Background: Resistant hypertension (RH) is defined as blood pressure (BP) that remains elevated (>140/90mmHg) despite being treated with an antihypertensive regimen of 3 or more medications from different classes, including a long-acting calcium channel blocker, an angiotensin converting enzyme inhibitor or angiotension receptor blocker and a diuretic. The prevalence of RH in South Africa is currently unknown, however, clinical reports suggest that it is not rare. Patients with RH are significantly predisposed to cardiovascular (CV) diseases compared to patients with controlled BP. Consequences of RH include left ventricular hypertrophy, heart failure, ischaemic heart disease, chronic kidney disease leading to end-stage renal disease, stroke, vascular dementia, CV death and peripheral arterial disease. A proportion of patients with RH who never achieve BP control despite maximal medical treatment, represent a potentially novel and distinctive phenotype which is different from RH patients whose BP canbe controlled. Recognising and categorising such patients becomes the initial and crucial step in stratifying phenotypes and defining mechanisms of treatment resistance. Objectives: The aim of this study was to identify patients with resistant uncontrolled hypertension (RUH) and compare phenotypes in these patients to resistant controlled hypertensives (RCH). Methods: We enrolled 50 patients from the Groote Schuur Hospital Hypertension Clinic: a teriary referral hospital for RH. Patients on 4 or more antihypertensive medication including a diuretic, with BP< 140/90mmHg were considered RCH, and those with BP ≥ 140/90 considered RUH. Assessments included clinical examination, electrocardiography, echocardiography, applanation tonometry, serum biomarkers and cardiovascular magnetic resonance (CMR - which included biventricular volumes and function, myocardial strain, tissue characteristics and late gadolinium enhancement - LGE). Results: Thirty were diagnosed with RUH and twenty with RCH. Patients with RUH were more likely to have a longer duration since diagnosis of hypertension (10.5±10.7 vs. 3.6±3.4, p=0.02) and more likely to be on treatment that included an ACE-inhibitor (90% vs. 58%, p=0.01). As expected, patients with RUH had significantly higher systolic BP (155.6±21.6 vs. 137.8±16.5 mmHg, p< 0.001), diastolic BP (88.4±14.5 vs. 77.5±13.6 mmHg, p= 0.03), mean arterial BP (115.4±17.2 vs 101±15.3 mmHg, p= 0.004) and pulse pressure (67.3±14.2 vs. 60.1±12.4 mmHg, p=0.001). Further, RUH patients had significantly lower large artery elasticity (12.5±4 vs 14.7±3.8ml/mmHgx100, p=0.08) and lower small artery elasticity (4.1±2.1 vs. 6.9±3.6ml/mmHgx100, p< 0.001). RUH patients also had a higher systemic vascular resistance (1754±418.4 vs. 1363±371.5dyneXsecXcm-5, p=0.002). On CMR, RUH patients had lower right ventricular (RV) end-systolic and end-diastolic volumes (p=0.02), as well as higher indexed left ventricular mass (LVMI) (61.6±17.6 vs 52.9±13.9 g/m2 , p= 0.06). There were no differences in native T1, extracellular volume quantification and LGE volume fraction between RUH and RCH patients. Conclusions: Patients with RUH have a greater involvement and more severe CV phenotype, that is likely to result in increased CV morbidity and mortality, including greater target end organ damage as a result of vascular adaptations and concentric remodeling.

Stroke is a leading cause of death and morbidity worldwide, and leaves stroke survivors with chronic disabilities. One of the key mechanisms that the brain triggers during stroke recovery is the sprouting of new axons and the formation of new neuronal connections. Meanwhile, studies have evidenced this phenomenon with methods using unspecific cell/axon markers. The dopamine (DA) system is thought to be implicated in stroke recovery. However, the specific contribution and remodeling of this system to enhance stroke recovery, and whether D1- class receptors play a role in this process, remain unclear. Using a mouse photothrombosis stroke model, immunohistochemical methods, imaging analysis of axonal fiber density and branching in the motor cortex, we demonstrated a specific dopaminergic axon remodeling in the periinfarct region, with or without DA agonist administration. Axonal remodeling of noradrenergic fibers was subtle. In mice subjected to saline IP injection and physical rehabilitation (running wheels), we observed an increase of only DA fiber density in the periinfarct area as compared to the contralateral (intact) side. However, mice treated with DHX for 7 days followed by physical rehabilitation did not show difference between the two hemispheres. Our results suggest a modulatory effect of DHX on axonal remodeling mainly in the contralateral side. Interestingly, treatment of naïve mice with DHX had no effect of DA axon remodeling suggesting that D1- mediated axonal remodeling is stroke-dependent. We also established the temporal profile of post-stroke DA axon remodeling in the absence of DHX and physical rehabilitation. At 4 days poststroke, there was a significant decrease in DA fiber density and a significant recovery was measured after 28 days relative to the contralateral side. Altogether, our data highlight a major remodeling of DA axons in motor cortex following stroke, and a potential role for D1-class receptors in improving post-stroke recovery. Understanding adaptations of the DA system following stroke will have a great impact on stroke recovery research. Aida Said Thesis submitted to the Faculty of Graduate and Postdoctoral Studies in partial fulfillment of the requirements for the Master of Science degree in Neuroscience Department of Cellular and Molecular Medicine Faculty of Medicine University of Ottawa August 30, 2019 © Aida Said, Ottawa, Canada, 2019   Abstract Stroke is a leading cause of death and morbidity worldwide, and leaves stroke survivors with chronic disabilities. One of the key mechanisms that the brain triggers during stroke recovery is the sprouting of new axons and the formation of new neuronal connections. Meanwhile, studies have evidenced this phenomenon with methods using unspecific cell/axon markers. The dopamine (DA) system is thought to be implicated in stroke recovery. However, the specific contribution and remodeling of this system to enhance stroke recovery, and whether D1-class receptors play a role in this process, remain unclear. Using a mouse photothrombosis stroke model, immunohistochemical methods, imaging analysis of axonal fiber density and branching in the motor cortex, we demonstrated a specific dopaminergic axon remodeling in the periinfarct region, with or without DA agonist administration. Axonal remodeling of noradrenergic fibers was subtle. In mice subjected to saline IP injection and physical rehabilitation (running wheels), we observed an increase of only DA fiber density in the periinfarct area as compared to the contralateral (intact) side. However, mice treated with DHX for 7 days followed by physical rehabilitation did not show difference between the two hemispheres. Our results suggest a modulatory effect of DHX on axonal remodeling mainly in the contralateral side. Interestingly, treatment of naïve mice with DHX had no effect of DA axon remodeling suggesting that D1-mediated axonal remodeling is stroke-dependent. We also established the temporal profile of post-stroke DA axon remodeling in the absence of DHX and physical rehabilitation. At 4 days post-stroke, there was a significant decrease in DA fiber density and a significant recovery was measured after 28 days relative to the contralateral side. Altogether, our data highlight a major remodeling of DA axons in motor cortex following stroke, and a potential role for D1-class receptors in improving post-stroke recovery. Understanding adaptations of the DA system following stroke will have a great impact on stroke recovery research.

Optimal response to drought is critical for plant survival and will affect biodiversity and crop performance during climate change. Mitotically heritable epigenetic and dynamic chromatin state changes have been implicated in the plant response to the drought stress hormone abscisic acid (ABA). The Arabidopsis SWI/SNF chromatin-remodeling ATPase BRAHMA (BRM) modulates response to ABA by preventing premature activation of stress response pathways during germination. Here, we show that the core ABA signalosome formed by ABA receptors, PP2Cs and SnRK2s physically interact with BRM to regulate BRM activity and post-translationally modify BRM by phosphorylation/dephosphorylation. Genetic evidence suggests that BRM acts downstream of SnRK2.2/2.3 kinases and biochemical studies identified evolutionary conserved SnRK2 phosphorylation sites in the C-terminal region of BRM. Our data suggest that SnRK2-dependent phosphorylation of BRM leads to its inhibition, and PP2CA-mediated dephosphorylation of BRM restores the ability of BRM to repress ABA response. ABA plays a key role to regulate germination and post-germination growth and the AP2-type ABI4 and bZIP-type ABI5 transcription factors (TFs) are required for ABA-mediated inhibition of post-germination growth when the embryo encounters water stress. The growth arrest induced by ABI4 and ABI5 involves ABA signaling and in the case of ABI5, it has been demonstrated that ABA inhibits the activity of BRM to induce ABI5 transcription. Loss of BRM activity leads to destabilization of a nucleosome involved in repression of ABI5 transcription. Therefore reduction of BRM activity in the brm-3 allele leads to enhanced expression of ABI5 in 2-d-old seedlings and enhanced sensitivity to ABA. Novel genetic evidence obtained in this work indicates that ABI4 is one of the redundant TFs regulated by BRM that mediate ABA response during germination and early seedling growth. Thus, the association of BRM with the ABI4 locus together with the observed derepression of ABI4 expression in brm-3 suggests that BRM directly regulates ABI4 expression. Finally, this work provides a direct link between the ABA signalosome and the chromatin-remodeling ATPase BRM, which enables ABA-dependent modulation of BRM activity as a possible mechanism to enhance plant drought tolerance. Additionally, we identified and characterized the promoter of PP2CA as a stress-inducible promoter and we have used it to drive the expression of ABA receptors from Arabidopsis and Solanum lycopersicum. This technology appears to be promising for the expression of ABA receptors in an inducible manner and to generate drought tolerant plants. / La respuesta óptima a la sequía es crítica para la supervivencia de las plantas y afectará a la biodiversidad y al rendimiento de los cultivos durante el cambio climático. Las modificaciones epigenéticas y los cambios dinámicos del estado de la cromatina han sido implicados en la respuesta de la planta al ácido abscísico (ABA), la conocida como la hormona del estrés hídrico. La ATPasa remodeladora de cromatina de tipo SWI/SNF de Arabidopsis, BRAHMA (BRM), modula la respuesta al ABA mediante la prevención de la activación prematura de las vías de respuesta al estrés durante la germinación. Aquí, mostramos que el núcleo del señalosoma de ABA formado por los receptores de ABA, las PP2Cs y las SnRK2s interaccionan físicamente con BRM para regular su actividad y modificarla post-traduccionalmente por mecanismos de fosforilación/desfosforilación. La evidencia genética sugiere que BRM actúa aguas abajo de las quinasas SnRK2.2/2.3 y los estudios bioquímicos identificaron la presencia en la región C-terminal de BRM de sitios de fosforilación de las SnRK2 que estaban conservados evolutivamente. Nuestros datos sugieren que la fosforilación de BRM que depende de las SnRK2 conduce a su inhibición, y que la desfosforilación de BRM mediada por PP2CA restaura la capacidad de BRM para reprimir la respuesta a ABA. El ABA juega un papel clave en la regulación de la germinación y el crecimiento post germinativo y los factores de transcripción de tipo AP2 como ABI4 y de tipo bZIP como ABI5, son necesarios para la inhibición del crecimiento post germinativo mediado por ABA cuando los embriones encuentran estrés hídrico. La detención del crecimiento inducida por ABI4 y ABI5 implica la señalización de ABA y en el caso de ABI5, se ha demostrado que el ABA inhibe la actividad de BRM para inducir la transcripción de ABI5. La pérdida de actividad de BRM conduce a la desestabilización de un nucleosoma implicado en la represión de la transcripción de ABI5. Por lo tanto, la reducción de la actividad de BRM en el alelo brm-3 conduce a una mayor expresión de ABI5 en plántulas de 2 días y una mayor sensibilidad a ABA. La nueva evidencia genética obtenida en este trabajo indica que ABI4 es uno de los factores de transcripción redundantes regulados por BRM que median la respuesta a ABA durante los estadios de germinación y crecimiento temprano de las plántulas. La asociación de BRM con el locus ABI4, junto con la desrepresión de la expresión de ABI4 observada en el mutante brm-3 sugiere que BRM regula directamente la expresión de ABI4. Por último, este trabajo proporciona una relación directa entre el señalosoma de ABA y la ATPasa remodeladora de cromatina BRM, que permite la modulación de la actividad de BRM de modo dependiente de ABA como un posible mecanismo para mejorar la tolerancia a sequía de las plantas. Además, hemos identificado y caracterizado el promotor de PP2CA como un promotor inducible por estrés y lo hemos utilizado para dirigir la expresión de los receptores de ABA de Arabidopsis y Solanum lycopersicum. Esta tecnología parece ser prometedora para la expresión de receptores de ABA de modo inducible y para generar plantas tolerantes a la sequía. / La resposta òptima a la sequera és crítica per a la supervivència de les plantes i afectarà la biodiversitat i al rendiment dels cultius durant el canvi climàtic. Les modificacions epigenètiques i els canvis dinàmics de l'estat de la cromatina han estat implicats en la resposta de la planta a l'àcid abscísic (ABA), la coneguda com hormona de l'estrès hídric. La ATPasa remodeladora de cromatina de tipus SWI/SNF d'Arabidopsis, BRAHMA (BRM), modula la resposta al ABA mitjançant la prevenció de l'activació prematura de les vies de resposta a l'estrès durant la germinació. Ací, mostrem que el nucli del senyalosoma d'ABA format pels receptors d'ABA, les PP2Cs i les SnRK2s interaccionen físicament amb BRM per regular la seva activitat i modificar-la post-traduccionalment per mecanismes de fosforilació/desfosforilació. L'evidència genètica suggereix que BRM actua aigües avall de les quinases SnRK2.2/2.3 i els estudis bioquímics van identificar la presència, a la regió C-terminal de BRM, de llocs de fosforilació de les SnRK2 que estaven conservats evolutivament. Les nostres dades suggereixen que la fosforilació de BRM que depèn de les SnRK2, condueix a la inhibició de BRM, i que la defosforilació de BRM mediada per PP2CA restaura la capacitat de BRM per reprimir la resposta a ABA. El ABA juga un paper clau en la regulació de la germinació i el creixement post-germinació i els factors de transcripció de tipus AP2 com ABI4 i de tipus bZIP com ABI5, són necessaris per a la inhibició del creixement post-germinació mediat per ABA quan els embrions pateixen estrès hídric. La detenció del creixement induïda per ABI4 i ABI5 implica la senyalització d'ABA i en el cas d'ABI5, s'ha demostrat que l'ABA inhibeix l'activitat de BRM per induir la transcripció d'ABI5. La pèrdua d'activitat de BRM condueix a la desestabilització d'un nucleosoma implicat en la repressió de la transcripció d'ABI5. Per tant, la reducció de l'activitat de BRM a l'al·lel brm-3 condueix a una major expressió d'ABI5 en plàntules de 2 dies i una major sensibilitat a l'ABA. La nova evidència genètica obtinguda en aquest treball indica que ABI4 és un dels factors de transcripció redundants que són regulats per BRM que medien la resposta a l'ABA durant els estadis de germinació i creixement primerenc de les plàntules. Per tant, l'associació de BRM amb el locus ABI4, juntament amb la desrepressió de l'expressió de ABI4 observada al mutant brm-3 suggereix que BRM regula directament l'expressió d'ABI4. Finalment, aquest treball proporciona una relació directa entre el senyalosoma d'ABA i l'ATPasa remodeladora de cromatina BRM, que permet la modulació de l'activitat de BRM de manera dependent d'ABA com un possible mecanisme per millorar la tolerància a sequera de les plantes. A més, hem identificat i caracteritzat el promotor de PP2CA com un promotor induïble per estrès i l'hem utilitzat per dirigir l'expressió dels receptors d'ABA d'Arabidopsis i Solanum lycopersicum. Aquesta tecnologia sembla ser prometedora per a l'expressió de receptors d'ABA de manera induïble i per generar plantes tolerants a la sequera. / Peirats Llobet, M. (2017). Fundamental and applied research in ABA signaling: Regulation by ABA of the chromatin remodeling ATPase BRAHMA and biotechnological use of the PP2CA promoter [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/82694 / TESIS / Premios Extraordinarios de tesis doctorales

Indiana University-Purdue University Indianapolis (IUPUI) / The Keap1-Nrf2 pathway regulates a wide range of cytoprotective genes, and has been found to serve a protective and beneficial role in many body systems. There is limited information available, however, about its role in bone homeostasis. While Nrf2 activation has been suggested as an effective method of increasing bone mass and quality, there have been conflicting reports which associate Keap1 deficiency with detrimental phenotypes. As Keap1 deletion is a common method of Nrf2 activation, further study should address the impacts of various methods of regulating Nrf2 expression. Also, little research has been conducted on the specific pathways by which Nrf2 activation improves bone quality. In this study, the effects of alterations to Nrf2 activation levels were explored in two specific and varied scenarios. In the first experiment, moderate Nrf2 activation was achieved via partial deletion of its sequestering protein, Keap1, in an aging mouse model. The hypothesis tested here is that moderate Nrf2 activation improves bone quality by affecting bone metabolism and response to mechanical loading. The results of this first experiment suggest a subtle, sex-specific effect of moderate Nrf2 activation in aging mice which improves specific indices of bone quality to varying degrees, but does not affect loading-induced bone formation. It is likely that the overwhelming phenotypic impacts associated with aging or the systemic effects of global Keap1 deficiency may increase the difficulty in parsing out significant effects that can be attributed solely to Nrf2 activation. In the second experiment, a cell-specific knockout of Nrf2 in the osteocytes was achieved using a Cre/Lox breeding system. The hypothesis tested here is that osteocyte-specific deletion of Nrf2 impairs bone quality by affecting bone metabolism and response to mechanical loading. The results of this experiment suggest an important role of Nrf2 in osteocyte function which improves certain indices of bone quality, which impacts male and female bones in different 7 ways, but did not significantly impact loading-induced bone formation. Further studies should modify the method of Nrf2 activation in an effort to refine the animal model, allowing the effects of Nrf2 to be isolated from the potential systemic effects of Keap1 deletion. Future studies should also utilize other conditional knockout models to elucidate the effects of Nrf2 in other specific cell types.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19606号 / 医博第4113号 / 新制||医||1015(附属図書館) / 32642 / 京都大学大学院医学研究科医学専攻 / (主査)教授 小池 薫, 教授 YOUSSEFIAN Shohab, 教授 川村 孝 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13213号 / 論医博第2163号 / 京都大学大学院医学研究科脳統御医科学系専攻 / (主査)教授 山下 潤, 教授 髙橋 良輔, 教授 木村 剛 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Indiana University-Purdue University Indianapolis (IUPUI) / Under normal conditions, smooth muscle cells do not replicate, or proliferate, and provide a means of contraction for many internal organs, including blood vessels and the gut. However, under abnormal or disease conditions, such as congenital heart disease and cancer, smooth muscle cells acquire the ability to replicate, to make extracellular matrix proteins and to migrate. Thus, determining how smooth muscle cells regulate these processes is crucial to understanding how the cells can switch between normal and diseased states. Serum response factor (SRF) is a widely expressed protein that plays a key role in the regulation of smooth muscle differentiation, proliferation and migration. It is generally accepted that one way that SRF can distinguish between these functions is through pathway-specific co-factor interactions. A novel SRF co-factor, chromodomain helicase DNA binding protein 8 (CHD8), was originally isolated from a yeast two-hybrid assay. CHD8 is widely expressed in adult tissues including smooth muscle. Data from in vitro binding assays indicate that the N-terminus of CHD8 can interact directly with the MADS domain of SRF. Co-immunoprecipitation assays verified the ability of these two proteins to interact within cells. Adenoviral-mediated shRNA knockdown of CHD8 in smooth muscle cells resulted in statistically significant 10-20% attenuation of expression of SRF-dependent, smooth muscle-specific genes. Similar experiments revealed that knockdown of CHD8 did not affect the SRF-dependent induction of immediate early genes required to promote proliferation. In contrast, knockdown of CHD8 in A10 vascular smooth muscle cells resulted in a marked induction in of apoptosis, characterized by increases in apoptotic markers such as phospho-H2A.X, cleaved PARP and activated caspase-3. These data suggest that CHD8 may play a specific role in modulating SRF’s activity toward anti-apoptotic genes, thereby regulating smooth muscle cell survival.

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