Thesis (Ph.D.)--Medical University of Ohio, 2005. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Sciences." Major advisor: L. John Greenfield, Jr. Includes abstract. Document formatted into pages: iv, 208 p. Title from title page of PDF document. Bibliography: pages 55-62,94-99,137-143,166-206.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 130-169).

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 111-139).

The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are ligand-gated glutamatergic ion channels that mediate most excitatory neurotransmission in the brain. Alterations in AMPAR synaptic accumulation mediate synaptic plasticity, including long-term potentiation, long-term depression and homeostatic synaptic plasticity. AMPAR abundance in neurons is determined by balanced processes of protein translation and degradation. Changes in AMPAR function and trafficking have direct impacts on synaptic transmission and cognitive functions. However, the molecular mechanisms regulating AMPAR expression and dynamics in neurons remain largely unknown. In this thesis, two molecular mechanisms that regulate AMPAR translation and protein stability through two different signaling pathways, 5' adenosine monophosphate-activated protein kinase (AMPK) and sirtuin 2 (SIRT2), are described. It is shown that SIRT2, a NAD+-dependent protein deacetylase, directly controls AMPAR stability by regulating AMPAR acetylation. For the first time, we discovered that AMPARs are subject to lysine acetylation, a novel form of post-translational modification for glutamate receptors. Under basal conditions, AMPARs are highly acetylated at their intracellular C termini, which protects against ubiquitination to antagonize AMPAR endocytosis and degradation, leading to prolonged receptor half-life. SIRT2 is also identified as the enzyme responsible for AMPAR deacetylation. Knockdown of SIRT2 led to elevated AMPAR acetylation and reduced ubiquitination, and consequently, increased AMPAR levels and synaptic transmission. SIRT2 knockout mice displayed weakened synaptic plasticity and impaired learning and memory. Resveratrol is a phytoalexin that has been shown to increase AMPAR expression and synaptic accumulation in neurons. The resveratrol effect on AMPAR expression is independent of sirtuin 1, the conventional target of resveratrol, but rather is mediated by AMPK and its downstream phosphoinositide 3-kinase (PI3K)/Akt pathway. Application of the AMPK activator, 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR), to neurons mimics the effects of resveratrol on both signaling and AMPAR expression. The resveratrol-induced increase in AMPAR expression results from elevated protein synthesis through the AMPK-PI3K pathway activation. These studies describe novel regulatory mechanisms responsible for the control of AMPAR protein amount and subcellular distribution in neurons, providing insights into our understanding of synaptic plasticity, brain function and neurological disorders. / 2017-12-06T00:00:00Z

Animal cells, as distinct as epithelia and migratory cells, have cell polarity that is defined by a common set of molecules. The Par complex polarizes the cortex of animal cells through the activity of atypical protein kinase C (aPKC). In this work, I aimed to determine the mechanism of aPKC substrate polarization and identify common characteristics of aPKC substrates that are polarized by phosphorylation. I found that several diverse Par-polarized proteins contain short highly basic and hydrophobic motifs that overlap with their aPKC phosphorylation sites. These Phospho-Regulated Basic and Hydrophobic (PRBH) motifs mediate plasma membrane localization by electrostatics-based phospholipid binding when unphosphorylated but are displaced into the cytoplasm when phosphorylated. To assess whether the Par complex polarizes other proteins by this mechanism, I developed an algorithm to identify potential PRBH motifs and score these linear motifs for basic and hydrophobic character, as well as the quality and number of aPKC phosphorylation sites. Using this algorithm, I identified numerous putative PRBH candidates in the fruit fly proteome and performed two screens of these candidates for Par-polarized proteins. The first screen focused on determining whether aPKC regulates cortical targeting of proteins that are reported to be polarized. This screen identified the Rho GAP crossveinless-c (cv-c) to be a novel aPKC substrate and found that aPKC is sufficient to polarize cv-c in a reconstituted polarity assay. The second screen characterized the localization of putative PRBH motif-containing proteins in vivo. This screen identified a previously uncharacterized protein, CG6454, to be basolateral in epithelia; however, ex vivo experiments found it to have a Ca2+-dependent and aPKC-independent membrane targeting mechanism. Overall this work identified a common mechanism for Par substrate polarization and used knowledge of this mechanism to identify a novel Par effector. This dissertation contains previously published coauthored materials as well as unpublished materials. / 2019-05-08

Mitochondria are key generators of cellular ATP, vital to complex life. Historically, mitochondrial generation of reactive oxygen species (ROS) was considered to be an unregulated process, produced by dysfunctional mitochondria. More recently, mitochondrial ROS generated by complex I, particularly by the process of reverse electron transfer (RET), has emerged as a potentially biologically relevant signal that is tightly-regulated and dependent on mitochondrial status. ROS production by RET is reported to play a role in the innate immune response and lifespan extension in fruit flies. One way in which mitochondrial ROS may behave as a signal is by altering the activity of AMP-activated protein kinase (AMPK), a key metabolic sensor and regulator of cell metabolism, which is activated when cellular ATP levels decrease during energy demand. Mitochondria can signal to AMPK via the magnitude of the cellular ATP/AMP and ATP/ADP ratios, which alter in response to mitochondrial function. Our view is mitochondria may also signal to AMPK via ROS. Important studies have helped to clarify the role of exogenous or cytosolic ROS in AMPK regulation. However, the effects of mitochondrial ROS on AMPK activity, specifically that generated by complex I, remain unclear and is the main focus of this thesis. I characterized the effects of exogenous H2O2 on cellular AMPK activity, ATP/ADP ratios and cellular redox state in a cell model. I then compounded this with selective mitochondria generated ROS by the mitochondria-targeted redox-cycler, MitoParaquat (MPQ). AMPK activity appeared to correlate with decreasing cell ATP/ADP ratios, indicating that both sources of ROS primarily activate AMPK in an AMP/ADP-dependent mechanism. In parallel, I developed an approach for analyzing the redox state of candidate proteins, an important step in determining if a protein is directly regulated by ROS. I also initiated development of a cell model for studying the downstream effects of mitochondrial ROS production by RET, by expressing alternative respiratory enzymes in a mammalian cell line.

xiii, 92 p. ; ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / Cellular polarity is a biological mechanism that is conserved across metazoa and is used in many different biological processes, one of which is stem cell self-renewal and differentiation. Stem cells generate cellular diversity during development by polarizing molecular determinants responsible for directing one daughter cell to maintain stem cell-like qualities and the other daughter cell to initiate a specific cell fate. The stem cell self-renewal versus differentiation choice is critical to avoid overproliferation of stem cells and tumor formation or underdevelopment of tissues and early animal death. Drosophila neural stem cells (neuroblasts) undergo asymmetric cell division (ACD) to populate the fly central nervous system and provide an excellent model system to study processes involving cellular polarity, ACD, stem cell self-renewal, and differentiation. Neuroblasts divide unequally to produce a large, apical self-renewing neuroblast and a small, basal ganglion mother cell that goes on to divide and form two neurons or glia. In this way, a small population of neuroblasts can give rise to thousands of neurons and glia to generate a functional central nervous system. Atypical Protein Kinase C (aPKC) is critical to establish and maintain neuroblast polarity, ACD, stem cell self-renewal, and differentiation. aPKC is part of the evolutionarily conserved Par complex, whose other members include Bazooka and Par-6, and they localize to the neuroblast apical cortex and function to restrict cell-fate determinants into one daughter cell. How aPKC is asymmetrically localized and how its activity translates into cell-fate specification are of incredible importance as apkc mutants where localization is disrupted no longer segregate cell-fate determinants. This work will show that Cdc42 recruits the Par-6/aPKC complex to the neuroblast apical cortex independent of Bazooka. Once there, aPKC phosphorylates the cell-fate determinant Miranda to exclude it from the apical cortex and restrict it basally. Par-6 and Cdc42 regulate aPKC kinase activity though inter- and intramolecular interactions that allow high aPKC kinase activity at the apical cortex and suppressed activity elsewhere. Cdc42 also functions to keep aPKC asymmetrically localized by recruiting the PAK kinase Mushroom bodies tiny to regulate cortical actin and provide binding sites for cortical polarity determinants. This dissertation includes previously published co-authored material. / Adviser: Kenneth Prehoda

Le kinome humain comprend 518 protéines. Elles participent au processus de phosphorylation des protéines qui joue un rôle important dans les voies de signalisation cellulaire. Leur dérégulation est connue comme étant une cause de nombreuses maladies graves telle que les cancers. Du fait de leur grande similarité structurale des protéines kinases, il est difficile de développer des inhibiteurs qui soient à la fois efficaces et sélectifs. L’absence de sélectivité conduit le plus souvent à des effets secondaires particulièrement néfastes pour l’organisme. Au cours de cette thèse, nous avons d’abord développé de nouvelles métriques dont le but est de déterminer la sélectivité d’inhibiteurs à partir de données d’inhibition. Elles présentent l’avantage, comparées à d’autres métriques, d’être applicables sur n’importe quel type de données. Dans un deuxième temps, nous avons développé une approche protéométrique dans le but de comprendre pourquoi certaines protéines kinases ne sont jamais inhibées par des inhibiteurs de Type II. Le modèle statistique mis en place nous a permis d’identifier plusieurs résidus discriminants dont certains déjà décrits expérimentalement dans la littérature. Dans un troisième temps, nous avons développé un nouveau descripteur 3D de protéines kinases avec lequel nous avons mis en place et validé des modèles protéo-chimiométriques visant à étudier et découvrir de nouveaux inhibiteurs. / The human kinome contains 518 proteins. They share a common mechanism of protein phosphorylation known to play an important role in cellular signaling pathways. Impaired kinase function is recognized to be involved in severe diseases like cancer. Due to high structural similarity between protein kinases, development of potent and selective kinase inhibitors is a challenging task. The selectivity of kinase inhibitors may lead to side effects potentially harmful. In this thesis, we first developed new selectivity metrics to determine inhibitor selectivity directly from biological inhibition data. Compared to existing metrics, the new selectivity scores can be applied on diverse inhibition data types. Second, we developed a proteometric approach in order to understand why some protein kinases are never inhibited by Type II inhibitors. The statistical model built for this purpose allowed us to identify several discriminant residues of which few of them correspond to experimentally described residues of interest. Third, using a new 3D protein kinase descriptor, we developed and validated novel proteo-chemometrics approaches to study and discover new kinase inhibitors.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2018-04-19T13:14:58Z No. of bitstreams: 1 texto completo.pdf: 2407879 bytes, checksum: 24a29868ff91347f5c8b65b662cb7870 (MD5) / Made available in DSpace on 2018-04-19T13:14:58Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2407879 bytes, checksum: 24a29868ff91347f5c8b65b662cb7870 (MD5) Previous issue date: 2018-02-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Serine/Arginine protein kinases (SRPKs) são componentes chave da maquinaria de splicing através da regulação por fosforilação das proteínas SR, as quais são cruciais para a seleção dos sítios de splicing alternativo. Entretanto, as SRPKs são frequentemente encontradas superexpressas ou com atividade alterada em diversos tipos de cânceres, inclusive em leucemias. Dessa forma, a busca por pequenas moléculas inibidores destas quinases são de potencial interesse para o delineamento de novas estratégias terapêuticas. Nesta tese, descreve­se a avaliação da inibição farmacológica de SRPKs pelo inibidor seletivo N­(2­ (piperidin­1­il)­5­(trifluorometil)fenil)isonicotinamida (SRPIN340) sobre a viabilidade de linhagens leucêmicas linfoides e mieloides. Além de mostrar atividade citotóxica significativa, o efeito dos tratamentos na regulação da fosforilação das proteínas SR e na alteração da expressão dos genes MAP2K1, MAP2K2, VEGF e FAS foram também identificados. Além disso, a inibição farmacológica de SRPKs foi capaz de desencadear eventos precoces e tardios de apoptose. Por último, estudos de fluorescência intrínseca de triptofano, docking molecular e dinâmica molecular foram analisados a fim de se obter informações estruturais acerca do complexo SRPK/SRPIN340. No segundo estudo, é descrita a síntese de uma série de vinte e duas trifluorometil arilamidas baseadas na estrutura molecular do SRPIN340, além da avaliação dos seus efeitos antileucêmicos. Alguns dos derivados apresentaram efeitos citotóxicos superiores contra linhagens de leucemia mieloide e linfoide em comparação com o SRPIN340. Em particular, os compostos N­(2­(4­bromofenilamino)­5­(trifluorometil)fenil)­2­cloronicotinamida (24), N­(2­(4­bromofenilamino)­5­(trifluorometil)fenil)nicotinamida (30), e N­(2­(4­ bromofenilamino)­5­(trifluorometil)fenil)benzamida (36) apresentaram valores de IC 50 na faixa de 6,0 – 35,7 μM (μmol L ­1 ). Estes três compostos também foram capazes de desencadear eventos de apoptose e autofagia, além de exibir efeito sinergístico em combinação com o agente quimioterápico vincristina. Além disso, o composto 30 se mostrou mais eficiente que o SRPIN340 na diminuição da fosforilação das proteínas SR bem como na diminuição da expressão de isoformas oncogênicas dos genes MAP2K1, MAP2K2, VEGF, e RON. Tomados conjuntamente, estes resultados sugerem que inibidores de SRPKs são capazes de suprimir o crescimento celular através da regulação dos eventos de splicing e podem ser considerados como ponto de partida importante para desenvolvimento de novas estratégias terapêuticas contra leucemias e outros tipos de cânceres. / Serine/Arginine protein kinase (SRPK) are key components of the splicing machinery trough the phospho­regulation of SR Proteins, which are crucial for exon selection in the alternative splicing. However, SRPK have frequently been found overexpressed or/and with altered activity in a number of cancers, including leukemias. Thus, the discovery of small molecule inhibitors against these kinases is of potential interest to identify novel therapeutic opportunities. Here, it is described the pharmacological inhibition of SRPK by N­(2­(piperidin­1­yl)­5­ (trifluoromethyl)phenyl)isonicotinamide (SRPIN340) on the viability of lymphoid and myeloid leukemia cell lines. Along with significant cytotoxic activity, the effect of treatments in regulating the phosphorylation of the SR protein family and in altering the expression of MAP2K1, MAP2K2, VEGF and FAS genes were also assessed. Furthermore, it was found that pharmacological inhibition of SRPKs can trigger early and late events of apoptosis. Finally, intrinsic tryptophan fluorescence emission, molecular docking and molecular dynamics were analyzed to gain structural information on the SRPK/SRPIN340 complex. In a second study, it is described the synthesis of a series of twenty­two trifluoromethyl arylamides based on the SRPIN340 scaffold and the evaluation of their antileukemia effects. Some derivatives presented superior cytotoxic effects against myeloid and lymphoid leukemia cell lines compared to SRPIN340. In particular, compounds N­(2­(4­bromophenylamino)­5­(trifluoromethyl)phenyl)­2­chloronicotinamide (24), N­(2­(4­bromophenylamino)­5­(trifluoromethyl)phenyl)nicotinamide (30), and N­(2­(4­ bromophenylamino)­5­(trifluoromethyl)phenyl)benzamide (36) presented IC 50 values within the 6.0 – 35.7 μM (μmol L ­1 ) range. In addition, these three compounds were able to trigger apoptosis and autophagy, and they exhibited synergistic effects in combination with the chemotherapeutic agent vincristine. Moreover, compound 30 was more efficient than SRPIN340 in impairing the intracellular phosphorylation status of SR proteins as well as the expression of MAP2K1, MAP2K2, VEGF, and RON oncogenic isoforms in leukemia cells. Taken together, these results suggest that SRPK pharmacological inhibitors may be considered for the development of novel therapeutic strategies against leukemias and other types of cancers.

Osteoblasts are highly active cells that are responsible for secreting bone forming components such as collagen type I and matricellular proteins that mediate collagen deposition and mineralisation. SPARC and osteopontin are matricellular proteins that are involved in bone regulation and cell-matrix interactions and are also upregulated in metastatic disease. Secretion of these proteins results in changes to the stromal environment that includes cell migration, angiogenesis, matrix degradation, matrix deposition, bone mineralisation and bone resorption. Signalling pathways not only lead to the expression of target proteins, but also have immediate early effects, for example, on cell adhesion. We asked if the ERK 1 and 2 module of the MAPK pathway was involved in the intracellular trafficking of SPARC and Osteopontin. Membrane trafficking is an essential process that ensures newly synthesised proteins pass from their site of synthesis to the extracellular environment. Using an inhibitor of ERK 1 and 2 activation (U0126), as well as siRNA directed against ERK 1 or 2 individually, a change in intracellular localisation of SPARC and osteopontin was observed in cells treated with U0126 and siRNA against ERK 2 alone, likely in or around the Golgi apparatus. Consistent with the observation above, analysis of protein secretion showed that there was a reduction of total protein secreted (30% reduction) when ERK 1 and 2 activation was prevented together or knock down of ERK 2 alone. A mechanism is proposed where ERK 2 is likely activating a substrate that is allowing SPARC and osteopontin to continue along the secretory pathway. This directly implicates ERK 2 as an important regulator of matricellular protein secretion in osteoblasts. In cancer, Ras mutations can lead to permanent activation of the MAPK pathway leading to cancer cell proliferation and survival, however, we propose another mechanism important in metastasis whereby ERK 2 activation is manipulated to facilitate secretion of matricellular proteins which can then mediate changes to the stromal environment that allow the tumour to metastasise successfully.