower limb OA with a lifetime risk of over 40%. It is a disease characterised by symptoms such as pain and loss of function. In addition there are typical structural features on both radiographs and MRI. Knee OA represents a spectrum of disease, ranging from early preclinical cartilage change to established full thickness disease. Anteromedial knee OA is a particular phenotype of knee OA where disease is confined to the medial compartment. Whilst end stage arthritis is treated reliably with joint arthroplasty, those with early stage disease are treated with a variety of non- surgical interventions with varying success. This thesis is concerned with understanding the disease of patients that have early radiographic changes but symptoms not controlled by conservative measures. Up to 150 of these patients a year present to the Nuffield Orthopaedic Centre, Oxford. They have been described as being in the “Treatment Gap”. A series of validation studies were performed to determine the optimal method for diagnosing cartilage defects within the knee. The three commonest diagnostic methods were examined for their validity. Arthroscopic assessments of cartilage lesions demonstrated a moderate level of intra and inter observer reliability. In contrast, radiographs and MRI demonstrated high levels of reliability. When using MRI as a criterion standard, both radiographs and arthroscopic assessment were found to have poor accuracy. Based on the work in this thesis a formal definition of the cartilage changes exhibited in early knee OA was proposed. A cross sectional cohort of 100 patients with the symptoms and radiological features of early knee OA were identified. Their pain and function profile was compared to two comparison groups of patients at the end stage of knee OA (defined by the need for partial or total arthroplasty). In up to 78% of individual cases those with early OA had pain and function profiles as bad as those with end stage disease. The cross sectional symptoms of early knee OA demonstrate a marked discordance with their mild radiographic changes. The same cohort was extended to 125 patients. They were followed over one year with monthly PROM assessments to determine how symptoms change over time. 43% of patients experience a clinical improvement over 12 months, 31% experience a clinical deterioration and 26% remain unchanged. The range in OKS variation over 12 months was on average 12 points, with clinically relevant variation occurring on 45% of monthly measurements. Patients with early knee OA can expect to experience considerable variation in their symptoms over 12 months and this must be considered when planning interventions. A number of patients with early knee OA were noticed to demonstrate medial meniscal extrusion. Using data from the Osteo Arthritis Initiative (OAI) a nested case control study was designed to determine how the presence of meniscal extrusion in an otherwise normal knee affects the risk of developing knee OA over the next 48 months. This demonstrated an Odds Ratio of 3.5, suggesting that meniscal extrusion is a considerable risk factor for the development of OA. The presence of a knee injury or operative intervention to the index meniscus was shown to increase this risk. Many phenotypes of OA are known to demonstrate familial aggregation. In an attempt to determine where the earliest structural changes occur in medial compartment knee OA, a cohort of patients selected only for their family history of the disease were developed. This cohort was compared to spouse controls for the presence of knee OA, as well as meniscal extrusion and long leg alignment. In addition, a functional analysis of their cartilage was performed. This cohort was not shown to be at increased risk of disease compared to controls. Discussion of the possible reasons for this finding is presented. Early knee osteoarthritis is a considerable clinical problem. This thesis has aided the understanding of the condition by firstly defining the radiological description of these patients. Secondly, their cross sectional and longitudinal symptom profile have been described for the first time. In addition, the presence of an extruded meniscus has been demonstrated as a substantial risk factor for the disease. Finally, family history has not been demonstrated as a risk factor for the disease within the limits of the study described here. Future work has been proposed.