No description available.

Thesis (M. S.)--Biology, Georgia Institute of Technology, 2008. / Committee Chair: Chernoff,Yury; Committee Member: Cairney,John; Committee Member: Choi,Jung; Committee Member: Doyle,Donald; Committee Member: Lobachev,Kirill. Part of the SMARTech Electronic Thesis and Dissertation Collection.

The misfolding of native, cellular prion protein (PrPc) to a conformationally altered pathogenic isoform, designated scrapie PrPsc, is the main molecular process involved in the pathogenesis of prion diseases. Prion diseases are marked by the accumulation of conformationally modified forms of cellular prion protein. An N-terminal portion of the prion protein, PrP (106-128), is a 23-residue peptide fragment and is characterized by an amphipathic structure with two domains: a hydrophilic N-terminal domain and a hydrophobic C-terminal domain. In this study, the aggregation characteristics of the PrP (106-128) peptide were investigated using a combination of biophysical approaches. We investigated the effect of different factors including concentrations, pH, and metal ions, on the aggregation of the peptide. Our results demonstrated that the peptide steadily aggregates at concentrations higher than 25 M. The aggregation propensity and fibril formation is higher at pH 7.4 and pH 8.1, and the aggregation is inhibited at pH lower than 6. Furthermore, our results indicate that the Cu2+ has much less effect on the peptide amyloidogenesis, while Zn2+ has a significant influence on the PrP (106-128) amyloidogenesis. We further presented a systematic analysis of the impact of phospholipid liposomes of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1’-racglycerol) (POPG) in the absence or presence of cholesterol, on the amyloidogenesis of PrP (106-128). The results showed that POPC vesicles does not significantly influence the aggregation kinetics of the peptide. However, the anionic lipid POPG delays the aggregation in a concentration-dependent manner, whereas the addition of POPG with the cholesterol shows fast kinetics of fibrillization, thus reducing the lag time of the aggregation kinetics. We also monitored the effect of cholesterol and its derivatives including cholesterol-SO4 and DC-cholesterol on PrP (106-128) amyloidogenesis. Our results showed that the cholesterol inhibits the peptide aggregation and delays the formation of fibrils in a concentration-dependent manner. Cholesterol-SO4 dramatically facilitates the aggregation at high concentrations but has the potential to slow down the fibrillization at low concentrations, whereas cationic DC-cholesterol vesicles can effectively inhibit peptide fibril formation at high concentrations. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2020. / FAU Electronic Theses and Dissertations Collection

An affinity based bio-sensing technique was developed using an anti-transmissible spongiform encephalopathy monoclonal antibody as a bio-recognition molecule. Fluorescein iso-thio-cynate (FITC), labeled with a prion epitope (QYQRES), was used as a decoy for prions. Experiments done in 0.1M phosphate buffer revealed that the dye fluorescence increased with the pH of the buffer and was influenced by solvent polarity. Binding studies conducted at pH 6, 7, and 8 showed that the optimum pH for the antibody-decoy binding was 7. Maximum differences between control and antibody samples were observed at pH 7. The optimum incubation time was found to be less than 4 hours for the control, antibody, and the prion samples at room temperature. Prion detection curves were established at 4 and 10 nM antibody decoy concentrations. The lowest detectable prion concentration in phosphate buffer was 8 nM. Experimental conditions determined in the phosphate buffer were used to implement the technique in gelatin and baby formula. Prion detection curves were generated in 0.01, 0.4, 1.0 and 2.0 mg/ml of gelatin solution. The gelatin interfered with the binding and the displacement reaction of antibody, decoy and prion. Addition of an anionic surfactant, sodium dodecyl sulfate (SDS) at 0.3 mg/ml to gelatin samples facilitated prion detection in gelatin. The lowest detectable concentration of prion in gelatin was 0.5 nM at 0.4mg/ml gelatin. The baby formula samples produced light scattering and the intrinsic peak of baby formula at 526nm interfered with the dye peak at 514nm. Serial dilutions of baby formula were done to reduce the interference. Prion detection curves were then obtained at 1.31 and 5.34 mg/ml baby formula and 0.454 mg/ml of Triton-X-100 was added to the baby formula samples. The lowest detectable concentration of prion was 2 nM for baby formula. This developed bio-sensing technique can be used to detect prion in gelatin and baby formula solutions. Addition of surfactants assisted prion detection in foods, while high concentrations of gelatin and baby formula had an adverse effect on the detection system.

The work described in this dissertation details the development of a biomimetic materials for use in sensors and therapeutics, based on new advances in material science. The sensors developed herein target neurodegenerative diseases. Two of the diseases, the transmissible spongiform encephalopathies (TSEs) and Alzheimer’s disease (AD), are diseases associated with the abnormal folding of a protein, thus detecting the disease is dependent upon developing structure specific sensor technologies. Both sensors developed in this work take advantage of the unique optical properties associated with nanoscale metal particles, however they use different types of spectroscopies for optical detection of the presence of the disease associated abnormal protein, and different types of recognition elements that bring the disease associated proteins close to the nanoscale metal particles. In the case of TSEs, the recognition element was a commercially available antibody. In the case of AD, the recognition element was a molecular scale self-assembled surface. A therapeutic for AD was developed based on the molecular scale materials developed for the AD biosensor. Mathematical models were developed that facilitated the rational design of the biosensors described in this work that could also be used in future biosensor development.

Thèse de doctorat : mathématiques appliquées : Ecole centrale de Paris : 2009. / Titre provenant de l'écran-titre. Bibliogr. 422 réf.

Prions are infectious, self-propagating protein aggregates that have been uncovered in evolutionary divergent members of the eukaryotic domain of life. It is not known whether prokaryotic organisms contain proteins that exhibit prion-like behavior. However, studies have shown that the E. coli cytoplasm can support conversion of the well-characterized Saccharomyces cerevisiae yeast prion protein Sup35 into the prion form and that this conversion, like in the yeast system, is dependent on the presence of amyloid aggregates of another yeast prion protein, a so-called PIN factor. It is interesting that the bacterial system recapitulates the in vivo requirements for Sup35 prion formation in the native yeast system despite the fact that bacteria diverged from eukaryotes ~2.2 billion years ago. In yeast, once formed, the Sup35 prion is stably propagated and this process is independent of the PIN factor. Using the same yeast prion protein, Sup35, in CHAPTER 2 we show that prion aggregates can be maintained for up to 90 generations in the bacterial cytoplasm and that these aggregates are still infectious when transformed into yeast.

No description available.

Prion diseases are invariably fatal proteinaceous neurodegenerative disorders of the central nervous system. The infectious agent is the host encoded prion protein which has undergone a post-translational refolding from a predominantly alpha-helical to highly beta-sheet containing structure. The mechanism of prion-induced neurotoxicity remains elusive in large part due to the absence of a sufficiently neurotoxic cell culture assay. A modern technique for identifying previously unrecognized mediators of a biological pathway is to screen a commercially available library of gene silencing molecules targeting all known open reading frames. Synthetic gene silencing molecules, such as short hairpin RNA (shRNA), employ the endogenous gene silencing pathway to inhibit protein synthesis. To date, no publication has described the implementation of a large-scale library to screen for genetic mediators of prion neurotoxicity. This project was aimed at developing a cell culture model of acute prion neurotoxicity and screening a library of shRNA molecules in order to identify previously unrecognized gene targets essential to prion-induced neurotoxicity. Using a fragment of the prion protein (PrP106-126 peptide) to mimic prion neurotoxicity, human neuroblastoma cells transduced with a retroviral shRNA library were screened for resistance. Involvement of a subset of library identified gene targets in prion disease was assessed in vivo by quantitative real-time PCR (qPCR) analysis. Validation of the protection conferred by reducing expression of a gene target of interest was accomplished using individual lentiviral vectors expressing shRNA. Of the approximately 54,000 shRNA sequences screened, 80 different shRNA sequences recovered from neurotoxic prion peptide-resistant cells were considered to be of interest. Of these, 49 corresponding gene targets were assessed in vivo by qPCR with the majority demonstrating significant differential expression in brains of prion infected mice. Validation of the protection conferred from knockdown of two identified genes, abcb4 and ube2cbp, was completed. Knockdown of either gene imparted significant protection against prion-induced neurotoxicity, with qPCR analysis confirming significantly reduced mRNA transcript levels. Overall, the validity of the novel assay system developed has been demonstrated, and the first comprehensive list of gene candidates involved in mediating acute prion neurotoxicity has been determined.

Les maladies à prion sont un cas unique de pathologie où l’agent infectieux, le prion, est une protéine. La protéine prion possède plusieurs caractéristiques qui la rendent particulière vis-à-vis d’autres protéines cellulaires, telles que sa capacité à agréger et à transmettre sa conformation agrégée à la protéine soluble ainsi que la transmission des agrégats de la protéine d’une cellule à l’autre et d’un organisme à un autre. De plus en plus, on associe l’agrégation de protéines à différentes maladies humaines, comme les maladies d’Alzheimer, de Parkinson et le diabète de type 2. Certaines protéines impliquées dans ces pathologies font partie des prionoïdes, une catégorie réservée aux protéines aux propriétés agrégatives qui démontrent certaines des caractéristiques associées aux prions. Récemment, la protéine p53, un facteur de transcription fortement impliqué dans le cancer, a été montrée comme étant capable d’agréger in vitro. Une accumulation de la protéine a également été observée dans des cellules tumorales, laissant croire que l’agrégation de p53 se produit également in vivo, et pourrait avoir un rôle dans le développement du cancer. Ces observations portent à croire que la protéine p53 pourrait elle aussi faire partie des prionoïdes. L’objectif de cette étude est donc de montrer que la protéine p53 possède certaines des caractéristiques des prions. Pour ce faire, la protéine p53 recombinante a été produite pour former des agrégats de p53 et ces agrégats ont été utilisés pour déterminer si la protéine démontre des caractères prionoïdes. Les résultats obtenus montrent une agrégation in vitro de p53WT pleine longueur ainsi que de sa forme tronquée, p53C. De plus, des cellules en culture sont capables d’internaliser ces agrégats, qui co-agrègent ensuite avec la protéine p53 endogène de ces cellules. Enfin, nos résultats montrent clairement que l’internalisation des agrégats par les cellules se fait par la macropinocytose. Nous avons donc réussi à prouver que la protéine p53 agit comme un prion puisqu’elle s’agrège spontanément, ses agrégats sont internalisés par des cellules en culture et sont capables de co-agréger avec la protéine soluble.