Structural and functional studies on nucleotide binding to AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is an enzyme that senses and regulates cellular energy balance thus playing a key role in homeostasis. As such it is a target for treatment of metabolic disorders such as type II diabetes. AMPK is a hetero-trimeric complex composed of an α, β and γ subunit. α contains the catalytic kinase domain, β is a scaffolding subunit that enables complex formation and γ monitors cellular energy via nucleotide binding to its CBS domains. AMPK is primarily activated by phosphorylation at Thr-172 on the activation loop in the kinase domain. It exerts its cellular effects via phosphorylation of a range of downstream targets involved in different aspects of energy production & utilization. The aim of this thesis is to characterize the mechanistic basis of energy regulation of mammalian AMPK via structural and binding measurements. Fluorescence studies have been facilitated by the use of N-methylanthraniloyl (mant) labelled AMP and of β-Nicotinamide adenine dinucleotide 2’-phosphate (NADPH) to monitor competition with AMP, ADP and ATP. A number of mutations in the γ subunit, which interfere with the normal function of AMPK and cause Wolff-Parkinson- White (WPW) syndrome, have also assessed for changes in nucleotide binding affinities and potential implications for the regulation of kinase activity. Thermal denaturation experiments are used to investigate the stabilizing effects of nucleotides and other small molecule ligands. This method was used in a low throughput screen against an enriched list of compounds selected from an in silico screen to try to identify novel activators. I have also determined the structure of the regulatory fragment of the enzyme bound to 5-aminoimidazole-4-carboximide riboside monophosphate (ZMP), an intermediate on the biosynthetic route to AMP, the fluorescence reporter mant-AMP and the WPW mutants Arg298\rightarrowGly, Arg69\rightarrowGln and His150\rightarrowArg. The structures of the mutants have revealed that nucleotide binding is impaired due to a reduced affinity for the nucleotides thus affecting the regulation of the kinase

Experimental evaluation of a localization algorithm for multiple acoustic sources in reverberating environments

Publication in the conference proceedings of EUSIPCO, Florence, Italy, 200

aPKC Inhibition by Par3 CR3 Flanking Regions Controls Substrate Access and Underpins Apical-Junctional Polarization

Atypical protein kinase C (aPKC) is a key apical-basal polarity determinant and Par complex component. It is recruited by Par3/Baz (Bazooka in Drosophila) into epithelial apical domains through high-affinity interaction. Paradoxically, aPKC also phosphorylates Par3/Baz, provoking its relocalization to adherens junctions (AJs). We show that Par3 conserved region 3 (CR3) forms a tight inhibitory complex with a primed aPKC kinase domain, blocking substrate access. A CR3 motif flanking its PKC consensus site disrupts the aPKC kinase N lobe, separating P-loop/αB/αC contacts. A second CR3 motif provides a high-affinity anchor. Mutation of either motif switches CR3 to an efficient in vitro substrate by exposing its phospho-acceptor site. In vivo, mutation of either CR3 motif alters Par3/Baz localization from apical to AJs. Our results reveal how Par3/Baz CR3 can antagonize aPKC in stable apical Par complexes and suggests that modulation of CR3 inhibitory arms or opposing aPKC pockets would perturb the interaction, promoting Par3/Baz phosphorylation

تطور الآراء النحوية عند ابن هشام الأنصاري "دراسة تحليلة نحوية"

هذا البحث يتكلم عن " تطور الآراء النحوية عند ابن هشام الأنصاري "دراسة تحليلية نحوية". و هذا البحث يحتوي على المشكلتين هما: (1) كيف تطور المظاهر النحوية عند ابن هشام. (2) كيف تطور الأراء النحوية عند ابن هشام الأنصاري. الأهداف من إجراءات هذا البحث هي: (1) أن يعرف الباحث أو القارئ تطور المظاهر النحوية عند ابن هشام. (2) أن يعرف الباحث أو القارئ تطور الأراء النحوية عند ابن هشا م الأنصاري. من نتائج البحث، هناك بعض الأمور التي حصل عليه الكاتب من خلال إجراءاته، مما يالي: 1) مظاهر النحوية عند ابن هشام محورين أساسين: الأول : التدرج النحوي في المسألة الواحدة. الثاني : مخالفة رأيه السابق والأدول أنه إلى رأي آخر. الثالث: نيابة الصفة عن المصدري في باب المفعول المطلق. 2) تطور الآراء النحوية عند ابن هشام الأنصاري تكون منها: الكلام اصطلاحا، الكلم والكلمة، علامات الاسم