Graduation date: 1997

Influenza virus has long been a major threat to public health worldwide. The virus can be highly deadly because of antigenic shift. Since the H5N1 outbreak in Hong Kong in 1997, avian flu is regarded as the next pandemic threat. For combating the disease, it is essential to investigate more on the influenza virus, in particular H5N1. Nucleoprotein (NP) is a major component of the ribonucleoprotein complex (RNP) in the influenza virus. NP exhibits both structural and functional roles for influenza virus assembly and propagation and is involved in mediating the transcription-replication process. The NP of the virus binds the RNA genome and acts as a key adapter between the virus and the host cell. It therefore plays important roles and represents an attractive drug target. Recently, the X-ray structure of H5N1 NP was solved to a resolution of 3.3 Å , which provides valuable clues on how NP carries out its functions. However, the N-terminal 1-20 residues were not resolved in the H5N1 NP crystal structure. This N-terminal region is thought to contain a nuclear localization signal (NLS), a cellular splicing factor BAT1/UAP56 binding site, and a nuclear export signal. It has been suggested that the N-terminal NLS binds to importin (a cytosolic protein) for the nuclear import of NP. In the present study, the solution structure of H5N1 NP N-terminal peptide (NP20) in membrane mimetic solvent condition was determined using Circular Dichroism (CD) and Nuclear Magnetic Resonance (NMR) spectroscopies. The CD results show that NP20 adopted an α-helical conformation. The NMR data indicate that NP20 formed a single α-helix spanning from residues Gly5 to Gly16. Surface electrostatic potentials further showed that the NP20 peptide is amphipathic in nature, which may be important for its binding with importin. NMR titration experiments have been carried out between NP20 and importin. Addition of importin into the solution of NP20 peptide caused significant broadening of the NMR signals of NP20 and progressive changes of the chemical shifts of NOE cross-peaks at increasing importin concentration confirm that NP20 could bind with importin. Therefore, the present study supports that NP20 region is the binding site of importin mediating the import of NP into the host cell nucleus. In conclusion, the knowledge gained from this study provides a better understanding on the structure of NP20 and its interaction with the host importin protein, and may serve as a template for the development of novel antiviral drug targeting NP with improved therapeutic index. / published_or_final_version / Chemistry / Master / Master of Philosophy

Cone snails are carnivorous marine mollusks, utilizing their neuropeptide-rich venom for prey capture. The venom of Conus brunneus, a wide-spread Eastern Pacific vermivore, has not been extensively studied. In the current work, peptides from the dissected venom were characterized and tested using preliminary bioassays. Six peptides (A-F) were isolated and tested. Three peptide identities were determined by comparison with previously reported data: bru9a (A), bru3a (F), and an a-conotoxin (E). Preliminary screening in a stroke-related model of induced glutamate excitotoxicity in primary neuronal cells and PC12 cell cultures indicated potential neuroprotective activity of peptide fractions A, D, and F. Further testing is necessary to determine and verify structure, activity, target, and mechanism of action of the promising peptides from C. brunneus, which may prove effective neuropharmacological agents to treat stroke. / by Rebecca A. Crouch. / Thesis (M.S.)--Florida Atlantic University, 2013. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Ce mémoire porte sur l'étude des structures et des interactions membranaires de peptides amyloïdes dérivés de la transthyrétine (TTR10-20), de la protéine de prion (PrP110-136) et de l'amyloïde-β (Aβl-42). Grâce aux spectroscopies IR-TF et RMN, complémentées par des techniques de microscopic nous avons étudié la structure et les interactions entre TTR10-20 et PrP110-136 et des membranes modèles zwitterioniques (DMPC) et anioniques (DMPG). Finalement, les spectroscopies UV-Vis et de fluorescence ont permis d'étudier la cinétique d'agrégation d'Aβi-42. Les résultats démontrent que TTR10-20 a tendance à former des agrégats non-fibrillaires qui affectent davantage la dynamique des têtes polaires de DMPC. D'un autre côté, PrP110-136 forme une hélice transmembranaire avec DMPC, mais cette hélice semble préférer interagir avec les têtes polaires des vésicules de DMPG. Dans la dernière étude, nous avons déterminé qu'Aβl-42 commence à former des structures fibrillaires après une phase latente de 500 à 600 minutes.

Cette thèse porte sur l'étude de la structure du peptide amyloïde-p (AP) (25-35) et de ses interactions avec des membranes. L'Ap (25-35) est la séquence cytotoxique de l'Ap (1-40/42), soit le peptide parent majoritairement impliqué dans la formation de plaque senile extracellulaire dans la maladie d'Alzheimer. L'Ap fut proposé être au centre de la problématique, reliée à l'agrégation pathogène. L'implication des membranes est une aire émergente et moins explorée jusqu'à maintenant. Les objectifs de cette thèse furent de caractériser la structure de l'Ap (25-35) en fonction de diverses conditions physico-chimiques, en absence et en présence de membranes. Nous avons également proposé un rôle protecteur du cholestérol dans ces interactions. Nous avons donc utilisé diverses méthodes biophysiques dans le but de mieux définir l'Ap (25-35) à titre de cible thérapeutique potentielle. • Nous avons montré que l'agrégation de l'Ap (25-35) dépend de la concentration et est favorisée à pH physiologique et faible force ionique. Le peptide interagit avec les membranes de PC, PG et PC/PG et une désagrégation partielle de l'Ap (25-35) en résulte. L'Ap (25-35) n'induit que peu de pores membranaires. Le peptide se présente majoritairement en structure de feuillets-p intermoléculaires antiparallèles et de protofilaments. La présence d'un coude-p de type II est proposée près des résidus en N-terminal. Le peptide se désagrège dans le temps, mais l'interaction membranaire prévient ce phénomène. L'Ap (25-35) montre une structure étendue dans son domaine hydrophobe et le registre des brins-p optimise l'interaction hydrophobe. Le peptide interagit différemment avec les membranes selon sa concentration. La présence d'une haute teneur en cholestérol atténue les effets membranaires de l'Ap (25-35) et une réduction protège tout autant les membranes. Le peptide interagit en surface, au niveau des têtes polaires des lipides et nous avons proposé que l'axe moléculaire des protofilaments soit parallèle au plan de la membrane. / This thesis reports the study of the structure of the amyloid-p (AP) (25-35) and its interactions with membranes. This peptide is the cytotoxic sequence of the parent peptide Ap (1-40/42), mostly found in the extracellular deposits responsible for the senile plaque formation typical of Alzheimer's disease. The Ap peptide was proposed to be at the core of the problematic related to pathogenic aggregation. The involvement of membranes is a new and less explorated area so far. The main objective of this thesis was to characterize the* structure of the AP (25-35) peptide as a function of many physico-chemical conditions, in the absence and in the presence of membranes. We have also explored the protective role of cholesterol in these interactions. To do so, we have used several biophysical techniques in the aim to better define the Ap (25-35) peptide as a potential therapeutic target. We have shown that the aggregation of the AP (25-35) peptide depends on concentration and is favored at physiological pH and at low ionic strength. The peptide interacts with membranes made of PC, PG and PC/PG and this interaction results in a partial disaggregation of the peptide. The Ap (25-35) peptide does not significantly induce pore formation in membranes. The peptide is structured mostly as intermolecular antiparallel p-sheets and as protofilaments. The presence of a type II p-turn is proposed in the N-terminal region. The peptide disaggregates as a function of time, but this trend is hindered by the interaction with membranes. The Ap (25-35) peptide shows an extended structure in its hydrophobic domain and the register of the p-strands optimizes the hydrophobic interactions. The peptide interacts differently with membranes as a function of concentration. The presence of a high amount of cholesterol attenuates the membrane effects of the peptide and a reduced amount also protects the membranes in the same extent. The AP (25-35) peptide interacts at the surface of the membranes, especially with the lipid polar head groups and we propose that the molecular axis of the protofilaments is basically parallel to the plane of the membrane.