No description available.

No description available.

No description available.

No description available.

A hallmark of Alzheimer's disease (AD) is the accumulation of insoluble senile plaques in the brain.[1] The major component of the insoluble plaques is the amyloid-β peptide (Aβ) that is produced through cleavage of the amyloid-β precursor protein (APP).[2] It is well understood that once the monomeric Aβ is generated, it has the potential to aggregate into soluble oligomers and further into insoluble fibrils. Recently it has been proposed that early oligomers are the main toxic species in the aggregation cascade.[3] However, it has been shown that the formation of toxic early oligomers is inhibited by several endogenous plasma proteins, including albumin and transferrin (Tf). In this investigation we are focusing on the mechanism of inhibition of the Aβ early oligomerization by Tf. Specifically, we have targeted the early stages of Aβ aggregation using a deletion mutant of the Aβ peptide, i.e. the Aβ12-28 fragment, which selectively stabilizes the early Aβ oligomers. Self-association of this peptide was controlled by adding-NaCl to filtered monomeric Aβ samples and the effect of Tf inhibition on these aggregates was probed by 1H relaxation NMR experiments.[4-7] Our data shows that Tf directly targets intermediary Aβ oligomers via a coating mechanism. 1. Kirkitadze, M.D., Condron, M.M. and Teplow, D.B, JMB 2001 312;1103-1119. 2. Stefan F. Lichtenthaler and Christian Haass, JCI 2004 113(10);1384-1387. 3. Necula M., Kayed R., Milton, S. and Glabe C.G, JBC 2007 282(14);10311-10324. 4. Klement K., Wieligmann K., Meinhardt J., Hortschansky P., Richter W., and Fändrich M., JMB 2007 373;1321-1333. 5. Huang H, Milojevic J, Melacini G. J Phys Chem B. 2008 112(18):5795-802. 6. Milojevic J, Esposito V, Das R, Melacini G. JACS. 2007 129(14):4282-90. 7. Milojevic J, Esposito V, Das R, Melacini G. J Phys Chem B. 2006 110(41):20664-70. / Thesis / Master of Science (MSc)

<p dir="ltr">Zeolites are microporous, crystalline aluminosilicates widely used in catalysis and separation. The substitution of Si⁴⁺ with Al³⁺ ([AlO_4/2]^-) creates a charge imbalance that can be compensated by a metal cation or complex (Mⁿ⁺) or a Brønsted acid proton (H⁺) within microporous voids and at external surfaces. Brønsted acid sites in aluminosilicates of diverse topologies have similar acid strength, but the diffusion of reactants and products can vary depending on the micropore size, tortuosity, and connectivity. The coupled effects of H⁺-site reactivity and diffusional constraints imposed by the inorganic zeolitic framework can be assessed by the diffusion parameter, which depends on the bulk proton density ([H⁺]) and the diffusion pathlength (L), derived from the Thiele modulus expression that relates reaction and diffusion rates within porous catalysts. This motivates synthetic approaches to control zeolite properties that influence diffusion and reactivity such as crystallite size and proton density. Prior synthetic methods have tried to minimize the diffusional constraints by decreasing the diffusion pathlength (L) by synthesizing zeolite crystallites at the nanometer scale or by increasing the effective diffusivity (D_e) by synthesizing hierarchical materials. However, these synthetic approaches may simultaneously influence multiple zeolite properties, such as the spatial distribution of acid sites throughout crystallites or at extracrystalline surfaces, convoluting the influence of these properties on the rates, selectivity, and deactivation of acid-catalyzed reactions.</p><p dir="ltr">Two types of spatial distribution of acid sites could be present within a zeolite. The first is the fraction at unconfined extracrystalline surfaces, and this property is often convoluted with the effect of crystallite size. Assuming acid sites are evenly distributed through the crystallite, as the crystallite size increases, the fraction of external acid sites decreases because the surface area-to-volume decreases. The second type of spatial distribution of acid sites is referred to as “zoning”—a concentration gradient of active sites from the external surface to its core, or vice versa. This type of spatial distribution of acid sites is challenging to quantify accurately. “Zoning” effects may also occur inadvertently during zeolite synthesize using conventional methods. In this work, we synthesize zeolitic materials (i.e., MFI) with controlled spatial distribution of acid sites independently of crystallite size and H⁺-site density to study their effects on propene oligomerization catalysis. A core@shell synthesis approach was used to passivate external MFI zeolite surfaces by an inert shell (Si-MFI) of short thickness relative to the size of the core crystallite. Although other passivation treatments can cause pore blockage or narrowing, transient sorption measurement showed no additional diffusional limitations were introduced by the growth of the Si-MFI shell. Propene dimerization rates (per H⁺, 503 K, 16 – 620 kPa C₃H₆) and transient behavior upon pressure step-changes persist reveal the influence of intrazeolite diffusional constraints on the Al-MFI core due to heavier oligomer products that accumulate inside micropores. On the contrary, dimerization rates did not reach a pseudo-steady-state on Al-MFI@Si-MFI and required high temperature caused by the formation of surface carbonaceous deposits in the absence of acid sites that otherwise assist in the cracking and desorption of coke precursor species. Thus, the passivation of the external surface imposes a transport limitation at the surface due to a carbonaceous layer that forms during the reaction, restricting the diffusion of products out of crystallites and shifting the selectivity towards a lighter product composition.</p><p dir="ltr">An inverted core@shell (Si-MFI@Al-MFI) material was also synthesized to investigate the effect of the spatial distribution of acid sites on the diffusion parameter, where the acid sites are preferentially located at the external surface and the core is inert (i.e. Si-MFI). The spatial distribution of acid sites was varied by growing an Al-MFI shell on a siliceous core and maintaining a similar bulk crystallite size. Mesitylene benzylation was used to quantify the fraction of external acid sites. Differences in measured propene dimerization rates (per H⁺) and product selectivity can be rationalized considering the thickness of the Al-rich shell in the core@shell material to an Al-MFI sample of similar crystallite size, evincing the dominant influence of the diffusion parameter on propene oligomerization catalytic behavior. Overall, this study demonstrated how zeolite synthetic methods can be used to isolate the effects of spatial distributions of Al from crystallite size and H⁺-site density and provide guidance for zeolite catalyst design efforts to control structural properties that influence reactions driven by coupled kinetic-transport phenomena.</p>

T-cell Intracellular Antigen-1 (TIA1) is a multi-domain RNA-binding protein involved in stress granule formation and implicated in neurodegenerative diseases. TIA1 contains three RNA recognition motifs (RRMs), which are capable of binding nucleic acids, and a C-terminal intrinsically disordered prion-related domain (PRD), which plays a role in promoting liquid-liquid phase separation. Motivated by our previous findings indicating that RRMs 2 and 3 exhibit a well-ordered structure in the oligomeric full-length form, whereas RRM1 and PRD demonstrate a propensity for phase separation, the present work in this dissertation aims to investigate the functional competence of the oligomeric state and its binding capabilities. Moreover, the study explores the effects of ligand binding on oligomerization dynamics and potential alterations in protein conformation primarily using solid-state NMR methods. The NMR data show that ssDNA binds to full-length oligomeric TIA1 primarily at RRM2, but also weakly at RRM3, and Zn2+ binds primarily to RRM3. The binding of Zn2+ and DNA was reversible and without the formation of amyloid fibrils. The addition of Zn2+ caused the TIA1:DNA complexes to collapse, indicating that Zn2+ may play a regulatory role by shifting the nucleic acid binding off RRM3 and onto RRM2 by occupying various “half” binding sites on RRM3 and introducing a mesh of crosslinks in the supramolecular complex. Furthermore, this dissertation presents an investigation into the interdomain interactions between RRM2 and RRM3, facilitated by the successful preparation of segmentally labeled protein samples using the trans-splicing approach. The results confirm the hypothesis that Zn2+ can bring RRM2 and RRM3 closer together by crosslinking different monomers, as evidenced by the observation of enhanced NMR signals from heteronuclear correlations around the Zn2+ binding sites. In conclusion, studying the structure of full-length TIA1 oligomers is expected to reveal the mechanisms by which an RNA regulatory protein assembles and binds to its biologically relevant ligands while preserving a highly ordered oligomeric structure.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Please refer to full text for abstract / AFRIKAANSE OPSOMMING: Sien volteks vir opsomming

C-terminal mutations of the phosphoprotein nucleophosmin (NPM) are the most frequent genetic aberration detected in adult acute myeloid leukemia (AML). I focused on characterization of type A, B and E of AML-related C-terminal mutations. The plasmids bearing fluorescently labeled wild type or mutated NPM have been constructed to characterize mutation-induced changes in the localization of NPM. Mammalian cell lines HEK293T, HeLa and NIH 3T3 were used for production of the chimeric proteins. The intracellular localization of the mutated forms of NPM was analyzed by immunofluorescence staining and fluorescence microscopy of the living cells. The localization of the mutNPM type A and B was almost identical and predominantly cytoplasmic, while mutNPM type E was detected in nucleolus and cytoplasm simultaneously. However localization of the mutated forms was greatly influenced by the used cell line. It has been demonstrated that the exogenous NPM interacts with the endogenous NPM and that they mutually affect their intracellular localization due to heterooligomer formation. Detailed analysis of the relationship between the C-terminal mutations and the localization of the mutated NPM improves understanding of specific mutation effect on the formation and progression of AML and also specifies its prognostic...

Les récepteurs couplés aux protéines G forment une grande famille de récepteurs transmembranaires. De nombreuses études montrent que ces récepteurs présenteraient une tendance à interagir entre eux et à former des oligomères. Ces structures sont toutefois sujettes à controverse. En effet, très peu d'éléments permettent d'affirmer que ces oligomères existeraient dans les tissus natifs, la plupart des caractérisations se faisant en systèmes hétérologues. Nous avons donc développé une approche basée d'une part sur l'utilisation de ligands fluorescents pour marquer les récepteurs dans leur environnement natif et d'autre part sur le FRET (Fluorescence Resonance Energy Transfer) en temps résolu en utilisant des cryptates de lanthanides, en particulier le Lumi4-Tb. Nous avons ainsi pu montrer et publier l'existence d'oligomères du récepteur de l'ocytocine dans la glande mammaire. Le protocole de cette étude a aussi été publié et a été validé pour la mise en évidence d'hétéro-oligomères, plus précisément entre les récepteurs V1a et V2 de la vasopressine. La poursuite de l'étude de ce phénomène dans les tissus natifs nous a poussés à développer notre propre dispositif de microscopie FRET en temps résolu. Ce dispositif est basé sur un microscope en champ large auquel nous avons ajouté une source laser pour l'excitation pulsée et une caméra CCD Multigate pour la détection. Nous en présentons ici les premiers résultats ainsi que sa validation pour l'utilisation de multiples fluorophores accepteurs avec une contamination minimale par le Lumi4-Tb. Enfin, nous proposons un modèle pharmacologique montrant l'utilisation de ligands bivalents pour étudier le couplage des oligomères. / G-protein coupled receptors form a very large family of transmembrane receptors. Numerous studies have shown that these receptors showed a tendency to interact and form oligomers. These structures are however the matter of great debate. Indeed, very few elements allow us to maintain that these oligomers could exist in native tissues, most studies being carried out in heterologous systems. We have therefore developed an approach based for one part on the use of fluorescent ligands to label receptors in their native environment, and on the other part on time-resolved FRET (Fluorescence Resonance Energy Transfer) by using lanthanide cryptates, more specifically Lumi4-Tb. We have thus been able to show and publish the existence of oxytocin receptor oligomers in the mammary gland. The protocol used for this study was also published and validated for the study of hetero-oligomers, more specifically between vasopressin V1a and V2 receptors. Following on our study of oligomers in native tissues, we have developed our own setup to perform time-resolved FRET microscopy. This setup is based on a wide field microscope to which we added a laser source for the pulsed excitation and a Multigate CCD camera for imaging. We are here presenting the first results as well as its validation for the use of multiple acceptor fluorophores with minimal bleed through from the Lumi4-Tb. Lastly, we propose a pharmacological model showing the use of bivalent ligands to study oligomer coupling.