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<p>Thrombosis is a common complication of chemotherapy for breast cancer patients. However, the specific mechanisms by which chemotherapy agents modulate these hemostatic pathways are not well understood. In this thesis, we investigated the mechanism(s) by which chemotherapy agents can upregulate procoagulant pathways (tissue factor (TF), phosphatidylserine exposure, and cell-free DNA (CFDNA) release) and impair the protein C (PC) anticoagulant pathway. We examined the effects of chemotherapy agents doxorubicin, epirubicin and the cyclophosphamide metabolite acrolein on cell surface procoagulant activity. We found that treatment of endothelial cells with the chemotherapy drugs increased phosphatidylserine exposure and TF activity on treated endothelial cells, blood monocytes and/or smooth muscle cells. This corresponded to an increase in thrombin generation on chemotherapy-treated cells exposed to recalcified, defibrinated plasma. We also found that found that doxorubicin and epirubicin can increase CFDNA release from breast cancer chemotherapy patients and healthy mice, which corresponds to an increase in thrombin-antithrombin levels. Treatment of venous whole blood and isolated neutrophils with doxorubicin and epirubicin increased CFDNA release. We found that exposure of recalcified plasma to CFDNA isolated from epirubicin-treated whole blood increased thrombin generation by activating the contact pathway. We investigated the effects of chemotherapy on the PC anticoagulant pathway. We found that acrolein decreased EPCR while increasing thrombomodulin expression on treated endothelial cells. A corresponding decrease in activated PC generation was measured on acrolein-treated endothelial cells exposed to recalcified, defibrinated plasma. Healthy mice treated with acrolein and cyclophosphamide increased PC antigen levels, but no measurable increase in plasma APC levels. Breast cancer chemotherapy drugs elevate thrombin generation by activating coagulation through the TF and contact pathways, and by promoting phosphatidylserine exposure, as well as by impairing PC activation EPCR expression. These studies provide insight into the mechanisms of breast cancer chemotherapy-induced hypercoagulation.</p> / Doctor of Philosophy (Medical Science)

Past studies of mammary tissue specific expression of transgenes using the murine whey acidic protein (WAP) promoter have shown widely variable, position-dependent and copy number-dependent expression. This study evaluates a series of three WAP transgenes containing the cDNA of human protein C (hPC) for the expression of human protein C in the milk of mice. In two of the transgenes studied, the cDNA of (hPC) was inserted at the translational start site of a 7.8 kbp mouse WAP genomic DNA Eco RI fragment containing 2.6 kbp of 5’ flanking, 3.9 kbp WAP coding (exons and introns), and 1.3 kbp 3’ untranslated region (UTR) and flanking sequences (designated WAPPC1 and WAPPC2). A third transgene consisted of only the 2.6 kbp of WAP 5’ UTR and flanking DNA, 1.4 kbp hPC cDNA, and 1.3 kbp of 3’ WAP UTR and flanking DNA with no linker sequences (designated WAPPC3). The WAPPC1 and WAPPC2 transgenes expressed up to about 10 μg/ml recombinant hPC in mouse milk while WAPPC3 expressed 30-300 (n=10, n=5, n=11, number of founder lines evaluated for each transgene, respectively). In contrast to past studies with WAP-cDNA fusion transgenes where the maximal expression was about 5% of endogenous WAP expression, the WAPPC3 transgene gave maximal expression which was about 30% of endogenous WAP expression. Thus, results from the combination in WAPPC3 of intact 5’ and 3’ WAP UTR with the cDNA of hPC suggests that introns are not necessary to enable high level expression in the mammary gland when using WAP regulatory elements. Relative specific transcript and protein levels in the transgenic animals studied suggest that the rates of translation initiation may be different for the mRNAs of each of the transgenes studied. / Ph. D.

Thesis (PhD ) -- Stellenbosch University, 2011. / Bibliography / ENGLISH ABSTRACT: The regulation of cardiac contractility is dependent on cooperative interaction between the thick and thin filaments, as well as their accessory proteins, within the cardiac sarcomere. Alteration in cardiac contractility due to a defective sarcomere typically results in cardiomyopathies, such as hypertrophic cardiomyopathy (HCM). One of the sarcomeric genes frequently mutated and which accounts for the second most common form of HCM encodes cardiac myosin binding protein C (cMyBPC), a thick filament accessory protein whose physiological function is poorly understood. However, studies have implicated cMyBPC in thick filament structure and function as well as in the regulation of contractility. The N-terminal region of cMyBPC houses the cMyBPC-motif, which contains three phosphorylation sites, between domains C1 and C2. The hierarchical phosphorylation of this motif, by first calcium/calmodulin kinase II (CamKII) and then by cyclic AMP-activated protein kinase (PKA), is cardinal in the role of cMyBPC in the regulation of cardiac contractility in response to ß-adrenergic stimulation. Moreover, phosphorylation of this motif is inversely correlated to cMyBPC proteolysis and has been shown to be cardioprotective. Thus, proteins that have an effect on cMyBPC function or turnover may also influence filament structure and hence affect contractility, which, in turn, affects the structure of the cardiac muscle. One such protein is the Copper metabolism MURR1-domain containing protein 4 (COMMD4), which was previously identified as a novel interactor of cMyBPC during a yeast two-hybrid (Y2H) library screen in our laboratory. COMMD4 binds specifically to the cMyBPC motif in a phosphorylation-dependent manner. The exact function of COMMD4 is unknown; however, it is a member of the COMM family of proteins that has been linked to copper metabolism as well as to the ubiquitin-proteasome pathway (UPS). Intriguingly, recent studies have shown that the UPS plays a role in cMyBPC-derived HCM, while dietary copper depletion is also known to cause cardiac hypertrophy. Based on these findings, COMMD4 was considered an interesting candidate regulator of sarcomeric function and contractility, and by extension, a candidate modifier of cardiac hypertrophy. Thus, the aim of the present study was two-fold. Firstly, COMMD4 was used as bait in a Y2H library screen to determine its distal ligands, with a view to further elucidate its function, particularly in the context of MyBPC functioning, and identified interactors were subjected to further in vitro and in vivo verification studies. Also, the phosphorylation-dependent nature of the interaction between COMMD4 and cMyBPC was further investigated using a domain/phosphorylation assay. Secondly, COMMD4 and its Y2H-identified putative interactors were assessed as possible modifiers of hypertrophy in a family-based association study, using three cohorts of South African HCM-families in which one of three founder mutations segregate. Six putative interactors, viz. cardiac actin (ACTC1), Down syndrome critical region 3 (DSCR3), enolase 1 (ENO1), F-box and leucine rich repeat protein 10 (FBXL10), legumain (LGMN) and sorting nexin3 (SNX3) were identified and confirmed as COMMD4 interactors using Y2H analyses, followed by in vitro and in vivo co-immunoprecipitation and 3D co-localisation assays. Moreover, as some COMMD protein family members and the newly-identified interactors of COMMD4 have previously been linked to the UPS, the functional effect of siRNA-mediated knockdown of COMMD4 on cMyBPC turnover was also investigated. Data revealed accumulation of cMyBPC in the endosomes upon COMMD4 knockdown, suggesting a functional role for COMMD4 in the turnover of cMyBPC. In addition, association analysis revealed strong evidence of association between various single nucleotide polymorphisms (SNPs) in SNX3 and a number of hypertrophy traits, thus suggesting a role for SNX3 as a candidate modifier of hypertrophy in HCM. No evidence of association was observed for any of the genes encoding the other COMMD4 interactors implicated in protein turnover. The present study demonstrates that COMMD4, a little understood member of the COMM family of proteins, binds to the cMyBPC motif of cMyBPC in a phosphorylation-dependent manner. Furthermore, based on the functions of its protein interactions, we hypothesise that COMMD4 plays a role in protein trafficking and turnover. More specifically, COMMD4 seems to help to facilitate formation of protein complexes with the Skp1-Cul1-Fbxl (SCF) E3 ubiquitin ligase and probably helps to stabilise the target substrate for subsequent ubiquitin-conjugation. As COMMD4 seems to affect the protein turnover of cMyBPC and possibly other sarcomeric proteins, such as actin, these results establish a novel association between the sarcomere, HCM and the UPS. In addition, identification of SNX3 as a hypertrophy modifier will allow for the improved understanding of HCM patho-aetiology. SNX3 thus adds to the growing body of sarcomeric modifier genes, which, eventually, may improve risk profiling in HCM. Furthermore, as genetic modifiers appear sufficient to completely prevent disease expression in some HCM carriers, the identification of SNX3 may point to the protein turnover pathway as a potential new target for intervention. / AFRIKAANSE OPSOMMING: Die regulering van kardiale kontraktiliteit is afhanklik van die koöperatiewe interaksie tussen die dik en dun filamente, asook hul geassosieerde proteïene, in die kardiale sarkomeer. Veranderinge in kardiale kontraktiliteit as gevolg van 'n defektiewe sarkomeer lei tot kardiomiopatieë soos hipertrofiese kardiomiopatie (HKM). Een van die sarkomeriese gene wat dikwels gemuteer is en wat verantwoordelik is vir die tweede algmeenste vorm van HKM,is dié van kardiale miosien-bindingsproteïen C (cMyBPC),'n proteïen geassosieer met die dik filament waarvan die fisiologiese funksie nog nie goed bekend is nie. Studies betrek cMyBPC in dik filament struktuur en funksie asook in die regulering van kontraktiliteit. Die N-terminale gebied van cMyBPC huisves die cMyBPC-motief, wat drie fosforilerings-setels tussen domeine C1 en C2 bevat. Die hiërargiese fosforilering van hierdie motief, eerstens deur kalsium/kalmodulien-gereguleerde kinase II (CamKII), gevolg deur siklies AMP-geaktiveerde proteïen kinase (PKA), is kardinaal in die rol van cMyBPC in die regulering van kardiale kontraktiliteit in reaksie op ß-adrenergiese stimulasie. Verder, fosforilering van hierdie motief is omgekeerd gekorreleer aan cMyBPC proteolise en is ook bewys om kardiobeskermend te wees. Dus, proteïene wat 'n uitwerking het op die funksie van cMyBPC mag ook filament struktuur en kontraktiliteit beïnvloed, wat op hul beurt die struktuur van die kardiale spier affekteer. Die koper metabolisme MURR1-domein bevattende protein 4 (COMMD4), was voorheen geïdentifiseer as 'n nuwe bindingsgenoot van cMyBPC tydens gis twee-hibried (G2H) analise in ons laboratorium. COMMD4 bind spesifiek aan die cMyBPC motief in 'n fosforilasie afhanklike wyse. Die presiese funksie van COMMD4 is onbekend; maar dit is 'n lid van die COMM domein familie van proteine wat geassosieerd is met koper metabolisme sowel as die “ubiquitin” proteosoom pad (UPP). Interesant genoeg, onlangse studies het bewys dat die UPP 'n rol speel in cMyBPC-afgeleide HKM, terwyl koper uitputting in die dieet ook bekend is om kardiale hipertrofie te veroorsaak. Gebaseer op hierdie bevindinge was COMMD4 oorweeg as 'n interessante kandidaat reguleerder van sarkomeries funksie en kontraktiliteit, asook 'n kandidaat modifiseerder van kardiale hipertrofie. Dus, die doel van die huidige studie was tweeledig. Eerstens, was COMMD4 as aas gebruik in 'n G2H biblioteek sifting om sy distale ligande te bepaal, met die oog om verdere lig te werp op sy funksie, veral in die konteks van MyBPC funksionering, en geïdentifiseerde bindingsgenote was onderwerp aan verdere 'in vitro’ en 'in vivo’ verifikasie studies. Daarbenewens was die fosforilering-afhanklike aard van die interaksie tussen COMMD4 en cMyBPC verder ondersoek met behulp van 'n domein/fosforilasie toets. Tweedens, COMMD4 en sy G2H-geïdentifiseerde vermeende bindingsgenote was geassesseer as moontlik modifiseerders van hipertrofie in 'n familie-gebaseerde assosiasie studie, met behulp van drie kohorte van Suid-Afrikaanse HKM-families waarin een van die drie stigter mutasies segregeer. Ses vermeende interaktors, nl. kardiale aktien (ACTC1), Down-sindroom kritiese streek 3 (DSCR3), enolase 1 (ENO1), F-boks en leusien ryke herhalings proteïen 10 (FBXL10), legumain (LGMN) en sorteer nexin3 (SNX3) is geïdentifiseer en bevestig as COMMD4 bindingsgenote deur G2H analises, gevolg deur in vitro en in vivo ko-immunopresipitasie en 3D ko-lokalisasie toetse. Die funksionele effek van siRNA-bemiddelde uitklop van COMMD4 op cMyBPC omset was ook ondersoek omdat 'n paar COMMD proteïen familielede, asook die nuut-geïdentifiseerde bindingsgenote van COMMD4, geassosieerd is met die UPP. Data toon ophoping van cMyBPC in die endosome by COMMD4 uitklop, wat dus aandui op 'n funksionele rol vir COMMD4 in die omset van cMyBPC. Daarbenewens, toon assosiasie analise sterk bewyse van assosiasie tussen die verskillende enkele nukleotied polimorfismes (SNPs) in SNX3 en 'n aantal hipertrofiese kenmerke,wat aandui op 'n rol vir SNX3 as 'n kandidaat modifiseerder van hipertrofie in HKM. Geen bewyse van assosiasie was waargeneem vir enige van die gene wat kodeer vir die ander COMMD4 bindingsgenote wat geïmpliseer word in die proteïen omset. Die huidige studie toon dat COMMD4, 'n min verstaande lid van die COMM familie van proteïene, aan die cMyBPC motief van cMyBPC in'n fosforilasie-afhanklike wyse bind. Verder, gebasseer op die funksies van die proteïen interaksies, hipotiseer ons dat COMMD4 'n rol speel in proteïen vervoer en omset. Meer spesifiek, COMMD4 blyk om die vorming van proteïene komplekse met die Skp1-Cul1-Fbxl (SCF) E3 "ubiquiti". ligase te fasiliteer en help waarskynlik om die teiken-substraat vir die daaropvolgende ubiquitin-konjugasie te stabiliseer. Omdat dit lyk asof COMMD4 die proteïen-omset van cMyBPC en moontlik ander sarkomeriese proteïene, soos aktien, ook beïnvloed, vestig die resultate dus 'n nuwe assosiasie tussen die sarkomeer, HKM en die UPP. Daarbenewens sal die identifisering van SNX3 as 'n hipertrofie modifiseerder voorsiening maak vir die verbeterde begrip van HKM pato-etiologie. SNX3 voeg dus by tot die groeiende ?getal van sarkomeriese modifiseerende gene, wat uiteindelik, die risiko-ontleding in HKM mag verbeter. Verder, omdat dit blyk dat genetiese modifiseerders voldoende is om die siekte-uitdrukking heeltemal te verhoed in sekere HKM draers, kan die identifikasie van SNX3 na die proteïen-omset roete dui as 'n potensiële nuwe teiken vir intervensie.

Cardiac myosin binding protein-C (cMyBP-C) is a structural and regulatory component of cardiac thick filaments. It is observed in electron micrographs as seven to nine transverse stripes in the central portion of each half of the A band. Its C-terminus binds tightly to the myosin rod and contributes to thick filament structure, while the N-terminus can bind both myosin S2 and actin, influencing their structure and function. Mutations in the MYBPC3 gene (encoding cMyBP-C) are commonly associated with hypertrophic cardiomyopathy (HCM). In cardiac cells there exists a population of myosin heads in the super-relaxed (SRX) state, which are bound to the thick filament core with a highly inhibited ATPase activity. This report examines the role cMyBP-C plays in regulating the population of the SRX state of cardiac myosin by using an assay that measures single ATP turnover of myosin. We report a significant decrease in the proportion of myosin heads in the SRX state in homozygous cMyBP-C knockout mice, however heterozygous cMyBP-C knockout mice do not significantly differ from the wild type. A smaller, non-significant decrease is observed when thoracic aortic constriction is used to induce cardiac hypertrophy in mutation negative mice. These results support the proposal that cMyBP-C stabilises the thick filament and that the loss of cMyBP-C results in an untethering of myosin heads. This results in an increased myosin ATP turnover, further consolidating the relationship between thick filament structure and the myosin ATPase. Crown Copyright (C) 2016 Published by Elsevier Ltd. All rights reserved.

Une coagulopathie aigue endogène (EAC) est présente chez 25% des patients de traumatologie dès leur arrivée. Des résultats d’études récentes montrent que cette EAC est liée à l’activation de la voie de la protéine C (aPC). Quelques heures après, se développe un état pro-coagulant associant un niveau abaissé d’aPC et un taux plasmatique élevé de l’inhibiteur de l’activateur du plasminogene (PAI-1). Nous trouvons que l’incidence des pneumopathies associées à la ventilation est significativement augmentée chez ces patients sans toutefois connaître le rôle exact de ces anomalies de coagulation. Basé sur cette hypothèse central de susceptibilité augmentée a l’infection et plus particulièrement aux pneumopathie a P.aeruginosa (PA) le but de ce travail est d’identifier les mécanismes par lesquels PAI-1 et aPC peuvent moduler la perméabilité de la barrière alveolo capillaire et ceci a travers 3 objectifs spécifiques1 – Objectif 1 : déterminer les mécanismes par lequel PA augmente la perméabilité endothéliale. 2 – Objectif 2 : déterminer le rôle d’aPC dans la modulation des effets de PA sur l’œdème pulmonaire lésionnel.3 – Objectif 3 : déterminer le rôle de PAI-1 dans la modulation des effets de PA sur l’œdème pulmonaire lésionnel.En utilisant un inhibiteur spécifique des petites GTPases nous démontrons le rôle centrale joué par RhoA dans le développement de l’œdème pulmonaire induit par PA. PAI-1 et aPC sont impliquées dans le mécanisme lésionnel pulmonaire. aPC et l’inhibition de la voie du RhoA attenue le développement de l’œdème pulmonaire et diminue la dissémination systémique bactérienne. Cependant le blocage invivo de la voie de PAI-1 est associé à une surmortalité et à une augmentation de la charge bactérienne suggérant un rôle de PAI-1 dans l’activation de la réponse inflammatoire nécessaire a l’éradication de PA / A clinically significant acute endogenous coagulopathy (EAC) is present in 25% of major trauma patients upon arrival in the emergency department, before any fluid resuscitation. Results from recent clinical studies indicate that EAC is primarily caused by the activation of the anticoagulant protein C pathway. Several hours later, there is the development of a systemic procoagulant activity associated with low plasma levels of activated protein C (aPC) and an inhibition of the fibrinolysis caused by elevated plasma levels of plasminogen activator inhibitor 1 (PAI-1). We have found that the incidence of ventilator-associated pneumonia (VAP) is significantly increased in trauma patients with these coagulation abnormalities [6, 9]. However, whether these coagulation abnormalities play a mechanistic role in the increased susceptibility to nosocomial lung infection observed after severe posttraumatic hemorrhage is unknown. Thus, the central hypothesis is that the increased susceptibility to P. aeruginosa (PA) pneumonia following severe trauma with tissue hypoperfusion is mediated in part by these posttraumatic coagulation abnormalities within the airspaces of the lung. Specifically, in this work, we will identify through 3 specific aims the mechanisms by which PAI-1 and aPC modulate PA–mediated increase in alveolar-capillary barrier permeability.1 - Specific Aim 1: To determine the mechanisms by which PA increases lung endothelial permeability.2 - Specific Aim 2 : To determine the Role of aPC in modulating the effect of PA on the lung endothelial barrier function3 - Specific Aim 3 : To determine the Role of PAI-1 in modulating the effect of PA on the lung endothelial barrier functionIn the present work, we demonstrated the central role small GTPases RhoA plays in the increase of permeability induced by pseudomonas infection. PAI-1 and aPC are deeply involved in the control of early lung inflammation. aPC and inhibition of the RhoA pathway attenuates the development of pulmonary edema and decrease in the systemic dissemination of P. aeruginosa. However, in vivo disruption of PAI-1 signalling is associated with higher mortality at 24 h and significant increase in the bacterial burden suggesting that PAI-1 is required for the activation of the innate immune response necessary for the eradication of PA from the distal airspaces of the lung