SH3 domains are excellent models for probing folding and protein interactions. This thesis describes NMR studies of several SH3 domains, including the N-terminal SH3 domain of the Drosophila adaptor protein Drk (drkN SH3 domain), the SH3 domain of the proto-oncogene tyrosine-kinase Fyn, and the SH3 domains of the human adaptor protein CIN85, involved in Cbl-mediated downregulation of epidermal growth factor receptor (EGFR) and other receptor tyrosine kinases (RTKs). The drkN SH3 domain is an ideal system for studying disordered states. The unique quality of this isolated domain is that it exists in an approximately 50/50 equilibrium between its folded and unfolded states under non-denaturating buffer conditions. Interestingly, the single T22G mutation dramatically stabilizes the domain. Here the NMR structures of the drkN SH3 domain and its T22G mutant are determined and compared in order to illuminate the causes of the marginal stability of the domain. Solvent exposure of the folded and the unfolded drkN SH3 domains are probed and compared with a novel NMR technique using molecular oxygen dissolved in solution as a paramagnetic probe. The changes in partial molar volume along the folding trajectories of the drkN SH3 and Fyn SH3 domains are also studied and analyzed here in terms of changes in protein hydration and packing accompanying folding. Finally, the interactions between the SH3 domains of CIN85 and ubiquitin are discussed. All three are shown to bind ubiquitin. The structure of the SH3-C domain in complex with ubiquitin is presented and the effect of disruption of ubiquitin binding on ubiquitination of CIN85 and EGFR in vivo is discussed. SH3 domains are easily amendable to a wide range of NMR approaches and provide a good system for development and testing of novel methods. Through the use of these approaches significant insights into details of SH3 domain structure, stability, mechanisms of folding and cellular function have been gained.

SH3 domains are excellent models for probing folding and protein interactions. This thesis describes NMR studies of several SH3 domains, including the N-terminal SH3 domain of the Drosophila adaptor protein Drk (drkN SH3 domain), the SH3 domain of the proto-oncogene tyrosine-kinase Fyn, and the SH3 domains of the human adaptor protein CIN85, involved in Cbl-mediated downregulation of epidermal growth factor receptor (EGFR) and other receptor tyrosine kinases (RTKs). The drkN SH3 domain is an ideal system for studying disordered states. The unique quality of this isolated domain is that it exists in an approximately 50/50 equilibrium between its folded and unfolded states under non-denaturating buffer conditions. Interestingly, the single T22G mutation dramatically stabilizes the domain. Here the NMR structures of the drkN SH3 domain and its T22G mutant are determined and compared in order to illuminate the causes of the marginal stability of the domain. Solvent exposure of the folded and the unfolded drkN SH3 domains are probed and compared with a novel NMR technique using molecular oxygen dissolved in solution as a paramagnetic probe. The changes in partial molar volume along the folding trajectories of the drkN SH3 and Fyn SH3 domains are also studied and analyzed here in terms of changes in protein hydration and packing accompanying folding. Finally, the interactions between the SH3 domains of CIN85 and ubiquitin are discussed. All three are shown to bind ubiquitin. The structure of the SH3-C domain in complex with ubiquitin is presented and the effect of disruption of ubiquitin binding on ubiquitination of CIN85 and EGFR in vivo is discussed. SH3 domains are easily amendable to a wide range of NMR approaches and provide a good system for development and testing of novel methods. Through the use of these approaches significant insights into details of SH3 domain structure, stability, mechanisms of folding and cellular function have been gained.

The search for efficient and economical ways to beneficiate fine coal remains an active research area. Recent developments have shown that the reflux classifier can successfully be used on Australian coals, and based on that, a number of pilot plant investigations have been done in South Africa. While pilot scale units are usually used to test the applicability of a new technology on specific coals, a need exists to gather more fundamental data at a laboratory scale in order to save manpower, costs and time. This study has aimed at introducing a way to pre-test material prior to pilot plant trials in the design chain. The study shows that a laboratory water only reflux classifier can be used as a density fractionator, which accurately produces washability data for coal – this was also investigated by Callen et al. (2008). There is also a linear correlation between density cut-point and fluid velocity within the plates. Only when looking at the model proposed in Walton (2011:68), does it become clear that the relationship is indeed slightly curved. Many investigations from laboratory and pilot tests accept the linear relationship, and describe it as slightly curved due to the settling being in the intermediate settling regime (Iveson et al., 2014; Galvin & Lui, 2011). The separation procedures that produce two products – an overflow and underflow – compare well with fractionation results produced. Thus, fractionation results can generate washability data and predict batch separation operations. The laboratory reflux classifier setup is also dependent on particle size, where individual size ranges achieve e.p.m. values of 0.012 and 0.030, while the combined separation efficiency is 0.039. It was, however, found that the respective laboratory scale reflux classifier that was designed and built was not suitable for continuous operation. The vertical fluidisation section was not high enough to enable a steady fluidised bed. This was necessary for density separation within the bed and to produce a significant pressure differential. It is also recommended to obtain a PID controller. / MIng (Chemical Engineering), North-West University, Potchefstroom Campus, 2015

The search for efficient and economical ways to beneficiate fine coal remains an active research area. Recent developments have shown that the reflux classifier can successfully be used on Australian coals, and based on that, a number of pilot plant investigations have been done in South Africa. While pilot scale units are usually used to test the applicability of a new technology on specific coals, a need exists to gather more fundamental data at a laboratory scale in order to save manpower, costs and time. This study has aimed at introducing a way to pre-test material prior to pilot plant trials in the design chain. The study shows that a laboratory water only reflux classifier can be used as a density fractionator, which accurately produces washability data for coal – this was also investigated by Callen et al. (2008). There is also a linear correlation between density cut-point and fluid velocity within the plates. Only when looking at the model proposed in Walton (2011:68), does it become clear that the relationship is indeed slightly curved. Many investigations from laboratory and pilot tests accept the linear relationship, and describe it as slightly curved due to the settling being in the intermediate settling regime (Iveson et al., 2014; Galvin & Lui, 2011). The separation procedures that produce two products – an overflow and underflow – compare well with fractionation results produced. Thus, fractionation results can generate washability data and predict batch separation operations. The laboratory reflux classifier setup is also dependent on particle size, where individual size ranges achieve e.p.m. values of 0.012 and 0.030, while the combined separation efficiency is 0.039. It was, however, found that the respective laboratory scale reflux classifier that was designed and built was not suitable for continuous operation. The vertical fluidisation section was not high enough to enable a steady fluidised bed. This was necessary for density separation within the bed and to produce a significant pressure differential. It is also recommended to obtain a PID controller. / MIng (Chemical Engineering), North-West University, Potchefstroom Campus, 2015

Anaphylaxis is a rapid, life-threatening hypersensitivity reaction. Until recently, it was mainly attributed to histamine released by mast cells activated by allergen crosslinking (XL) of FcεRI-bound allergen-specific IgE. However, recent reports established that anaphylaxis could also be triggered by basophil, macrophage and neutrophil secretion of platelet activating factor subsequent to FcγR stimulation by IgG/Ag complexes. I have investigated the contribution of Fyn and Lyn tyrosine kinases to FcγRIIb and FcγRIII signaling in the context of IgG-mediated passive systemic anaphylaxis (PSA). I found that mast cell IgG XL induced Fyn, Lyn, Akt, Erk, p38 and JNK phosphorylation. Additionally, IgG XL of mast cells, basophils and macrophages resulted in Fyn- and Lyn-regulated mediator release in vitro. FcγR–mediated activation was enhanced in Lyn-deficient (KO) cells, but decreased in Fyn KO cells, compared to wild type cells. More importantly, Lyn KO mice displayed significantly exacerbated PSA features while no change was observed for Fyn KO mice, compared to wild type littermates. Intriguingly, this work establishes that mast cells account for the majority of serum histamine in IgG-induced PSA. Taken together, these findings establish pivotal roles for Fyn and Lyn in the regulation of PSA and highlight their unsuspected functions in IgG-mediated pathologies.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 81-97).

Thesis (M.Sc.). / Written for the Dept. of Chemistry. Title from title page of PDF (viewed 2009/06/23). Includes bibliographical references.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24508号 / 医博第4950号 / 新制||医||1064(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 平井 豊博, 教授 武藤 学, 教授 中島 貴子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No / Recently published research indicates that soluble oligomers of beta-amyloid (Abeta) may be the key neurotoxic species associated with the progression of Alzheimer's disease (AD) and that the process of Abeta aggregation may drive this event. Furthermore, soluble oligomers of Abeta and tau accumulate in the lipid rafts of brains from AD patients through an as yet unknown mechanism. Using cell culture models we report a novel action of Abeta on neuronal plasma membranes where exogenously applied Abeta in the form of ADDLs can be trafficked on the neuronal membrane and accumulate in lipid rafts. ADDL-induced dynamic alterations in lipid raft protein composition were found to facilitate this movement. We show clear associations between Abeta accumulation and redistribution on the neuronal membrane and alterations in the protein composition of lipid rafts. In addition, our data from fyn(-/-) transgenic mice show that accumulation of Abeta on the neuronal surface was not sufficient to cause cell death but that fyn is required for both the redistribution of Abeta and subsequent cell death. These results identify fyn-dependent Abeta redistribution and accumulation in lipid rafts as being key to ADDL-induced cell death and defines a mechanism by which oligomers of Abeta and tau accumulate in lipid rafts.

Les sécrétines peptidiques de l’hormone de croissance (GHRPs) constituent une classe de peptides synthétiques capables de stimuler la sécrétion de l’hormone de croissance (GH). Cette activité est médiée par leur liaison à un récepteur couplé aux protéines G : le récepteur des sécrétines de l’hormone de croissance (GHS-R1a), identifié subséquemment comme le récepteur de la ghréline. La ghréline est un peptide de 28 acides aminés sécrété principalement par les cellules de la muqueuse de l’estomac, qui exerce de nombreux effets périphériques indépendamment de la sécrétion de l’hormone de croissance. Les effets indépendants de la sécrétion de GH incluent, entre autres, des actions sur le contrôle de la prise de nourriture, le métabolisme énergétique, la fonction cardiaque, le système immunitaire et la prolifération cellulaire. L’étude de la distribution périphérique des sites de liaison des GHRPs nous a permis d’identifier un second site, le CD36, un récepteur scavenger exprimé dans plusieurs tissus dont le myocarde, l’endothélium de la microvasculature et les monocytes/macrophages. Le CD36 exprimé à la surface du macrophage joue un rôle clé dans l’initiation du développement de l’athérosclérose par la liaison et l’internalisation des lipoprotéines de faible densité oxydées (LDLox) dans l’espace sous-endothélial de l’artère. L’hexaréline, un analogue GHRP, a été développé comme agent thérapeutique pour stimuler la sécrétion de l’hormone de croissance par l’hypophyse. Sa propriété de liaison aux récepteurs GHS-R1a et CD36 situés en périphérie et particulièrement sa capacité d’interférer avec la liaison des LDLox par le CD36 nous ont incité à évaluer la capacité de l’hexaréline à moduler le métabolisme lipidique du macrophage. L’objectif principal de ce projet a été de déterminer les effets de l’activation des récepteurs CD36 et GHS-R1a, par l’hexaréline et la ghréline, le ligand endogène du GHS-R1a, sur la physiologie du macrophage et de déterminer son potentiel anti-athérosclérotique. Les résultats montrent premièrement que l’hexaréline et la ghréline augmentent l’expression des transporteurs ABCA1 et ABCG1, impliqués dans le transport inverse du cholestérol, via un mécanisme contrôlé par le récepteur nucléaire PPARγ. La régulation de l’activité transcriptionnelle de PPARγ par l’activation des récepteurs CD36 et GHS-R1a se fait indépendamment de la présence du domaine de liaison du ligand (LBD) de PPARγ et est conséquente de changements dans l’état de phosphorylation de PPARγ. Une étude plus approfondie de la signalisation résultant de la liaison de la ghréline sur le GHS-R1a révèle que PPARγ est activé par un mécanisme de concertation entre les voies de signalisation Gαq/PI3-K/Akt et Fyn/Dok-1/ERK au niveau du macrophage. Le rôle de PPARγ dans la régulation du métabolisme lipidique par l’hexaréline a été démontré par l’utilisation de macrophages de souris hétérozygotes pour le gène de Ppar gamma, qui présentent une forte diminution de l’activation des gènes de la cascade métabolique PPARγ-LXRα-transporteurs ABC en réponse à l’hexaréline. L’injection quotidienne d’hexaréline à un modèle de souris prédisposées au développement de l’athérosclérose, les souris déficientes en apoE sous une diète riche en cholestérol et en lipides, se traduit également en une diminution significative de la présence de lésions athérosclérotiques correspondant à une augmentation de l’expression des gènes cibles de PPARγ et LXRα dans les macrophages péritonéaux provenant des animaux traités à l’hexaréline. L’ensemble des résultats obtenus dans cette thèse identifie certains nouveaux mécanismes impliqués dans la régulation de PPARγ et du métabolisme du cholestérol dans le macrophage via les récepteurs CD36 et GHS-R1a. Ils pourraient servir de cibles thérapeutiques dans une perspective de traitement des maladies cardiovasculaires. / Growth hormone-releasing peptides (GHRPs) are a class of small synthetic peptides known to stimulate GH release through their binding to a G protein-coupled receptor identified as GHS-R1a, later recognized as the ghrelin receptor. Ghrelin is an acetylated 28 amino acid hormone initially identified from the stomach, which induces the release of growth hormone (GH) from the pituitary but also regulates food intake, energy homeostasis, cardiovascular function, immune system and cell proliferation. In documenting the peripheral distribution of GHRPs binding sites, we uncovered the presence of another binding site for GHRPs, identified as CD36, a class B scavenger receptor. CD36 is expressed among several tissues, including myocytes, endothelial cells of the microvasculature and monocytes/macrophages. The macrophage CD36 contributes to excessive lipid loading and atherogenic formation of foam cells through uptake of oxidized low-density lipoprotein (oxLDL) in the subendothelial space of the artery. The properties of hexarelin, a ligand for GHS-R1a and CD36, which features overlapping binding sites with that of oxLDL binding domain on CD36, and thus interfering with the binding of oxLDL on CD36, have prompted us to evaluate the potential of hexarelin, as well as that of the endogenous ligand ghrelin in the modulation of macrophage cholesterol metabolism. We demonstrate here the ability of hexarelin and ghrelin to enhance the expression of ATP-binding cassette A1 and G1 transporters through a PPARγ-dependent mechanism. The hormone binding domain of PPARγ is not required to mediate PPARγ transcriptional activation by CD36 and GHS-R1a. Both hexarelin and ghrelin promotes phosphorylation of PPARγ in THP-1 macrophages. A more detailed study of GHS-R1a-initiated signaling revealed an intricate and complex signalling interplay triggered by ghrelin that involves modulation of Src-dependent Dok-1/ERK1/2 and Src-independent Gαq/PI3-K/Akt pathways, leading to PPARγ-dependent transcriptional competence in the macrophages. The central role of PPARγ on cholesterol metabolism in the macrophages has been demonstrated using peritoneal macrophages from PPARγ heterozygote mice whose response to hexarelin on PPARγ-LXRα-ABC transporters pathway was strongly impaired. Treatment of apolipoprotein E-null mice fed on a lipid-rich diet with hexarelin resulted in a significant reduction in atherosclerotic lesions, concomitant with an enhanced expression of PPARγ and LXRα target genes in peritoneal macrophages. The results presented in this thesis feature novel mechanisms by which the beneficial regulation of PPARγ and cholesterol metabolism in macrophages could be regulated by both CD36 and ghrelin receptor. The downstream effects following the activation of these receptors might be potential targets in the treatment of human coronary artery disease.