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).

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 This dissertation contains previously published coauthored materials as well as unpublished materials.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

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

A insuficiência cardíaca é uma síndrome clínica de mau prognóstico caracterizada por disfunção cardíaca associada à intolerância aos esforços, retenção de fluído e redução da longevidade. Dentre as serina/treonina quinases associadas às alterações funcionais e estruturais cardíacas observadas na progressão da insuficiência cardíaca, a família das proteínas quinase C (PKC) composta por 12 diferentes isoformas parece modular a contratilidade miocárdica e o remodelamento cardíaco. No presente estudo, caracterizamos o fenótipo cardíaco e o perfil de ativação das diferentes isoformas de PKC na progressão da insuficiência cardíaca de etiologia isquêmica em ratos. Além disso, estudamos o efeito da inibição sustentada da isoforma PKCβII sobre a sobrevida, o remodelamento cardíaco e a função ventricular em modelo de insuficiência cardíaca de etiologia isquêmica. Conseguinte, identificamos possíveis substratos cardíacos da PKCβII envolvidos na progressão da insuficiência cardíaca. Para isso, avaliamos os efeitos agudo e crônico da inibição da PKCβII sobre o transiente de cálcio e a contratilidade de cardiomiócito isolados de ratos adultos com insuficiência cardíaca. Por fim, testamos as inibições específicas das PKCβII e PKCβI na progressão da hipertrofia cardíaca compensada para a insuficiência cardíaca em modelo animal de hipertensão arterial sustentada. Nossos resultados sugerem que a inibição sustentada da PKCβII reverte o quadro de insuficiência cardíaca, melhorando a função ventricular, o remodelamento cardíaco e a sobrevida dos diferentes modelos de insuficiência cardíaca estudados, constituindose em uma estratégia terapêutica celular promissora / Heart failure is a common endpoint for many forms of cardiovascular disease and a significant cause of morbidity and mortality worldwide. Protein kinase C isozymes emerge as important potential therapeutic targets in chronic cardiovascular disease. However, individual PKC isozymes play different roles in the pathogenesis of cardiac diseases. Here, we characterized the cardiac phenotype as well as the different PKC isozyme activation profile during myocardial-induced heart failure progression in rat. Furthermore, we evaluated the role of selective PKCβ II inhibition on survival, left ventricle remodeling and cardiac function in myocardial-induced heart failure. Moreover, we identified the cardiac PKCβII substrates related to heart failure. Finally, PKCβII and PKCβI specific inhibitors were chronically delivered to hypertensive-induced heart failure rats and the cardiac phenotype was evaluated. Our data suggest that 6-wks of PKCβII inhibition, but not PKCβI, improved animal survival by restoring cardiac function and promoting cardiac anti-remodeling effect in both myocardial infarctioninduced heart failure and hypertensive-induced heart failure rats. The improved cardiac function and anti-remodeling effect of PKCβII inhibition seems to be associated with increased contractility of cardiac myocytes, improved miofilaments/Ca2+ sensitivity and decreased cardiac inflammatory response. Altogether, the results provide evidence for beneficial effects of PKCβII specific intracellular inhibition on cardiac function and remodeling, which may be a promising cellular therapy for heart failure treatment

Orientador: Carlos Amílcar Parada / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T13:34:40Z (GMT). No. of bitstreams: 1 Prado_FilipeCesardo_M.pdf: 428700 bytes, checksum: 1f8f2df5d5cae548c5b0d1a6a66947f7 (MD5) Previous issue date: 2010 / Resumo: Enquanto a hiperalgesia inflamatória depende da liberação de prostaglandinas e/ou de aminas simpatomiméticas que sensibilizam os neurônios aferentes primários, nosso grupo demonstrou recentemente que o bloqueio do receptor P2X3 no tecido periférico previne a hiperalgesia induzida pela carragenina.. No entanto, o mecanismo pelo qual a ativação dos receptores P2X3 neuronais contribui para a hiperalgesia inflamatória não está completamente estabelecido. O presente estudo verifica se a ativação do receptor P2X3 dos neurônios aferentes primários contribui para a hiperalgesia mecânica induzida pela prostaglandina E2 ou pela dopamine no tecido periférico. A co-administração de A317491 (60 µg / paw), um antagonista seletivo do receptor P2X3, ou o prétratamento com dexametasona (1 mg / mL / kg), preveniu a hiperalgesia mecânica medida 3 horas depois da administraçao de carragenina (300 µg / paw) na pata posterior de ratos. A administração de ??meATP (50 µg /paw) induziu hiperalgesia mecânica 1 hora, mas não 3 horas, depois da sua administração, que foi prevenida pela dexametasona ou pelo A317491. Doses sublimiares de PGE2 (4 ng / paw) ou dopamina (0.4 µg / paw) que não induzem hiperalgesia por si só, induziram hiperalgesia, 3 horas depois, quando administradas logo depois de ??meATP ou carragenina em ratos tratados com dexametasona. Esses estados de hiperalgesia ("priming") revelados pelas doses sublimiares de PGE2 ou dopamine foram prevenidos pelo A317491 ou pelo tratamento com administração intraganglionar (DRG-L5) de ODN antisense, mas não pelo ODN mismatch, contra o receptor P2X3 (40 µg /5µL once a day for 4 days). ODN antisense, mas não o ODN mismatch, reduziu a expressão dos receptores P2X3 no nervo safeno e no DRG-L5. Para verificar se a PKC? media esse estado de hiperalgesia, inibidor de translocação de PKC? (1 µg/paw) foi administrado no tecido periférico 45 minutos antes do ??meATP ou PGE2 (100 ng/paw). O inibidor de PKC? preveniu o estado de hiperalgesia induzido pelo ??meATP ("priming"), mas não a hiperalgesia mecânica induzida pela PGE2 (100 ng/paw). Dessa maneira, os resultados desse estudo sugerem que a hiperalgesia inflamatória depended a ativação dos receptores P2X3 neuronais e da subsequente translocação da PKC? , que aumenta a susceptibilidade dos neurônios aferentes primários (priming) à ação de outros mediadores inflamatórios como a PGE2 e as aminas simpatomiméticas / Abstract: While inflammatory hyperalgesia depends on the release of prostaglandins and/or sympathetic amines that ultimately sensitize the primary afferent neurons, we have recently demonstrated that blockade of P2X3 receptor in the peripheral tissue completely prevents carrageenan-induced hyperalgesia. However, the mechanism by which the activation of neuronal P2X3 receptor contributes to the inflammatory hyperalgesia is not completely clear. The present study verifies whether the activation of P2X3 receptor on primary afferent neurons contributes to the mechanical hiperalgesia induced by prostaglandin E2 or dopamine in the peripheral tissue. Co-administration of A317491(60 µg / paw), a selective P2X3,2/3 receptor antagonist, or pre-treatment with dexamethasone (1 mg / mL / Kg), prevented the mechanical hyperalgesia measured 3 hours after the administration of carrageenan (300 µg / paw) in the rat's hind paw. The administration of ??meATP (50 µg /paw) induced mechanical hiperalgesia 1 hour, but not 3 hours, after its administration, which also was prevented by dexamethasone or A317491. Sub-threshold doses of PGE2 (4 ng / paw) or dopamine (0.4 µg / paw) that do not induce hyperalgesia by themselves, induced maximal hyperalgesia, 3 hours after, when administrated Just following ??meATP or carrageenan in rats treated with dexamethasone. These hyperalgesic states ("priming") revealed by sub-threshold doses of PGE2 or dopamine were prevented by A317491 or treatment with ganglionar administrations (DRG-L5) of ODN antisense, but not ODN mismatch, against P2X3 receptor (40 µg /5µL once a day for 4 days). ODN antisense, but not ODN mismatch reduced the expression of P2X3 receptors in the saphenous nerve and in DRG-L5. To verify whether PKC? mediates this hyperalgesic state, PKC? translocation inhibitor (1 µg/paw) was administrated in peripheral tissue 45 min. before ??meATP or PGE2 (100 ng/paw). PKC? inhibitor inhibited the hyperalgesic state induced by ??meATP ("priming"), but not the mechanical hyperalgesia induced by PGE2 (100 ng/paw). Briefly, the findings of this study suggest that the inflammatory hyperalgesia depends on neuronal activation of P2X3 receptor and the subsequent PKC? translocation, which increases the susceptibility of primary afferent neurons (priming) to others inflammatory mediators such as PGE2 and symphatetic amines / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

Orientador: Elenice Aparecida de Moraes Ferrari / Disertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T18:30:13Z (GMT). No. of bitstreams: 1 Dias_ElayneVieira_M.pdf: 1520883 bytes, checksum: 05673dcedfdc35b300eb07144edbd5bb (MD5) Previous issue date: 2008 / Resumo: O condicionamento clássico aversivo é utilizado para investigar os mecanismos celulares e moleculares na formação da memória em diferentes espécies de animais. Estes envolvem processos sinápticos que desencadeiam mecanismos de sinalização intracelular com ativação de diferentes quinases em momentos específicos. A ativação da PKC é um dos mecanismos moleculares da plasticidade sináptica subjacente à formação de memória. O presente trabalho investigou o envolvimento da PKCá/âIl no condicionamento clássico aversivo em pombos. No Experimento 1, o inibidor da PKC, calfostina C foi administrado i.c.v. em um grupo de pombos (GCdCa, n=6; 5ml de solução 60mg/ml, DMSO 2%), 1h antes do condicionamento. Outro grupo recebeu veículo (GCdVe, n=5; DMSO 2% em salina). A sessão de condicionamento teve 20 min de duração e 3 pareamentos som-choque (treino). O teste ao contexto ocorreu 24h após o treino. O Experimento 2 usou grupos de pombos expostos ao contexto experimental (GCC), som e choque não pareados (GCR) ou som-choque pareados (GCd) para investigar a ativação da PKCá/âII no hipocampo 2h após o treino, por meio de Western blot. No Experimento 3, grupos de pombos não treinados (GC, n=6) ou sacrificados em diferentes tempos após o treino - G1min (n=6), G1h (n=6), G2h (n=6) e G24h (n=6) - foram utilizados para investigar o curso temporal da ativação da PKCá/âII e da fosforilação do substrato da PKC, GAP-43, no hipocampo. Todas as sessões foram gravadas em vídeo para posterior análise dos dados comportamentais. No Experimento 1 o GCdCa teve menor expressão da resposta condicionada de congelamento (freezing) ao contexto em comparação ao GCdVe (p<0,05), indicando que a administração da calfostina C prejudicou a memória aversiva contextual. Não ocorreram diferenças significativas na ativação da PKC á/âII entre os diferentes grupos (Experimentos 2 e 3;p>0,05), mas houve maior imuno-marcação da GAP-43 fosforilada no G1min quando comparado ao GC (Experimento 3; p<0,05). Esses dados indicam o envolvimento da PKC em mecanismos de aprendizagem e memória aversiva em pombos, e sugerem que outras isoformas além da PKCá/âII podem participar desses processos. / Abstract: The classical aversive conditioning is used to investigate cellular and molecular mechanisms of memory formation in different animal species. Those mechanisms involve synaptic processes that trigger intracellular signaling with activation of different kinases at specific time points. The PKC activation is one of the molecular mechanisms of synaptic plasticity underlying memory. This study investigated the involvement of PKCá/âIl in classical aversive conditioning in pigeons. In Experiment 1, the PKC inhibitor, calphostin C was administered i.c.v. in one group of pigeons (GCdCa, n=6; 5ml solution 60mg/ml, DMSO 2%), 1h before the conditioning. Another group received vehicle (GCdVe, n=5; DMSO 2% in saline). The session of conditioning had 20 min duration and 3 tone-shock pairings (training). The test to the context occurred 24h after training. Experiment 2 investigated with Western blot analysis the PKCá/âII activation in the hippocampus 2h after the training in groups of pigeons that were exposed to unpaired (GCR) or paired (GCd) tone-shock presentations or to the experimental context only (GCC). In Experiment 3, groups of pigeons naive (GC, n=6) or sacrificed at different times after the training - G1min (n=6), G1h (n=6), G2h (n=6) and G24h (n=6) - were used to investigate the time course of the PKCá/âII activation and phosphorylation of PKC substrate, GAP-43, in the hippocampus. All sessions were video recorded for analysis of behavioral data. In Experiment 1 GcdCa had lower expression of conditioned freezing response to the context in comparison to GCdVe (p<0.05), indicating that calphostin C administration impaired contextual aversive memory. No significant differences in the PKCá/âII activation were observed among the groups (Experiments 2 and 3; p>0.05) but the immunolabeling of phosphorylated GAP-43 in G1min was higher as compared to GC (Experiment 3; p<0.05). These data indicate the involvement of PKC in mechanisms of aversive learning and memory in pigeons and suggest that other isoforms besides PKCá/âII may play a role in those processes. / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

Abstract Cardiac myocytes react to increased workload and hypertrophic neurohumoral stimuli by increasing protein synthesis, reinitiating expression of fetal forms of structural genes, α-skeletal actin (α-SkA) and β-myosin heavy chain (β-MHC), and by increasing expression and secretion of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP). Initially, the response is beneficial, but when prolonged, it leads to pathological cardiomyocyte hypertrophy. In this study, cardiomyocyte hypertrophy was initiated by hypertrophic agonists, endothelin-1 (ET-1) and phenylephrine (PE), and by increased stretching of atrial wall. Transcription factor GATA4 was studied to identify the mechanism leading to increased gene expression of BNP. In BNP promoter, GATA4 binds to cis elements mediating hypertrophic response. Eliminating GATA4 binding by using the decoy approach, basal BNP gene expression was reduced. To identify mechanisms regulating GATA4, the roles of mitogen-activated protein kinases (MAPKs) were studied. Activation of p38 MAPK increased GATA4 binding to BNP gene and led to increased GATA4 dependent BNP gene expression. p38 MAPK was required for ET-1 induced GATA4 binding, whereas extracellular signal-regulated kinase (ERK) was required for maintaining basal GATA4 binding activity. PE and ET-1 activated protein kinase C (PKC) signaling in cardiac myocytes. Antisense oligonucleotide inhibition of PKCα markedly reduced PE induced ANP secretion and ET-1 induced BNP secretion, whereas gene expression of natriuretic peptides was not affected. Antisense PKCα treatment inhibited PE induced expression of α-SkA, while increased protein synthesis or β-MHC gene expression were not affected. Sretching of the perfused rat atria increased BNP, c-fos and BNP gene expression via mechanism involving p38 MAP kinase activation of transcription factor Elk-1. In cultured neonatal rat atrial myocytes stretch induced BNP gene expression was dependent upon transcription factor Elk-1 binding sites within the BNP gene promoter. In conclusion, hypertrophic signaling in cardiac myocytes involves multiple signaling cascades. Activation of p38 MAPK is required for the development of ET-1 induced hypertrophic phenotype and GATA4 mediated BNP gene expression in cultured ventricular myocytes, and for stretch induced Elk-1 dependent BNP gene expression in atrial myocytes. PKCα is involved in PE induced hypertrophic response and PE induced switch in gene programming inducing expression of α-SkA, the fetal form of cardiac α-actin.

Dopamine receptors belong to the G protein-coupled receptor (GPCR) superfamily and are classified into two families: D1-like (D1R and D5R) and D2-like (D2R, D3R and D4R), based on their ability to stimulate or inhibit adenylyl cyclase (AC). Classically, GPCRs (including D2R and D3R) are desensitized by the activation of the serine/threonine protein kinase C (PKC) upon phorbol-12-myristate-13-acetate (PMA) treatment. Previous studies demonstrate that while human D5R (hD5R) is also strongly desensitized upon PMA treatment, the human D1R (hD1R) undergo a robust PMA-induced sensitization. The aim of this PhD thesis was to explore how the canonical PKC- or phorbol ester-linked pathway can control the responsiveness of two similar GPCRs like hD1R and hD5R in an opposite fashion. Our data indicate that hD1R sensitization and hD5R desensitization are not mediated by a direct modulation of AC activity by PKC. Using a chimeric approach, we identified the third intracellular loop (IL3) as the key structural determinant controlling in an opposite manner the PMA-mediated regulation of hD1R and hD5R. To delineate the potential PKC phosphorylation sites, a series of mutation of serine (Ser) and threonine (Thr) located into IL3 of hD1R and hD5R were used. No hD1R mutation decreased the PMA-mediated sensitization. This suggests that hD1R phosphorylation is not required for PMA-induced sensitization. In contrast, our results indicate that PMA-mediated hD5R desensitization occurs through a hierarchical phosphorylation of Ser260, Ser261, Ser271 and Ser274. Notably, these hD5R mutants exhibited a PMA-induced sensitization, reminiscent of the PMA-induced hD1R sensitization. Additionally, using short hairpin RNAs (shRNAs), we showed that PKCε is the potentiating PKC while the desensitizing isoform is δ. Overall, our work suggests the presence or absence of specific Ser residues on IL3 of hD1-like receptors dictate if phosphorylation-dependent desensitization (through PKCδ) or phosphorylation-independent potentiation (via PKCε) will occur.

Cardiovascular diseases, particularly acute myocardial infarction, are one of the leading causes of death in developed countries. It is well known that adaptation to chronic intermittent hypobaric hypoxia (IHH) confers long-lasting cardiac protection against acute ischemia/reperfusion injury. Protein kinase C (PKC) appears to play a role in its cardioprotective mechanism since the administration of general PKC inhibitor completely abolished the improvement of ischemic tolerance in IHH hearts. However, the involvement of individual PKC isoforms remains unclear. Therefore, the primary aim of this study was to investigate the potential involvement of PKCδ and PKCε, the most prevalent PKC isoforms in rat heart, in the mechanism of IHH-induced cardioprotection. We showed that IHH up- regulated PKCδ protein in left ventricle, enhanced its phosphorylation on Ser643 and increased its co-localization with markers of mitochondrial and sarcolemmal membranes. PKCδ subcellular redistribution induced by IHH as well as the infarct size-limiting effect of IHH was reversed by acute treatment with PKCδ inhibitor rottlerin. These data support the view that PKCδ plays a significant role in IHH-induced cardioprotection. On the other hand, adaptation to IHH decreased the PKCε total protein level without affecting its...