Thesis (MSc (Physiological Sciences))--University of Stellenbosch, 2009. / Physiological cardiac hypertrophy is characterized by the heart’s ability to increase mass in a reversible fashion without leading to heart failure. In contrast, pathological cardiac hypertrophy leads to the onset of heart failure. For this study, we investigated a model of physiological hypobaric hypoxia-mediated right ventricular (RV) hypertrophy (RVH). Here our hypothesis was that the hypertrophic response and associated changes triggered in the RV in response to chronic hypobaric hypoxia (CHH) (increased RV mass, function and respiratory capacity) are reversible. To test our hypothesis we exposed male Wistar rats to 3 weeks of CHH and thereafter removed the hypoxic stimulus for 3 and 6 weeks, respectively. Adaptation to 3 weeks of CHH increased the RV to left ventricle (LV) plus interventricular septum ratio by increased (223.5 ± 7.03 vs. 397.4 ± 29.8, p<0.001 versus normoxic controls), indicative of RVH. Hematocrit levels, RV systolic pressure and RV developed pressure (RVDP) were increased in parallel. Mitochondrial respiratory capacity was not significantly altered when using both carbohydrate and fatty acid oxidative substrates. After the 3-week normoxia recovery period, the RV to LV ratio was increased but to a lesser extent compared to the 3-week hypoxic time-point, i.e. 244.7 ± 11.2 vs. 349.64 ± 3.8, p<0.001 versus normoxic controls. Moreover, hematocrit levels were completely normalized. However, the RV systolic pressure and the functional adaptations, i.e. increased RVDP induced by CHH exposure still persisted in the 3-week recovery (3HRe) group. Also, pyruvate utilization was increased versus matched controls (p<0.04 vs. matched controls). Interestingly, we found that at the 6-week recovery time point functional parameters were largely normalized. However, the RV to LV ratio was still increased by 269.3 ± 14.03 vs. 333.9 ± 11.7, p<0.0001 vs. matched controls. Furthermore, palmitoylcarnitine utilization was increased (p<0.03 vs. matched controls). In conclusion, we found that exposure to CHH resulted in various adaptive physiological changes, i.e. enhanced hematocrit levels, increased RV mass linked to greater RV contractility and respiratory function. It is important to note that all these changes only occurred in the RV and not in the LV. Furthermore, when a normoxic recovery period (3 and 6 weeks, respectively) were initiated, these physiological parameters largely normalized. Together, the findings of this thesis clearly show the establishment of a reversible model of RV physiological hypertrophy. Our future work will focus on disrupting signaling pathways underlying this process and to thereafter ascertain whether reversibility is abolished. Elucidation of such targets should provide a unique opportunity to develop novel therapeutic agents to treat patients and thereby reduce the burden of heart disease.

Transition of the cardiopulmonary circulation at birth requires functional closure of the ductus arteriosus (DA). The DA is an arterial shunt that is vital in the fetus for diverting the pulmonary circulation away from the uninflated lungs. Failure of the vessel to functionally close after birth is known as patent DA, which is the second most common congenital heart disease. Patent DA may seriously compromise neonatal health and current pharmacological treatments are often limited by serious complications or a significant failure rate, thereby increasing the necessity for surgical intervention. Recently, we were the first to show that genetic or pharmacological inactivation of cyclooxygenase (COX) -2 produces postnatal patent DA in mice. We also demonstrated that the DA expresses high levels of COX-2 during normal closure after birth, suggesting novel contractile actions of COX-2-dependent prostanoids in the DA. In humans, patent DA is more common in preterm infants than those born at full-term, however, mechanism(s) responsible for the reduced DA closure have not been identified. In the current studies, we examined COX-1 and COX-2 expression in the DA at multiple stages of gestation to determine whether alterations in the expression of these enzymes contribute to patent DA in preterm mice. Using real-time PCR, analysis of the time-course of COX-2 mRNA in the fetal mouse DA indicated that COX-2 expression significantly increased with advancing gestational age. The preterm (day 17.5) neonatal mouse DA showed attenuated COX-2 expression, as compared to the full-term (day 19.5) neonatal DA at 3 hours after birth. Furthermore, the DA of preterm neonatal mice showed incomplete closure after 3 hours of birth, a time-point when the DA of full-term neonates was completely remodeled. These data indicate a correlation between reduced DA closure and attenuated COX-2 expression. Additionally, COX-2 expression was significantly attenuated in the DA of mice deficient in the prostanoid receptor EP4, which also show a patent DA phenotype, suggesting the importance of this receptor for the induction of COX-2 required for DA closure. Overall, these studies suggest that attenuated expression of COX-2 may contribute to increased patent DA at preterm gestation.

<p>Chromatin is a highly dynamic, regulatory component in the process of transcription, repair, recombination and replication. The BRG1 and SNF2H proteins are ATP-dependent chromatin remodeling proteins that modulate chromatin structure to regulate DNA accessibility for DNA-binding proteins involved in these processes. The BRG1 protein is a central ATPase of the SWI/SNF complexes involved in chromatin remodeling associated with regulation of transcription. SWI/SNF complexes are biochemically hetero-geneous but little is known about the unique functional characteristics of the various forms. We have shown that SWI/SNF activity in SW13 cells affects actin filament organization dependent on the RhoA signaling pathway. We have further shown that the biochemical composition of SWI/SNF complexes qualitatively affects the remodeling activity and that the composition of biochemically purified SWI/SNF complexes does not reflect the patterns of chromatin binding of individual subunits. Chromatin binding assays (ChIP) reveal variations among subunits believed to be constitutive, suggesting that the plasticity in SWI/SNF complex composition is greater than suspected. We have also discovered an interaction between BRG1 and the splicing factor Prp8, linking SWI/SNF activity to mRNA processing. We propose a model whereby parts of the biochemical heterogeneity is a result of function and that the local chromatin environment to which the complex is recruited affect SWI/SNF composition.</p><p>We have also isolated the novel B-WICH complex that contains WSTF, SNF2H, the splicing factor SAP155, the RNA helicase II/Guα, the transcription factor Myb-binding protein 1a, the transcription factor/DNA repair protein CSB and the RNA processing factor DEK. The formation of this complex is dependent on active transcription and links chromatin remodeling by SNF2H to RNA processing.</p><p>By linking chromatin remodeling complexes with RNA processing proteins our work has begun to build a bridge between chromatin and RNA, suggesting that factors in chromatin associated assemblies translocate onto the growing nascent RNA.</p>

The rate limiting step of phagocytosis is the binding of collagen to specific receptors, which include β1 integrins and the discoidin domain receptor 1 (DDR1). While these two receptors may interact, the functional nature of these interactions is not defined. We examined the effects of DDR1 over-expression on β1 integrin function and determined that DDR1 over-expression enhanced cell attachment through β1 integrins. These data are consistent with data showing that DDR1 over-expression enhanced cell-surface, but not total, β1 integrin expression and activation. As shown by experiments with endoglycosidase H, DDR1 over-expression increased glycosylation of the β1 integrin subunit. Collectively these data indicate that DDR1 enhances β1 integrin interactions with fibrillar collagen, possibly by affecting the processing and trafficking of β1 integrins to the cell surface. Our data provide insight into the mechanisms by which fibrotic conditions such as cyclosporine A-induced gingival overgrowth are regulated.

The rate limiting step of phagocytosis is the binding of collagen to specific receptors, which include β1 integrins and the discoidin domain receptor 1 (DDR1). While these two receptors may interact, the functional nature of these interactions is not defined. We examined the effects of DDR1 over-expression on β1 integrin function and determined that DDR1 over-expression enhanced cell attachment through β1 integrins. These data are consistent with data showing that DDR1 over-expression enhanced cell-surface, but not total, β1 integrin expression and activation. As shown by experiments with endoglycosidase H, DDR1 over-expression increased glycosylation of the β1 integrin subunit. Collectively these data indicate that DDR1 enhances β1 integrin interactions with fibrillar collagen, possibly by affecting the processing and trafficking of β1 integrins to the cell surface. Our data provide insight into the mechanisms by which fibrotic conditions such as cyclosporine A-induced gingival overgrowth are regulated.

<p>Primary cilia are evolutionarily conserved, acetylated microtubule-based organelles that transduce mechanical and chemical signals. Primary cilium assembly is tightly controlled and its deregulation causes a spectrum of human diseases. Formation of primary cilium is a collaborative effort of multiple cellular machineries, including microtubule, actin network and membrane trafficking. How cells coordinate these components to construct the primary cilia remains unclear. In this dissertation research, we utilized a combination of cell biology, biochemistry and light microscopy technologies to tackle the enigma of primary cilia formation, with particular focus on isoform-specific roles of non-muscle myosin II family members. We found that myosin IIB (Myh10) is required for cilium formation. In contrast, myosin IIA (Myh9) suppresses cilium formation. In Myh10 deficient cells, Myh9 inactivation significantly restores cilia formation. Myh10 antagonizes Myh9 and increases actin dynamics, permitting pericentrosomal preciliary complex formation required for cilium assembly. Importantly, Myh10 is upregulated upon serum starvation-induced ciliogenesis and this induction requires Atat1/Mec-17, the microtubule acetyltransferase. Our findings suggest that Atat1/Mec17-mediated microtubule acetylation is coupled to Myh10 induction, whose accumulation overcomes the Myh9-dependent actin cytoskeleton, thereby activating cilium formation. Thus, Atat1/Mec17 and myosin II coordinate microtubules and the actin cytoskeleton to control primary cilium biogenesis.</p> / Dissertation

Chromodomain Helicase DNA-Binding Protein 4 (CHD4) is an ATPase that alters the phasing of nucleosomes on DNA and has recently been implicated in DNA double stranded break (DSB) repair. Here, we show that depletion of CHD4 in Acute Myeloid Leukemia (AML) blasts induces a global relaxation of chromatin that renders cells more susceptible to DSB formation, while concurrently impeding their repair. Furthermore, CHD4 depletion renders AML blasts more sensitive both in vitro and in vivo to genotoxic agents used in clinical therapy: daunorubicin (DNR) and cytarabine (ara-C). Sensitization to DNR and ara-C is mediated in part by activation of the ATM pathway, which is preliminarily activated by a Tip60-dependent mechanism in response to chromatin relaxation and further activated by genotoxic-agent induced DSBs. This sensitization preferentially affects AML cells, as CHD4 depletion in normal CD34+ hematopoetic progenitors does not increase their susceptibility to DNR or ara-C. Unexpectedly, we found that CHD4 is necessary for maintaining the tumor formatting behavior of AML cells, as CHD4 depletion severely restricted the ability of AML cells to form xenografts in mice and colonies in soft agar. Taken together, these results provide evidence for CHD4 as a novel therapeutic target whose inhibition has the potential to enhance the effectiveness of genotoxic agents used in AML therapy.

Le cycle réplicatif du HIV-1 aboutit à la formation de virions qui s’assemblent dans des microdomaines spécifiques localisés à la membrane plasmique ou sur des compartiments intracellulaires particuliers, nommés VCC pour « Virus-Containing Compartments ». Selon les cas, ces virions sont ensuite relâchés par bourgeonnement ou exocytose. Ces étapes nécessitent un remodelage membranaire via le cytosquelette d’actine, ce qui est régulé par des voies de signalisation contrôlées par les RhoGTPases. Certains résultats suggèrent l’implication de ces protéines dans la biogénèse du HIV-1. Cependant, il reste à caractériser les mécanismes moléculaires spécifiquement impliqués dans la régulation cellulaire de l’assemblage viral.L’objectif de cette thèse consistait donc à identifier les RhoGTPases et les effecteurs des voies de signalisation spécifiquement requis durant la biogénèse virale. Cette étude a porté sur les GTPases Rac1, Cdc42 et RhoA car elles ont un rôle majeur dans la régulation du cytosquelette d’actine et de la dynamique membranaire. Elle a été réalisée sur les lymphocytes T (LT) Jurkat, cellules modèles pour l’infection HIV-1 où les virions s’assemblent à la membrane plasmique ; et les cellules adhérentes HeLa où les virions peuvent aussi s’assembler au niveau des VCC. Nos résultats ont révélé le rôle de la voie de signalisation Rac1-IRSp53-Wave2 dans l’assemblage de Gag à la membrane plasmique des LT Jurkat, et un rôle pour RhoA dans la régulation de l’assemblage viral suggéré au niveau des VCC. Ce travail améliore la compréhension des voies de signalisation cellulaires sollicitées lors de l’assemblage du HIV-1, en particulier dans les lymphocytes T, cibles du virus. / During the last steps of HIV-1 replication cycle, the Gag proteins come together in particular microdomains located at the plasma membrane or in some intracellular compartments, named “Virus-containing compartments”. Then, the viral particles are released by budding or exocytosis. All these steps involve membrane and actin cytoskeleton remodeling which is regulated by the RhoGTPases. In fact, some data suggest the implication of such proteins in HIV-1 biogenesis, but molecular mechanisms underlying this effect is not yet understood. During this thesis, our aim was to characterize the RhoGTPases and the effectors of cell signaling pathways which are specifically required during HIV-1 particle biogenesis. We focused our study on the GTPases Rac1, Cdc42 and RhoA because their influence on membrane and actin cytoskeleton was essential. Moreover, this work was accomplished on Jurkat T lymphocytes which are model cells to HIV-1 infection where the Gag proteins assemble at the plasma membrane, and on HeLa cells where the Gag proteins can also assemble on virus-containing compartments. Our results showed the requirement of the Rac1-IRSp53-Wave2 signaling pathway for HIV-1 Gag assembly at the plasma membrane of Jurkat T cells, and a role for RhoA GTPase in the regulation of viral particle assembly on virus-containing compartments in HeLa cells. This study improved understanding of cell signaling pathways required during the HIV-1 particle biogenesis and release, particularly in T cells which are the main host cell for HIV-1.

Le peptide natriurétique de type B (BNP) est une hormone cardiaque fortement impliquée dans l'insuffisance cardiaque (IC). En clinique, le BNP est un bio-marqueur de diagnostic et de pronostic de l'IC; son taux sanguin augmentant avec la progression du remodelage cardiaque, de la dysfonction du ventricule gauche (VG) et des altérations du couplage excitation-contraction (CEC). Administré sous une forme recombinante, le nésiritide, pour ses effets hémodynamiques vasorelaxants, chez des patients en IC aiguë décompensée, il a montré des effets controversés avec un risque d'augmentation de la mortalité. Pour comprendre l'implication du BNP dans le remodelage cardiaque post-ischémique et son efficacité thérapeutique, nous avons étudié les effets cardiaques in vivo et sur le CEC d'un traitement chronique au BNP (0,03 µg/kg/min) sur deux groupes expérimentaux : i) des souris saines, Sham, pendant 14 jours et ii) des souris PMI (Post-Myocardial Infarction) en stade précoce, après ligature de l'artère coronaire gauche (pendant 7 et 14 jours). Chez les Sham, le BNP a engendré une suractivation du système nerveux sympathique, une hypertrophie cardiaque, des altérations électrophysiologiques in vivo et des mouvements calciques dans les cardiomyocytes du VG, à l'origine d'arythmies cellulaires et cardiaques. Les effets délétères du BNP ont été prévenus par une combinaison de ce traitement avec un β-bloqueur : le métoprolol. Chez les PMI, la supplémentation en BNP a réduit l'inotropisme positif précoce acquis par une modification des mouvements calciques, a accéléré l'apparition des altérations des protéines du CEC et la survenue des arythmies. Le BNP accélère la décompensation cardiaque et pourrait être à déconseiller, du moins en monothérapie, en traitement clinique. / BNP is a natriuretic peptide released in excess in the blood during heart failure (HF) to reduce blood volume and pressure. A recombinant form of human BNP, nesiritide, is used for patients with acute decompensated HF but deleterious cardiac effects have been reported. We aimed to investigate the cardiac effects of chronic BNP supplementation (0.03 µg/kg/min) on: i) healthy mice (Sham) for 14 days and ii) mice subjected to myocardial infarction (MI) induced by coronary artery ligation (PMI) for 7 (D7) and (D14) 14 days. Sham treated animals (Sham-BNP) showed an increase in ventricular arrhythmias occurrence in consequence of sympathetic tone increase and Ca2+ handling alterations observed at cellular and protein levels in the left ventricle (LV). Most of these effects were reduced in Sham-BNP by the selective beta1-adrenergic blocker metoprolol. In the PMI group, at early stages after ligation, we showed an increase in cardiomyocytes contraction and Ca2+ handling at D7 and D14. But this response was attenuated in PMI-BNP. In PMI, alterations in Ca2+ handling proteins, particularly SERCA2a and RyR2 appeared at D14 but not D7. But BNP supplementation accelerated these alterations which appeared since D7 in the PMI-BNP group, in parallel with spontaneous arrhythmias occurrence at D7. BNP infusion attenuates compensatory "inotropism" intrinsic to the LV cardiomyocytes and promotes arrhythmias. Our results show that BNP, at least in monotherapy, may be hazardous for the treatment of patients with decompensated HF.

The extracellular matrix (ECM) offers a protective shelter for cells and provides signaling paths important for cell to cell communication. ECM consists of basement membranes (BM) and interstitial matrix. BMs provide mechanical support for parenchymal cells, influence cell proliferation, survival, migration and differentiation. They are also important for tissue integrity. Laminins (LM) are the major non-collagenous component of BMs. Cell-ECM interactions, mediated by receptors, are indispensable during embryonic development, wound healing, remodeling and homeostasis of tissues. The integrins are the major cell-adhesion receptors. The expression of alpha11 integrin chain in the cornea is of great interest, as it is part of the alpha11beta1 integrin receptor for collagen type I, the predominant component of the corneal stroma. The aims were to thoroughly characterize the ECM in the developing and adult human eye, with particular focus on the cornea, LM and alpha11 integrin chains, and to examine alpha11 integrin chain in an animal model of corneal wound healing and remodeling. Human fetal eyes, 9-20 weeks of gestation (wg), and adult human corneas with different diagnosis were treated for immunohistochemistry with specific antibodies against LM and alpha11 integrin chains. Normal and knockout (ko) mice were treated with laser surgery to create a deep wound in the corneal stroma. The wound healing process was followed at different time points. The cellular source of alpha11 integrin chain was studied in cell cultures. In the fetal eyes, the BM of the corneal epithelium, the Descemet’s membrane (DM) and the Bruch’s membrane each had their specific combinations of LM chains and time line of development, whereas the lens capsule and the internal limiting membrane showed constant LM chain patterns. The epithelial BMs of normal and diseased adult corneas contained similar LM chains. The normal morphology of the epithelial BM was altered in the different diseases, particularly when scarring was present. In the scarred keratoconus corneas there were excessive LM chains. The majority of keratoconus corneas also expressed extra LM chains in the DM. At 10-17 wg alpha11 integrin chain was present in the human corneal stroma, especially in the anterior portion, but it was scarce at 20 wg, in normal adult corneas and in Fuchs’ endothelial dystrophy. In contrast, it was increased in the anterior portion of the stroma in keratoconus corneas with scarring. Alpha11 integrin ko mice had a defective healing with subsequent thinner corneas. Alpha11 integrin expression correlated to the presence of alpha-smooth muscle actin in vivo as well as in vitro. The distinct spatial and temporal patterns of distribution for alpha11 integrin and each of the LM chains suggest that they play an important role in human ocular differentiation. The selectively affected LM composition and the novel expression of alpha11 integrin chain in scarred keratoconus corneas as well as the pathologic healing in ko mice, indicate that alpha11 integrin and LM chains also play an important role in the process of corneal healing, remodeling and scarring and might participate in the pathogenesis of corneal disease. This knowledge is of practical importance for future topical therapeutic agents capable of modulating the corneal wound healing processes.