Dissertations / Theses on the topic 'Sup45p'

Le domaine C-terminal très conservé de la protéine Sup35, impliquée dans la terminaison de la traduction, possède un site potentiel de phosphorylation par la PKA au niveau de la Thréonine341. Nous avons recherché si ce résidu était phosphorylé in vivo et s’il était impliqué dans la régulation fonctionnelle de la protéine. Dans les conditions testées, aucune phosphorylation de Sup35p n’a pu être mise en évidence in vivo mais nous avons montré que le résidu T341 était critique pour la fonctionnalité de la protéine et pourrait être impliqué dans des interactions fonctionnelles entre les domaines N et C terminaux. Le domaine N de Sup35p est responsable du phénotype prion [PSI+]. Jusqu’à présent, les seules mutations caractérisées influençant les propriétés prion de cette protéine ont été localisées dans ce domaine N-terminal. Nous avons identifié une mutation dans le domaine C-terminal qui modifie les capacités d’agrégation de la protéine. Cette observation apporte de nouveaux éléments pour la compréhension du mécanisme de conversion de Sup35p vers un état agrégé. IMP3 est un gène essentiel codant une protéine impliquée dans la biogenèse des ribosomes. Nous avons construit une souche capable d’exprimer de façon endogène un allèle mutant hypomorphe du gène IMP3. Cette souche présente d’importants défauts de biogenèse de la petite sous unité 40S du ribosome. Nous avons montré que la fidélité de la traduction est affectée dans cette souche : l’efficacité de décalage du cadre lecture en +1 est augmentée. Nos expériences montrent que cette protéine pourrait être également impliquée dans la réparation de l’ADN et le contrôle de la taille des télomères
All Sup35 homologs share a potential phosphorylation site at threonine 341, suggesting a functional role for this residue. We investigated whether this residue is actually phosphorylated in yeast and if it is involved in the termination activity of the protein. In the conditions we tested, no phosphorylation of the Sup35 protein in vivo was detected. However our results point to a new critical residue involved in the translation termination activity of Sup35p and in functional interaction between the N- and C-domains of the protein. The N-terminal domain of Sup35p is required for prion propagation, driving the switch from the soluble, functional [psi-] state to the insoluble [PSI+] prion state. To date, all the critical elements for prion induction and propagation have been mapped to the N domain of the protein. Here we report for the first time a mutation in the C-terminal domain of Sup35p which alters the aggregation properties of Sup35p. This observation has important consequence for understanding the mechanism of prion conversion. The essential IMP3 gene encodes a component of the SSU processome, a large ribonucleoprotein required for processing of small subunit rRNA precursors. We constructed and analysed a mutant of the IMP3 gene able to sustain cell growth. A strain expressing this hypomorphic allele displayed ribosome biogenesis defects characteristic of a depletion in Imp3p. We demonstrated the +1 frameshifting was increased in the mutant strain. Our further characterization revealed involvement of the Imp3 protein in DNA repair and telomere length control, two pathways that are not directly related to ribosome biogenesis

Polyglutamine-expanded fragments, derived from the human huntingtin protein, are aggregation-prone and toxic in yeast cells, bearing endogenous QN-rich proteins in the aggregated (prion) form. Attachment of the proline-rich region targets polyglutamine aggregates to the large perinuclear deposit (aggresome). Aggresome targeting ameliorates polyglutamine cytotoxicity in the presence of the prion form of Rnq1 protein, however, aggresome-forming construct remains toxic in the presence of the prion form of translation termination (release) factor Sup35 (eRF3). Disomy by chromosome II partly ameliorates polyglutamine toxicity in the strains containing Sup35 prion. The chromosome II gene, coding for another release factor, and interaction partner of Sup35, named Sup45 (eRF1), is responsible for amelioration of toxicity. Plasmid-mediated overproduction of Sup45, or expression of the Sup35 derivative that lacks the QN-rich domain and is unable to be incorporated into prion aggregates, also ameliorate polyglutamine toxicity. Protein analysis indicates that polyglutamines alter aggregation patterns of the Sup35 prion and promote aggregation of Sup45, while excess Sup45 counteracts these effects. In the absence of Sup35 prion, disomy by chromosome II is still able to decrease polyglutamine toxicity. However, SUP45 is no longer the gene responsible for such an effect. Taken together with the finding that the presence of both the Rnq1 prion and the Sup35 prion has an additive effect on polyQ toxicity, one gene or few genes on chromosome II are able to ameliorate polyQ toxicity through a SUP45-independent pathway. The identification of such a gene is currently ongoing. Monosomy by chromosome VIII in diploid heterozygous by AQT (Anti-polyQ Toxicity mutants that are disomic by chromosome II) counteracted the effect of AQT. Similarly, deletion of the arg4 gene in chromosome VIII in AQT haploid was able to eliminate the AQT effect. Moreover, analysis of genes involved in the arginine and polyamine synthesis indicated that loss of genes in later stages of arginine biosynthesis causes increase of polyglutamine toxicity. Deletion of genes arg1, arg4, arg8 (arginine pathway) and spe1 (polyamine pathway) all suppressed the Sup35 prion phenotype expression in the nonsense suppression system. Further analysis regarding the mechanisms behind those effects is needed. Our data uncover the mechanisms by which genetic and epigenetic factors may influence polyglutamine toxicity, and demonstrate that one and the same type of polyglutamine deposits could be cytoprotective or cytotoxic, depending on the prion composition of a eukaryotic cell.

Chez S. Cerevisiae, les gènes SUP45 et SUP35 codant respectivement eRF1 et eRF3 sont essentiels. Nous avons isolé 16 mutants viables ayant une mutation faux sens ou non sens dans le gène SUP45. Toutes les mutations faux sens sont localisées dans la région de la protéine eRF1 qui reconnaît le codon STOP, elles n'altèrent ni le faux de la protéine, ni son interaction avec eRF3. Toutes les mutations non sens conduisent à un phénotype de suppression omnipotente et à la viabilité (testée dans 3 souches différentes). Pour tous ces mutants, la quantité d’eRF1 est réduite par rapport à la souche sauvage et elle est corrélée à la perte d'efficacité de la terminaison de la traduction. De plus, la quantité de certaines tRNA qui ont la capacité potentielle de lire des codons de terminaison était plus élevée chez ces mutants, ce qui pourrait expliquer le phénomène de translecture.

Les protéines prions sont associées à une classe de maladies neurodégénératives, dont l'encéphalopathie spongiforme transmissible (EST) est la mieux connue. La protéine prion Sup35p représente un tel modèle car elle est non associée à une maladie. Sup35p se compose de trois domaines : un domaine N-terminal qui est responsable de la formation de prion, d'un domaine de milieu (M) qui affiche un degré élevé de flexibilité, et un domaine C-terminal fonctionnel et globulaire. Le fragment Sup35pNM est souvent utilisé comme modèle pour documenter l'assemblage et les propriétés infectieuses de Sup35p. Les études de Sup35p et Sup35pNM par RMN du solide ont révélé d'étonnantes différences structurelles entre les deux cœurs amyloïdes de Sup35p et Sup35pNM. Nos résultats sur Sup35p apportent un nouvel éclairage sur le monde étonnamment diversifié des prions où la variabilité conformationnelle joue un rôle énorme et perturbant. Ils reflètent l'image émergente que les prions sont des unités structurelles complexes. En effet, même s'il affiche une structure très définie, un domaine donné peut adopter des conformations différentes selon les circonstances (en isolation, dans le contexte d'un fragment ou la protéine entière) ou de l'environnement (conditions de tampon, présence de chaperonnes). Nos résultats donnent une explication au niveau moléculaire pour la contractante propension à l'assemblage et l'infectiosité de Sup35pNM et Sup35p, et soulignent l'importance primordiale d'une caractérisation structurale au niveau moléculaire des agrégats utilisés dans des études fonctionnelles
Prion proteins are associated with a class of neurodegenerative diseases, including transmissible spongiform encephalopathy (TSE) which is the best known. The prion protein Sup35p displays a model system because it is not associated with disease. Sup35p consists of three domains: an N-terminal domain which is responsible for the prion formation, a middle domain (M) that displays a high degree of flexibility, and a functional C-terminal domain. Sup35pNM the fragment is often used as a model to document for the assembly and infectious properties of Sup35p. Solid-state NMR studies of Sup35p and Sup35pNM fibrils showed amazing structural differences between the two amyloid cores. Our results shed new light on the surprisingly diverse world of prions where conformational variability plays a huge role. They reflect the emerging picture that prions are complex structural units. Even if it displays a very defined structure, a given field may adopt different conformations depending on the circumstances (in isolation, in the context of the whole protein or fragment) or the environment (buffer conditions, presence of chaperones). Our results provide an explanation at the molecular level for the contrasting propensity assembly and infectivity Sup35pNM and Sup35p, and emphasize the central importance of a structural characterization at the molecular level

La synthèse protéique est un processus essentiel qui repose sur un complexe macromoléculaire présent dans toutes les cellules : le ribosome. La synthèse d’une nouvelle protéine par un ribosome, à partir d’un ARNm, est suivie d’une étape cruciale de libération de cette protéine. Chez la levure, la terminaison de la traduction requiert la présence du facteur de terminaison de classe I : eRF1, capable de reconnaître les trois codons stop, et du facteur de classe II : eRF3, dont l’activité GTPase est essentielle pour la terminaison. Les facteurs de classe I bactériens ont évolué indépendamment du facteur eucaryote, mais un petit tripeptide essentiel : GGQ, est strictement conservé dans ces protéines. Lors de la terminaison, ce tripeptide situé au cœur du centre peptidyl-transférase du ribosome procaryote, participe à l’hydrolyse de la liaison peptidique, qui permet la libération de la protéine. Chez S. Cerevisiae, ce motif subit une modification post-traductionnelle de la glutamine en N5-méthyl-glutamine, catalysée par un hétérodimére : Mtq2p/Trm112p. Nous avons reconstitué, in vitro, le système de méthylation des facteurs eRF1 humain et murin. Comme chez la levure la méthylation fait intervenir un hétérodimère, respectivement HEMK2α/hTrm112 et Pred28α/mTrm112, et nécessite la présence d’eRF3 lié au GTP. Chez S. Cerevisiae, la délétion ou la mutation de MTQ2, entraînent la perte de la modification d’eRF1, mais contrairement à ce qui a été montré chez E. Coli, cela n'affecte pas l’efficacité de terminaison in vivo. Cependant, les deux sous-unités de la méthyltransférase, Mtq2p et Trm112p, sont impliquées par des voies indépendantes dans le métabolisme du ribosome.

Thesis advisor: Jianmin Gao
Protein aggregation can be highly detrimental to organisms, and has been associated with diseases including Alzheimer's, Huntington's, type II diabetes, and transmissible spongiform encephalopathies such as Mad Cow disease. There is no single amino acid sequence responsible for aggregation into amyloid-like structures, but rather a large range of amyloidogenic peptides have been discovered. A fragment of the yeast Sup35 prion, GNNQQNY, has been found to aggregate using a "dry, steric zipper" structure. This study looks at mutants of GNNQQNY in order to elucidate the exact contributions of various amino acids to the aggregation process
Thesis (BS) — Boston College, 2008
Submitted to: Boston College. College of Arts and Sciences
Discipline: Chemistry
Discipline: College Honors Program

Microsatellite repeats are a phenomenon found in DNA where a short sequence, usually 1-6bps, is repeated dozens to hundreds of times. Microsatellite repeats that are able to be transcribed are termed expanded tandem repeat-containing RNA (xtrRNA) [1]. xtrRNA have been associated with many diseases, such as Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD), which are both caused by a repeat in the C9ORF72 gene. Recent research has been focused on trying to provide treatments for patients with these diseases. This study focuses on creating a drug screening process for therapeutics targeting transcription by stopping or slowing the transcription of C9ORF72 repeat expansions. One project has focused on interrupting the interaction of two transcription factors, SUPT5H and SUPT4H1, to slow transcription. Another project has focused on slowing transcription by using transcriptional inhibitors or nucleoside analogs at low concentrations. Our hypothesis is that if transcription rates are slowed enough, pausing or arrest of RNA polymerase will be induced at complex sequences, including GC-rich regions and repeats. This should reduce synthesis of xtrRNA and provide a starting point for therapeutic development.

Lors de ce travail nous avons obtenu et caractérisé des mutants suppresseurs dans le gène essentiel SUP 35 qui code pour le facteur de terminaison eRF3 chez la levure Saccharomyces cerevisiae. Nous avons identifié 5 mutations "missense" et 9 mutations "nonsense". Les souches correspondant aux mutations "nonsense" dans le gène SUP35 sont caractérisées par une diminution de quantité de protéine eRF3, par contre le niveau de mRNA SUP35 est oinchangé. Ces mutations "nonsens" ne sont pas léthals pour différentes souches (fonds génétique différents) même en abscence de mutation dans les gènes codant pour les tRNAs. Nous avons montré que les souches contenant les mutations dans les gènes SUP35 ou SUP45 sont caractérisées par des altérations du NMD ("Nonsense Mediated mRNA Decay), mécanisme qui élimine les ARNm contenant un codon stop en début de phase codante.

Proteinaceous infectious particles, termed 'prions' are self-perpetuating protein isoforms that transmit neurodegenerative diseases in mammals and phenotypic traits in yeast. Each conformational variant of a prion protein is faithfully propagated to a homologous protein in the same cell environment. However, a reduction in the efficiency of prion transmission between different species is often observed and is termed "species barrier". Prion transmission to a heterologous protein may, in some cases, permanently change the structure of the prion variant, and divergent proteins may interfere with prion propagation in a species-specific manner. To identify the importance of both protein sequence and the cell environment on prion interference and cross-species transmission, we employed heterologous Sup35 proteins from three Saccharomyces sensu stricto species: Saccharomyces cerevisiae (Sc), Saccharomyces paradoxus (Sp), and Saccharomyces bayanus (Sb). We performed our experiments in two different cell environments (Sc and Sp). Our data show that Sup35 from one species can form a prion in another, and we employed a transfection procedure to perform cross-species transfer of the prion. Using a shuffle procedure, we demonstrate that the specificity of prion transmission is determined by the protein itself rather than the cell environment. Interestingly, we noted that variant-specific prion patterns can be altered irreversibly during cross-species transmission through S. bayanus module II. We further show that prion interference does not always correlate with cross-species prion transmission, and the identity of particular regions or even a specific amino acid, rather than the overall level of PrD homology is crucial for determining cross-species transmission and interference. Lastly we provide evidence to suggest that prion interference is specific to the cell environment.

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L-Asparaginase type II enzymes catalyze the hydrolysis of L-asparagine to L-aspartate and ammonia, and to a lesser extent the hydrolysis of L-glutamine. Escherichia coli and Erwinia chrysanthemi are the main sources of L-asparaginases II used as therapeutic agents in the treatment of acute childhood lymphoblastic leukaemia. However, their glutaminase activity has been implicated in causing serious side effects. Fortunately, L-asparaginase II from Erwinia carotovora has significantly lower glutaminase activity and may represent an important alternative therapy. Here we describe cloning, expression, purification and determination of steady-state kinetic parameters for two constructs of E. carotovora Lasparaginase II: with (AspSP) and without the signal peptide (AspMP). AspMP was purified to homogeneity by a single-step protocol with 81 % yield, and AspSP by a two-step protocol with 35 % yield. The Km and kcat values for L-asparaginase and L-glutaminase activities were determined for both proteins. Analysis of specificity for competing substrates indicates that Lasparagine is a better substrate than L-glutamine for AspSP as compared to AspMP. However, the latter is produced by a simpler purification protocol and at higher yield. This process can be amenable to large scale production and be of interest to researchers and biopharmaceutical companies.
As enzimas L-asparaginase do tipo II catalisam a hidrólise de L-asparagina a Laspartato e amônia e, em menor quantidade, a hidrólise de L-glutamina. Escherichia coli e Erwinia chrysanthemi são as principais origens das L-asparaginases II utilizadas como agentes terapêuticos no tratamento da leucemia linfoblástica aguda infantil. Entretanto, sua atividade de glutaminase tem sido relatada, causando graves efeitos colaterais. Felizmente, a L-asparaginase II obtida de Erwinia carotovora possui baixa atividade de glutaminase, representando uma importante terapia alternativa. Neste trabalho é descrita a clonagem, expressão, purificação e determinação dos parâmetros cinéticos em estado estacionário para duas construções de L-asparaginase II de E. carotovora: com (AspSP) e sem peptídeo-sinal (AspMP). AspMP foi purificada à homogeneidade por um protocolo de uma etapa com 81% de rendimento, enquanto que AspSP por um protocolo de duas etapas com 35% de rendimento. Os valores de Km e kcat para as atividades de L-asparaginase e de L-glutaminase foram determinados para ambas as proteínas. A análise de especificidade para os substratos indicou que a L-asparagina é o melhor substrato que a L-glutamina para a AspSp quando comparado à AspMP. Entretanto, a AspMP é produzida por um protocolo de purificação simples que fornece um alto rendimento da enzima. Este processo pode ser adaptado para uma produção em larga escala e ser interessante para as pesquisas e companhias biofarmacêuticas.

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As enzimas L-asparaginase do tipo II catalisam a hidr?lise de L-asparagina a Laspartato e am?nia e, em menor quantidade, a hidr?lise de L-glutamina. Escherichia coli e Erwinia chrysanthemi s?o as principais origens das L-asparaginases II utilizadas como agentes terap?uticos no tratamento da leucemia linfobl?stica aguda infantil. Entretanto, sua atividade de glutaminase tem sido relatada, causando graves efeitos colaterais. Felizmente, a L-asparaginase II obtida de Erwinia carotovora possui baixa atividade de glutaminase, representando uma importante terapia alternativa. Neste trabalho ? descrita a clonagem, express?o, purifica??o e determina??o dos par?metros cin?ticos em estado estacion?rio para duas constru??es de L-asparaginase II de E. carotovora: com (AspSP) e sem pept?deo-sinal (AspMP). AspMP foi purificada ? homogeneidade por um protocolo de uma etapa com 81% de rendimento, enquanto que AspSP por um protocolo de duas etapas com 35% de rendimento. Os valores de Km e kcat para as atividades de L-asparaginase e de L-glutaminase foram determinados para ambas as prote?nas. A an?lise de especificidade para os substratos indicou que a L-asparagina ? o melhor substrato que a L-glutamina para a AspSp quando comparado ? AspMP. Entretanto, a AspMP ? produzida por um protocolo de purifica??o simples que fornece um alto rendimento da enzima. Este processo pode ser adaptado para uma produ??o em larga escala e ser interessante para as pesquisas e companhias biofarmac?uticas.

The word "Prion" refers to self-perpetuating protein aggregates that cause neurodegenerative diseases in mammals. It is a protein isoform that has undergone a conformational change which converts the normal form of the protein into the infectious form with the same amino acid sequence. Yeast [PSI+] prion is the prion isoform of Sup35 protein, a translation termination factor eRF3. It has been suggested that prion [PSI+] is controlled by the ensemble of chaperones with Hsp104 playing the major role. The previous work performed in the Chernoffs lab showed that the defective GET pathway caused by get led to the defect in [PSI+] curing by excess Hsp104. The GET pathway is a system responsible for transporting newly synthesized TA-protein to the ER membrane, and the components which have been proven to be involved in this pathway include: Get1, Get2, Get3, Get4, Get5 and Sgt2. In this study we describe the mechanism underlying the effect of the defective GET pathway on [PSI+]. We demonstrate that Sgt2, one of the components of GET pathway, interacts with Sup35 in both [PSI+] and [psi-] strains through its prion domain. Overproduction of Sgt2 and Hsp70-Ssa is triggered by the defective GET pathway and leads to the protection of [PSI+] aggregates from curing by excess Hsp104. We show that the direct interaction between Sgt2 and Hsp70-Ssa is not required for this protective effect.

錯誤折疊並聚集的促澱粉樣變蛋白和多肽分子通常以β折疊含量豐富的纖維狀澱粉態存在，這種纖維狀澱粉態被認為與多種神經退行性疾病的發病有關，例如老年癡呆症，多聚穀氨醯胺症以及傳染性海綿狀腦病。澱粉態沉積物作為多種神經退行性疾病的顯著標誌，促澱粉樣變蛋白和多肽發生錯誤折疊並聚集進而導致神經毒性的機理仍未被闡明。在當前的研究中，我們選擇酵母感染性蛋白Sup35作為探索促澱粉樣變蛋白聚集機理的模型。Sup35是一種存在於釀酒酵母細胞中的感染性蛋白，作為一種翻譯終止因子，它可以通過改變自身構象，進而形成不溶的纖維狀澱粉態沉澱。根據位置和功能的不同，Sup35可被劃分為3個結構域，即N，M和C。作為控制其感染性的區域，Sup35-NM被廣泛接受為一種用於研究促澱粉樣變蛋白的模型。研究人員已經針對Sup35的聚集機理開展了很多研究，其中最為廣泛接受的是Lindquist 等人提出的β螺旋模型。在這個模型中，相鄰的氨基酸片段形成了一種頭對頭和尾對尾的構象。我們的研究目的就是要探究這種聚集機理模型是否正確。如果不正確，我們將對聚集機理提出一種新的假設。
作為探索促澱粉樣變蛋白聚集過程的重要前提，研究人員必須首先製備出只含有單獨的蛋白單體的樣品溶液。否則，相關的動力學過程研究將被干擾。我們通過動態激光光散射研究發現，使用現有的多種用於溶解促澱粉樣變蛋白和多肽的實驗方法並不能製備出真正的蛋白溶液，得到的樣品中總含有微量的、尺寸大約為10-10² nm的聚集體。這些聚集體會極大地影響聚集的動力學過程。這也可以在一定程度上解釋為什麼在不同的文獻報導中，同一種蛋白在相同的環境中卻表現出差異巨大的動力學過程。在當前的研究中，我們將傳統方法與我們實驗室新進開發的超濾法相結合，發展出了一套可以用於製備真正的、不含有聚集體的促澱粉樣變蛋白或多肽溶液的方法。製備出的溶液可以保持其中的蛋白或多肽處於單體狀態至少一個星期，這為研究在生理條件下蛋白的聚集過程提供了重要的基礎。
為了研究Sup35不同亞基之間的相互作用，我們分別在其N結構域的頭，腰和尾做了半胱氨酸點突變，並用兩種相互獨立的方法研究亞基之間的相互作用。第一種方法是在突變位點引入空間位阻，從而減弱所謂的頭對頭尾對尾的相互作用。我們的想法很直接，如果Lindquist等人提出的機理是正確的，那麼突變後的蛋白將無法形成纖維狀澱粉態沉澱。第二種方法是通過形成二硫鍵在不同蛋白的半胱氨酸突變位點之間引入連接分子，共有兩種連接分子，一種長約2 Å，另一種長約11 Å。選擇這兩種連接分子的原因是，聚集體中兩條Sup35蛋白鏈之間的距離通常約4.7 Å，連接分子長於或短于這個距離應會對聚集產生不同影響，從而反映出聚集體的結構資訊。
在這篇博士論文中，首先，我將介紹促澱粉樣變蛋白研究的背景和激光光散射測量的原理以及研究中用到的主要實驗方法。然後，我將闡述如何將傳統方法和我們實驗室新進發展出的超濾法相結合，從而製備出真正的、不含聚集體的蛋白溶液。接下來，我還將證明通過動態和靜態激光光散射相結合，我們可以得到更多關於促澱粉樣變蛋白的微觀參數，包括分子量，蛋白單體和聚集體的流體力學半徑等。最後，我將針對不同Sup35突變體的聚集動力學過程來研究其亞基之間的相互作用並提出Sup35的聚集模型。
Misfolding and aggregation of amyloidogenic protein/peptide are frequently found in a β-sheet-rich fibrillar protein conformation known as amyloids, which are related to the onset of neurodegenerative diseases, ranging from Alzheimer and polyglutamine diseases to transmissible spongiform encephalopathies. While amyloid deposits are hallmarks of many neurodegenerative diseases, the mechanism by which these proteins/peptides gain their neurotoxic function upon misfolding and aggregation remains unclear. In the current study, we choose the yeast prion Sup35 as a model system to investigate the aggregation mechanism of amyloidogenic protein. The Sup35 protein is a yeast (Saccharomyces cerevisiae) prion protein, a translation termination factor that can convert into insoluble amyloid fibril. The structure of Sup35 protein can be divided into three regions; namely, N, M, and C based on their positions and different functions. Being the prion-determining region, Sup35-NM has been widely accepted as a model to study the amyloidogenic proteins. Many studies have been focused on the aggregation mechanism. The β-helix model proposed by Lindquist and her coworkers is mostly accepted. In such a model, Sup35-NM is folded to form a “head and a “tail region in the N region and different Sup35-NM chains aggregate together via a cooperative Head-to-Head and Tail-to-Tail stacking. The aim of our current study is to check whether this proposed mechanism is valid.
To gain insight into the mechanism of aggregation process, one must start with a solution that contains only individual (monomeric) protein chains. Otherwise, the kinetic study would be compromised. Our dynamic laser light scattering (LLS) study shows that the existing protocols of dissolving amyloidogenic protein/peptide do not result in a true solution; namely, there always exist a trace amount of interchain aggregates with an average size of ~10-10² nm, which greatly affect the association kinetics, partially explaining why different kinetics were reported even for a solution with identical protein and solvent. In this thesis study, by using a combination of the conventional dissolution procedure and our newly developed ultra-filtration method, we have developed a novel protocol to prepare a true solution of amyloidogenic protein/peptide without any interchain aggregates. The resultant solutions remain in their monomeric state for more than one week, which is vitally important for further study of the interchain association under the physiological conditions
To investigate the inter-subunit relationship, cysteine variants mutated at “Head, Waist, Tail" of the N region have been constructed. Two independent assessments have been proposed to study the inter-subunit interaction. One is to provide steric hindrance to the mutated sites so that the so called “Head-to-Head and Tail-to-Tail" interaction will be attenuated. Our strategy is quite straightforward, if the mechanism proposed by Lindquist and her coworkers is valid, the modified protein should lose its ability to form amyloid fibrils. The other strategy is to introduce disulfide cross-linkage between different mutation sites. Two types of disulfide cross-linkage have been chosen, one with a bond length of ~2 Å and the other, ~11 Å. The reason for such choices is that Sup35-NM has a characteristic inter-strand distance of ~4.7 Å. The disulfide bond length shorter or longer than this distance is supposed to play a different role in the protein aggregation, shedding light on the structural information.
In this Ph.D. thesis, we first introduce the background of amyloidogenic protein research and present the principle and instrumentation of laser light scattering, the main technique applied in our study. Next we show how to obtain a true solution of amyloidogenic protein with no oligomeric aggregates by combining a conventional dissolution procedure and our newly developed ultra-filtration method. We also show how to combine static and dynamic laser light-scattering measurements in the study of protein solutions, which leads to more microscopic parameters, such as the molar mass and the hydrodynamic sizes of individual protein chains and their aggregates. Our focus is on the aggregation kinetics of modified Sup35-NM variants and on the investigation of the inter-subunit interaction. Finally, we propose a model for the aggregation of Sup35-NM prion protein.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Diao, Shu.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.
Includes bibliographical references (leaves 99-101).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.
ABSTRACT (in Chinese) --- p.i
ABSTRACT --- p.iii
Table of content --- p.v
Acknowledgement --- p.viii
Chapter Chapter 1 --- Introduction and background --- p.1
Chapter 1.1 --- The biological role of amyloidogenic protein --- p.1
Chapter 1.1.1 --- The role of amyloidogenic protein in human disease --- p.1
Chapter 1.1.2 --- The functional role of amyloidogenic protein in living system --- p.2
Chapter 1.1.3 --- The role of amyloidogenic protein asnonchromosomal genetic elements --- p.3
Chapter 1.2 --- The structure of amyloid fibrils --- p.4
Chapter 1.2.1 --- Macromolecular structure of amyloid fibrils --- p.5
Chapter 1.2.2 --- Structure models for protofilament --- p.6
Chapter 1.2.3 --- The polymorphism of amyloid fibrils --- p.9
Chapter 1.3 --- Aggregation mechanism of amyloidogenic protein --- p.10
Chapter 1.3.1 --- The nucleated polymerization mechanism --- p.11
Chapter 1.3.2 --- Multiple conformations adopted by amyloidogenic protein chains --- p.13
Chapter 1.3.3 --- Sequence effect on amyloid formation --- p.15
Chapter 1.4 --- The pathogenesis of amyloid diseases --- p.16
Chapter 1.4.1 --- Prefibrillar aggregates may be the real culprits --- p.16
Chapter 1.4.2 --- Strategies to prevent amyloid diseases --- p.17
Chapter 1.5 --- References and Notes --- p.19
Chapter Chapter 2 --- Principle of Laser Light Scattering and Instrumentation --- p.27
Chapter 2.1 --- Introduction --- p.27
Chapter 2.2 --- Static Laser Light Scattering --- p.28
Chapter 2.2.1 --- Scattering by a small particle --- p.28
Chapter 2.2.2 --- Scattering by many small-particle system --- p.30
Chapter 2.2.3 --- Scattering by real systems --- p.31
Chapter 2.3 --- Dynamic Laser Light Scattering --- p.37
Chapter 2.3.1 --- Power spectrum of scattered light --- p.37
Chapter 2.3.2 --- Siegert relation --- p.39
Chapter 2.3.3 --- Translational diffusions --- p.40
Chapter 2.3.4 --- Analysis of the correlation function profile --- p.42
Chapter 2.4 --- Combination of Static and Dynamic Light Scattering --- p.44
Chapter 2.5 --- Practice of Laser Light Scattering --- p.45
Chapter 2.5.1 --- Light source --- p.45
Chapter 2.5.2 --- Optics and cell design --- p.46
Chapter 2.5.3 --- Detector --- p.47
Chapter 2.5.4 --- Sample Preparation --- p.47
Chapter 2.5.5 --- Differential refractometer --- p.48
Chapter 2.6 --- References and Notes --- p.49
Chapter Chapter 3 --- How to obtain a true solution of amyloidogenic protein/peptide with no oligomeric aggregates --- p.51
Chapter 3.1 --- Introduction --- p.51
Chapter 3.2 --- Experimental section --- p.53
Chapter 3.3 --- Results and discussion --- p.59
Chapter 3.4 --- Conclusion --- p.68
Chapter 3.5 --- References and Notes --- p.71
Chapter Chapter 4 --- Aggregation mechanism investigation of the Yeast prion protein Sup35-NM --- p.73
Chapter 4.1 --- Introduction --- p.73
Chapter 4.2 --- Experimental section --- p.75
Chapter 4.3 --- Results and discussion --- p.82
Chapter 4.3.1 --- Aggregation kinetics of Sup35-NM protein initiated from monomeric state --- p.82
Chapter 4.3.2 --- Does Sup35-NM protein aggregate in a head-to-head and tail-to-tail fashion? --- p.87
Chapter 4.3.2.1 --- The effect of dimerization on Sup35-NM aggregation --- p.88
Chapter 4.3.2.2 --- Inter-subunit investigation by Pyrene excimer fluorescence assay --- p.92
Chapter 4.3.2.3 --- The effect of PEGylation on Sup35-NM aggregation --- p.94
Chapter 4.4 --- Conclusion --- p.98
Chapter 4.5 --- References --- p.100
Publications --- p.102

Hsp104 is a protein remodeling factor that is crucially important for induced thermotolerance and prion propagation in yeast. Recent work demonstrates that Hsp104 is able to directly recognize and interact with synthetic polypeptide substrates, and that this interaction is dependent on the amino acid composition or sequence (Lum et al., 2008). Here this concept is applied to the in vivo substrate Sup35. Sup35, a translation termination factor, also forms the yeast prion [PSI+]. The maintenance of the prion is critically dependent on the expression levels of Hsp104. Over-expression of Hsp104 leads to the loss of prions, as does inhibition of this protein remodeling factor. As part of this thesis, an in vitro assay was established in which spontaneous nucleation, the event preceding of fiber formation, was suppressed. Fibrilization itself then becomes strictly dependent on the chaperones Hsp104, huHsp70p and Ydj1. In line with in vivo observations, Hsp104 mutants that fail to propagate [PSI+] also fail to overcome nucleation inhibition in this assay. Following this, the next part of this work established that the middle (M) domain of Sup35 inhibited this process, while not affecting spontaneous fibrilization under non-inhibitory conditions. This finding was reproduced in vivo, as middle domain over-expression also led to curing of weak [PSI+]. This suggested that the M-domain contains an Hsp104 binding site. This hypothesis is supported by data presented in this thesis which show that a small segment 129-148 within the Middle domain has enhanced Hsp104 binding properties. Deletion of this 20-mer peptide also reduced the Hsp104 ability to interact with this prion substrate; it also results in the destabilization of the prion and enhanced curing by the prion curing agent guandidinium hydrochloride. This represents the first ever Hsp104 binding site identified within a natural substrate.

碩士
國立陽明大學
生命科學系暨基因體科學研究所
101
Prions are infectious and self-propagating amyloid aggregates found in mammals and yeasts. The mammalian prion PrPSc causes fatal neurodegenerative diseases, such as bovine spongiform encephalopathy (BSE) in cattle. In fungi, prions alter the relationship between genotype and phenotype in a heritable way. Here, we use the S. cerevisiae [PSI+] prion, an amyloid conformer of the yeast translation termination factor Sup35, to study the polymerization and propagation of prions. The polypeptide fragment consisting of the first 40 amino acid residues of Sup35 (Sup(1-40)) was linked in tandem to construct fusion proteins. In vitro, the rate and yield of fiber formation were not increased by linking more Sup(1-40) units, suggesting that linkage of Sup(1-40) units might introduce unexpected structural and kinetical barriers. [PSI+] amyloids can have different structures, referred to as strains. Three [PSI] strains ―VH, VK, and VL― were characterized previously. The in vitro formed fibers of Sup(1-40) repeats displayed low infectivity in yeast and most of the few yeast transformants were of the VK strain type. Nevertheless, a novel [PSI+] strain, designated W8, was discovered in two of the transformants. The linked Sup(1-40) units might favor the fiber to adopt the novel W8 structure which is then faithfully propagated in yeast by the native Sup35 protein. W8 infectivity can be propagated in vitro using a bacterially expressed, simple Sup35(1-61) fragment. Strain competition experiments further established that W8 was dominant over VK and VL, but weaker than the VH strain in the 5V-H19 yeast genetic background.

博士
國防醫學院
生命科學研究所
103
Strains of the yeast prion [PSI] are different folding patterns of the same Sup35 protein, which stacks up periodically to form a prion fiber. Chemical cross-linking is employed here to probe different fiber structures assembled with a mutant Sup35 fragment. The photo-reactive cross-linker, p-benzoyl-ʟ-phenylalanine (pBpa), was biosynthetically incorporated into bacterially prepared recombinant Sup(1-61)-GFP, containing the first 61 residues of Sup35, followed by the green fluorescent protein. Four methionine (Met) substitutions and two alanine (Ala) substitutions were introduced at fixed positions in Sup(1-61) to allow cyanogen bromide cleavage to facilitate subsequent mass spectrometry analysis. Amyloid fibers of pBpa and Met/Ala substituted Sup(1-61)-GFP were nucleated from purified yeast prion particles of two different strains, namely VK and VL, and shown to faithfully transmit specific strain characteristics to yeast expressing the wild type Sup35 protein. Intra- and inter-molecular cross-linking were distinguished by tandem mass spectrometry analysis on fibers seeded from solutions containing equal amount of 14N and 15N-labeled protein. Fibers propagating the VL strain type exhibited inter-molecular cross-linking between amino acid residues 3 and 28, as well as intra- and inter-molecular linking between 32 and 55. Inter- and intra-molecular cross-linking between residues 32 and 55 were detected in fibers propagating the VK strain type. Adjacencies of amino acid residues in space revealed by cross-linking were used to constrain possible chain-folds of different [PSI] strains.

碩士
國立陽明大學
生化暨分子生物研究所
102
Huntington’s disease (HD) is an inherited neurological disorder caused by abnormal expansion of CAG repeats, which encode a long stretch of polyglutamine, in Huntingtin (HTT) gene. While the expression of mutant HTT contributes a gain-of-function for pathogenesis of HD, the biological activity of normal HTT is essential for neuron development and survival. For this reason, it is an optimal goal having HD treatments that inhibit the expression of HTT alleles in a selective manner. Currently, effective treatment is not available for this disorder and the development of therapeutic compounds is urgently needed. In our previous study, we demonstrated that Supt4h down-regulation can selectively reduce mutant HTT gene expression without an overt impact on the expression of wild-type HTT. Furthermore, the function of Supt4h in supporting RNA polymerase II transcribing through CAG expansion region is dependent on the formation of Supt4h/5h complex. Here, based on Bimolecular Fluorescence Complementation (BiFC), we established a cell-based assay system to monitor the interaction between Supt4h and Supt5h, and also applied this system to identify small chemical compounds that could interfere with the Supt4h/Supt5h complex formation. Among 60,000 compounds screened, 7 hits were identified with an inhibitory effect on the fluorescence signal of BiFC. These compounds were subjected to further characterization to assure their activities on Supt4h/5h protein-protein interaction.

S. cerevisiae Sup35p inhabits two metastable states: functional translation termination factor; and prion-like aggregate [PSI+], which propagates by converting soluble Sup35p to its own misfolded form. Once initiated, Sup35p polymerization in [PSI+] cells is spontaneous, but [PSI+] prion inheritance depends on the Hsp104p disaggregase. To identify Hsp104-interacting sequences, Sup35p was subjected to a systematic deletion screen. [PSI+] maintenance by mutant Sup35p was assessed in both presence and absence of plasmid-encoded WT Sup35p in haploid sup35 cells. Large deletions abolished [PSI+], implying perturbations of prion structure, while others imparted [PSI+]-dependent toxicity. Removal of a single 25aa segment destabilised [PSI+] inheritance, resulting in enhanced rates of prion loss. This is consistent with the expected prion propagation defect in response to reduced Hsp104p interaction. However, several mutants containing this 25aa segment share the destabilised prion phenotype, suggesting chaperone/prion interactions are strongly context-dependent, and no one Sup35p region is solely responsible for Hsp104p recognition.

Στην παρούσα διατριβή μελετήθηκε ο ρόλος της έλικας h44 του 18S rRNA του Saccharomyces cerevisiae επί διαφόρων παραμέτρων της πρωτεϊνικής σύνθεσης. Η μελέτη διεξήχθη με την βοήθεια των σημειακών μεταλλάξεων A1491G (rdn15) και U1495C (rdnhyg1), οι οποίες εντοπίζονται στην Α-θέση του ριβοσώματος. Η μετάλλαξη rdn15 επιδρά ήπια στον ρυθμό ανάπτυξης των κυττάρων ενώ τα rdn15 ριβοσώματα επιτελούν την πρωτεϊνοσύνθεση με αυξημένη ακρίβεια. Η έλλειψη σοβαρών επιπτώσεων παρουσία της rdn15 φανερώνει ότι το νουκλεοτίδιο 1491 δεν παίζει καθοριστικό ρόλο στην λειτουργία του ριβοσώματος. Τα κύτταρα ζύμης που φέρουν την μετάλλαξη rdnhyg1 αναπτύσσονται βραδύτερα από τα κύτταρα αγρίου τύπου, ενώ τα rdnhyg1 ριβοσώματα πρωτεϊνοσυνθέτουν με ελαφρώς αυξημένη συχνότητα λάθους. Η μετάλλαξη αυξάνει επίσης την συγγένεια της Α-θέσης του ριβοσώματος για το αμινοακυλο-tRNA και επιδρά αρνητικά στο στάδιο της μετατόπισης, χωρίς να επηρεάζει την ενεργότητα πεπτιδυλοτρανσφεράσης. Η επίδραση της μετάλλαξης rdnhyg1 επί διαφόρων παραμέτρων της πρωτεϊνοσύνθεσης δικαιολογεί την συντήρηση της U1495 κατά την εξέλιξη. Η μετάλλαξη sup45-R2ts εντοπίζεται στο γονίδιο που κωδικοποιεί τον παράγοντα τερματισμού eRF1 και οδηγεί στην αντικατάσταση της προλίνης 86 από αλανίνη. Η μετάλλαξη δεν επηρεάζει τις περισσότερες από τις λειτουργίες του ριβοσώματος που εξετάστηκαν, αλλά μειώνει την μεταφραστική πιστότητα. Σε κύτταρα που φέρουν ταυτόχρονα την μετάλλαξη sup45-R2ts και την ριβοσωματική μετάλλαξη rdn15, η συχνότητα λάθους αυξάνεται σε βαθμό μεγαλύτερο από την αθροιστική επίδραση των δύο επιμέρους μεταλλάξεων, επιβεβαιώνοντας μια ιδιαίτερη αλληλεπίδραση του μεταλλαγμένου παράγοντα eRF1 με τα rdn15 ριβοσώματα, που, όπως προκύπτει, αντιστρέφει τον υπερακριβή χαρακτήρα των μεταλλαγμένων ριβοσωμάτων. Όταν η μετάλλαξη sup45-R2ts συνυπάρχει με την ριβοσωματική μετάλλαξη rdnhyg1 η συχνότητα λάθους δεν επηρεάζεται σημαντικά. Η rdnhyg1 φαίνεται να ελαχιστοποιεί την επίδραση του μεταλλαγμένου παράγοντα eRF1 ενισχύοντας την δράση GTPάσης του eRF3. Τα παραπάνω αποτελέσματα φανερώνουν επιπλέον ότι η sup45 δύναται να μεταβάλει τις ιδιότητας ορισμένων μεταλλάξεων κατά την ριβοσωματική λειτουργία. Το στέλεχος που φέρει την μετάλλαξη rdn15 είναι πολύ ευαίσθητο έναντι της παρομομυκίνης αλλά και έναντι της τομπραμυκίνης, αν και σε μικρότερο βαθμό. Τα αποτελέσματα αυτά αποδίδονται στην ικανότητα της μετάλλαξης να αυξάνει την συγγένεια της Α-θέσης του ριβοσώματος για τα εν λόγω αντιβιοτικά. Αντίθετα, η μετάλλαξη rdn15 προσδίδει ανθεκτικότητα στην υγρομυκίνη, φανερώνοντας ότι ο τρόπος πρόσδεσης και δράσης του συγκεκριμένου αμινογλυκοζίτη διαφοροποιείται. Το στέλεχος που φέρει την μετάλλαξη rdnhyg1 είναι ανθεκτικό και στα τρία αντιβιοτικά σε σύγκριση με το αγρίου τύπου, φανερώνοντας ότι η U1495 είναι καθοριστική για την πρόσδεση των αμινογλυκοζιτών στο ριβόσωμα. Τα κύτταρα που φέρουν την εξωριβοσωματική μετάλλαξη sup45-R2ts είναι πιο ευαίσθητα από τα αντίστοιχα αγρίου τύπου έναντι και των τριών αμινογλυκοζιτών. Ωστόσο η μετάλλαξη sup45-R2ts, δεν επηρεάζει την ικανότητα των αντιβιοτικών αυτών να προσδένονται στα αγρίου τύπου και μεταλλαγμένα ριβοσώματα και να επάγουν άμεσα τα μεταφραστικά λάθη. Η μελέτη επίδρασης των αμινγλυκοζιτών επιβεβαίωσε ότι η παρομομυκίνη και η υγρομυκίνη αναστέλλουν την ανάπτυξη των κυττάρων ζύμης, ενώ η τομπραμυκίνη δεν έχει καμία επίδραση. Το γεγονός αυτό συνδυάζεται με την αδυναμία της τομπραμυκίνης να αναστείλει την πρόσδεση του υποστρώματος στην Α-θέση των ριβοσωμάτων. Ωστόσο η τομπραμυκίνη, όπως η παρομομυκίνη και η υγρομυκίνη, είναι ικανή να αυξήσει την συχνότητα λάθους και την σύνθεση πολυφαινυλαλανίνης.
In present study, we investigated the role of helix h44 of 18S rRNA of Saccharomyces cerevisiae on several parameters of protein synthesis. For this purpose we employed mutations A1491G (rdn15) and U1495C (rdnhyg1) which are located in the A-site of the ribosome. The rdn15 mutation slightly delays cell growth, while rdn15 ribosomes translate proteins with higher fidelity. The lack of severe impairment of ribosomal function by mutation rdn15 indicates that the nature of nucleotide 1491 is not essential for ribosomal function. Yeast cells carrying the rdnhyg1 mutation grow slower than wild-type cells, while their ribosomes possess a slightly increased error rate. This mutation also increases the affinity of the A-site for aminoacyl-tRNA and renders ribosomes less efficient in translocation without affecting peptidyltransferase activity. The effect of mutation rdnhyg1 on several parameters of protein synthesis explains why U1495 is evolutionarily conserved. Mutation sup45-R2ts is located in the gene encoding eukaryotic Release Factor 1 (eRF1) and results in the substitution of proline 86 by alanine. This mutation leaves unaffected most ribosomal functions but it decreases translational fidelity. When ribosomal mutation rdn15 is introduced in cells already carrying sup45-R2ts mutation, the error frequency is increased to a degree which is higher than the additive effect of the two mutations, testifying to a previously reported special interaction of eRF1 with rdn15 ribosomes, which in this case reverses the hyperaccurate character of rdn15 ribosomes. When mutation sup45-R2ts is expressed in cells also carrying ribosomal mutation rdnhyg1, the error frequency is not significantly altered. Mutation rdnhyg1 seems to minimize the effect of the mutant factor eRF1 on ribosomal function by enhancing GTPase activity of eRF3. The results obtained with rdn15 and rdnhyg1 alone or in combination with sup45-R2ts show for the first time that the presence of sup45 may result in significant changes in the properties of the mutations under study. The strain carrying mutation rdn15 exhibits extremely high sensitivity toward paromomycin and also increases sensitivity of yeast ribosomes to tobramycin but to a lesser degree. These results demonstrate the ability of this mutation to increase affinity of the A-site for aminoglycosides. In contrast, mutation rdn15 causes resistance to hygromycin, revealing that binding and possibly action of hygromycin is differentiated from the other two aminoglycosides. The strain carrying mutation rdnhyg1 is resistant to all three antibiotics tested compared to wild type, indicating that U1495 participates in aminoglycoside binding to the ribosome. Cells carrying the extraribosomal mutation sup45-R2ts are more sensitive toward all three antibiotics compared to their wild type cells. Nevertheless, mutation sup45-R2ts does not affect the ability of these antibiotics to bind to the ribosome and directly induce translational errors. The study of amingolycoside action confirmed that paromomycin and hygromycin inhibit cells growth, while no such effect is observed during cell growth in the presence of tobramycin. This fact is combined with the inability of tobramycin to inhibit substrate binding to the ribosomal A-site Nevertheless, it was shown that tobramycin, like paromomycin and hygromycin, is effective both in inducing translational errors and increasing polyphenylalanine synthesis in wild-type and mutant ribosomes.

9 ABSTRACT For proper function proteins should have a native conformation. If their conformation is impaired due to environmental stress or genetic mutation, proteins become prone to aggregation. There exist various types of protein aggregates. Stable non-membraneous inclusions can form which can serve for clearance of aberrant proteins from place where they can interfere with essential cellular processes. Another type of aggregates can serve as transient deposits of proteins thus protecting them from stress conditions. Stress granules (SG) are a such example of transient granules. Their formation is induced by heat shock for example. SGs contain mRNA, components of translation machinery, and other proteins. One of these proteins is Mmi1, small highly conserved protein with unknown function. Association of Mmi1 with stress granules and partial co-localization with chaperon Cdc48 and proteasom indicates Mmi1 can mediate heat stress damaged protein degradation. We have uncovered that yeast prion protein Sup35 is a component of stress granules as well. With regard to its aggregation capability there existed an assumption that prion domain of Sup35 could serve as scaffold for SG assembly. However as we show deletion of prion domain of Sup35 protein does not affect stress granules formation dynamics. Yeast...

Αντικείµενο της διατριβής είναι η ανάπτυξη νέων µεθοδολογιών εκµάθησης και σύγκλισης των Ασαφών Γνωστικών ∆ικτύων που προτείνονται για τη βελτίωση και προσαρµογή της συµπεριφοράς τους, καθώς και για την αύξηση της απόδοσής τους, αναδεικνύοντάς τα σε αποτελεσµατικά δυναµικά συστήµατα µοντελοποίησης. Τα νέα βελτιωµένα Ασαφή Γνωστικά ∆ίκτυα, µέσω της εκµάθησης και προσαρµογής των βαρών τους, έχουν χρησιµοποιηθεί στην ιατρική σε θέµατα διάγνωσης και υποστήριξης στη λήψη απόφασης, καθώς και σε µοντέλα βιοµηχανικών συστηµάτων που αφορούν τον έλεγχο διαδικασιών, µε πολύ ικανοποιητικά αποτελέσµατα. Στη διατριβή αυτή παρουσιάζονται, αξιολογούνται και εφαρµόζονται δύο νέοι αλγόριθµοι εκµάθησης χωρίς επίβλεψη των Ασαφών Γνωστικών ∆ικτύων, οι αλγόριθµοι Active Hebbian Learning (AHL) και Nonlinear Hebbian Learning (NHL), βασισµένοι στον κλασσικό αλγόριθµό εκµάθησης χωρίς επίβλεψη τύπου Hebb των νευρωνικών δικτύων, καθώς και µια νέα προσέγγιση εκµάθησης των Ασαφών Γνωστικών ∆ικτύων βασισµένη στους εξελικτικούς αλγορίθµους και πιο συγκεκριµένα στον αλγόριθµο Βελτιστοποίησης µε Σµήνος Σωµατιδίων και στον ∆ιαφοροεξελικτικό αλγόριθµο. Οι προτεινόµενοι αλγόριθµοι AHL και NHL στηρίζουν νέες µεθοδολογίες εκµάθησης για τα ΑΓ∆ που βελτιώνουν τη λειτουργία, και την αξιοπιστία τους, και που παρέχουν στους εµπειρογνώµονες του εκάστοτε προβλήµατος που αναπτύσσουν το ΑΓ∆, την εκµάθηση των παραµέτρων για τη ρύθµιση των αιτιατών διασυνδέσεων µεταξύ των κόµβων. Αυτοί οι τύποι εκµάθησης που συνοδεύονται από την σωστή γνώση του εκάστοτε προβλήµατος-συστήµατος, συµβάλλουν στην αύξηση της απόδοσης των ΑΓ∆ και διευρύνουν τη χρήση τους. Επιπρόσθετα µε τους αλγορίθµους εκµάθησης χωρίς επίβλεψη τύπου Hebb για τα ΑΓ∆, αναπτύσσονται και προτείνονται νέες τεχνικές εκµάθησης των ΑΓ∆ βασισµένες στους εξελικτικούς αλγορίθµους. Πιο συγκεκριµένα, προτείνεται µια νέα µεθοδολογία για την εφαρµογή του εξελικτικού αλγορίθµου Βελτιστοποίησης µε Σµήνος Σωµατιδίων στην εκµάθηση των Ασαφών Γνωστικών ∆ικτύων και πιο συγκεκριµένα στον καθορισµό των βέλτιστων περιοχών τιµών των βαρών των Ασαφών Γνωστικών ∆ικτύων. Με τη µεθοδο αυτή λαµβάνεται υπόψη η γνώση των εµπειρογνωµόνων για τον σχεδιασµό του µοντέλου µε τη µορφή περιορισµών στους κόµβους που µας ενδιαφέρουν οι τιµές των καταστάσεών τους, που έχουν οριστοί ως κόµβοι έξοδοι του συστήµατος, και για τα βάρη λαµβάνονται υπόψη οι περιοχές των ασαφών συνόλων που έχουν συµφωνήσει όλοι οι εµπειρογνώµονες. Έτσι θέτoντας περιορισµούς σε όλα τα βάρη και στους κόµβους εξόδου και καθορίζοντας µια κατάλληλη αντικειµενική συνάρτηση για το εκάστοτε πρόβληµα, προκύπτουν κατάλληλοι πίνακες βαρών (appropriate weight matrices) που µπορούν να οδηγήσουν το σύστηµα σε επιθυµητές περιοχές λειτουργίας και ταυτόχρονα να ικανοποιούν τις ειδικές συνθήκες- περιορισµούς του προβλήµατος. Οι δύο νέες µέθοδοι εκµάθησης χωρίς επίβλεψη που έχουν προταθεί για τα ΑΓ∆ χρησιµοποιούνται και εφαρµόζονται µε επιτυχία σε δυο πολύπλοκα προβλήµατα από το χώρο της ιατρικής, στο πρόβληµα λήψης απόφασης στην ακτινοθεραπεία και στο πρόβληµα κατηγοριοποίησης των καρκινικών όγκων της ουροδόχου κύστης σε πραγµατικές κλινικές περιπτώσεις. Επίσης όλοι οι προτεινόµενοι αλγόριθµοι εφαρµόζονται σε µοντέλα βιοµηχανικών συστηµάτων που αφορούν τον έλεγχο διαδικασιών µε πολύ ικανοποιητικά αποτελέσµατα. Οι αλγόριθµοι αυτοί, όπως προκύπτει από την εφαρµογή τους σε συγκεκριµένα προβλήµατα, βελτιώνουν το µοντέλο του ΑΓ∆, συµβάλλουν σε ευφυέστερα συστήµατα και διευρύνουν τη δυνατότητα εφαρµογής τους σε πραγµατικά και πολύπλοκα προβλήµατα. Η κύρια συνεισφορά αυτής της διατριβής είναι η ανάπτυξη νέων µεθοδολογιών εκµάθησης και σύγκλισης των Ασαφών Γνωστικών ∆ικτύων προτείνοντας δυο νέους αλγορίθµους µη επιβλεπόµενης µάθησης τύπου Hebb, τον αλγόριθµο Active Hebbian Learning και τον αλγόριθµο Nonlinear Hebbian Learning για την προσαρµογή των βαρών των διασυνδέσεων µεταξύ των κόµβων των Ασαφών Γνωστικών ∆ικτύων, καθώς και εξελικτικούς αλγορίθµους βελτιστοποιώντας συγκεκριµένες αντικειµενικές συναρτήσεις για κάθε εξεταζόµενο πρόβληµα. Τα νέα βελτιωµένα Ασαφή Γνωστικά ∆ίκτυα µέσω των αλγορίθµων προσαρµογής των βαρών τους έχουν χρησιµοποιηθεί για την ανάπτυξη ενός ∆ιεπίπεδου Ιεραρχικού Συστήµατος για την υποστήριξη λήψης απόφασης στην ακτινοθεραπεία, για την ανάπτυξη ενός διαγνωστικού εργαλείου για την κατηγοριοποίηση του βαθµού κακοήθειας των καρκινικών όγκων της ουροδόχου κύστης, καθώς και για την επίλυση βιοµηχανικών προβληµάτων για τον έλεγχο διαδικασιών.
The main contribution of this Dissertation is the development of new learning and convergence methodologies for Fuzzy Cognitive Maps that are proposed for the improvement and adaptation of their behaviour, as well as for the increase of their performance, electing them in effective dynamic systems of modelling. The new improved Fuzzy Cognitive Maps, via the learning and adaptation of their weights, have been used in medicine for diagnosis and decision-making, as well as to alleviate the problem of the potential uncontrollable convergence to undesired states in models of industrial process control systems, with very satisfactory results. In this Dissertation are presented, validated and implemented two new learning algorithms without supervision for Fuzzy Cognitive Maps, the algorithms Active Hebbian Learning (AHL) and Nonlinear Hebbian Learning (NHL), based on the classic unsupervised Hebb-type learning algorithm of neural networks, as well as a new approach of learning for Fuzzy Cognitive Maps based on the evolutionary algorithms and more specifically on the algorithm of Particles Swarm Optimization and on the Differential Evolution algorithm. The proposed algorithms AHL and NHL support new learning methodologies for FCMs that improve their operation, efficiency and reliability, and that provide in the experts of each problem that develop the FCM, the learning of parameters for the regulation (fine-tuning) of cause-effect relationships (weights) between the concepts. These types of learning that are accompanied with the right knowledge of each problem-system, contribute in the increase of performance of FCMs and extend their use. Additionally to the unsupervised learning algorithms of Hebb-type for the FCMs, are developed and proposed new learning techniques of FCMs based on the evolutionary algorithms. More specifically, it is proposed a new learning methodology for the application of evolutionary algorithm of Particle Swarm Optimisation in the adaptation of FCMs and more concretely in the determination of the optimal regions of weight values of FCMs. With this method it is taken into consideration the experts’ knowledge for the modelling with the form of restrictions in the concepts that interest us their values, and are defined as output concepts, and for weights are received the arithmetic values of the fuzzy regions that have agreed all the experts. Thus considering restrictions in all weights and in the output concepts and determining a suitable objective function for each problem, result appropriate weight matrices that can lead the system to desirable regions of operation and simultaneously satisfy specific conditions of problem. The first two proposed methods of unsupervised learning that have been suggested for the FCMs are used and applied with success in two complicated problems in medicine, in the problem of decision-making in the radiotherapy process and in the problem of tumor characterization for urinary bladder in real clinical cases. Also all the proposed algorithms are applied in models of industrial systems that concern the control of processes with very satisfactory results. These algorithms, as it results from their application in concrete problems, improve the model of FCMs, they contribute in more intelligent systems and they extend their possibility of application in real and complex problems. The main contribution of the present Dissertation is to develop new learning and convergence methodologies for Fuzzy Cognitive Maps proposing two new unsupervised learning algorithms, the algorithm Active Hebbian Learning and the algorithm Nonlinear Hebbian Learning for the adaptation of weights of the interconnections between the concepts of Fuzzy Cognitive Maps, as well as Evolutionary Algorithms optimizing concrete objective functions for each examined problem. New improved Fuzzy Cognitive Maps via the algorithms of weight adaptation have been used for the development of an Integrated Two-level hierarchical System for the support of decision-making in the radiotherapy, for the development of a new diagnostic tool for tumour characterization of urinary bladder, as well as for the solution of industrial process control problems.