Despite the development and use of specialised cardiac preservation solutions, the quality of the donor heart may still be compromised by its obligatory exposure to periods of ischaemia (both cold and warm) followed by reperfusion upon reintroduction of the recipient circulation. This is reflected in Transplant Registry data showing increased primary allograft failure as a function of increasing ischaemic time. The research described in this thesis is designed to further the understanding of the mechanisms by which the donor heart may be adapted to these prolonged periods of ischaemia and reperfusion by the activation of endogenous pro-survival signalling pathways by the addition of pharmacological agents to Celsior, a clinical preservation solution. Studies were conducted in an isolated working rat heart model of donor heart preservation. The first study investigated the cardioprotective effects of a novel inhibitor of poly(ADP-ribose) polymerase 1, INO-1153. Maximum protective effect (after a 6 hour storage period) was observed when the PARP inhibitor was administered prior to cardiac arrest and storage and when the agent was added to the Celsior cardioplegic / storage solution. This protective affect was associated with activation of the Akt signalling pathway and could be prevented by inhibition of Akt phosphorylation and activation. The second study examined functional protection and pro-survival signalling pathway activation in hearts arrested and stored for 6 hours in Celsior supplemented with glyceryl trinitrate (an exogenous source of nitric oxide) and Cariporide (an inhibitor of sodium hydrogen exchange). Here, cardiac protection was accompanied by activation of the ERK 1/2 pro-survival pathway as well as a decrease in apoptosis. The third study examined the cardioprotective effect of supplementation of Celsior with all three agents after an extended (10 hour) period of hypothermic storage. Significant recovery of function was only observed in the triply supplemented hearts, being accompanied by activation of both the Akt and ERK pathways. These studies demonstrate for the first time the feasibility of recruitment of endogenous pro-survival pathways as an approach to increasing the post-storage function of the donor heart. Importantly, for the logistics of clinical transplantation, these pathways can be recruited by addition of appropriate pharmacological agents to the arresting and storage solution.

Ischaemic stroke is a major cause of death and disability worldwide. Cerebral autoregulation, which can be impaired during acute stroke, and collateral flow to brain tissue through the circle of Willis, both play a role in preventing tissue infarction. The configuration of the arterial circle varies between individuals. Thus, personalised modelling of the cerebral arterial network, to determine the potential for collateral flow, can be of significant value in the clinical context of stroke. The interaction between autoregulation and collateral flow remains poorly understood. In this study, steady-state physiological models of the cerebral arterial network, including several common variants of the circle of Willis, were coupled to a spatially variable mathematical representation of cerebral autoregulation. The resulting model was used to simulate various arterial occlusions, as well as bilateral and unilateral impairment of autoregulation, in each structural variant. The work identified few circle of Willis variants that present either particularly high-risk or particularly low-risk of cerebral ischaemia. Instead it was found that most variants are dependent upon the bilateral function of autoregulation to facilitate collateral flow and preserve cerebral blood flows. When autoregulation was impaired unilaterally, downstream of an occlusion, blood flows in the contralateral hemisphere were preserved at the expense of the ipsilateral tissue at risk. Arterial network models have in the past been personalised using structural, rather than functional, angiography measurements. This thesis presents a novel model-based method for absolute blood volume flow rate quantification in short arterial segments using dynamic magnetic resonance angiography data. The work also investigated the additional information that can be obtained from such functional angiography. The flow quantification technique was found to accurately estimate flows in shorter arterial segments than an existing technique. However, improvements to noise performance, and strategies for rejection of contaminating signals from overlapping vessels within the imaging plane, are required before the technique can be applied to personalised cerebral arterial network modelling.

Die extrakorporale Zirkulation unter Einsatz einer Herz-Lungen-Maschine kann postoperativ zu kontraktilen ventrikulären Funktionsstörungen führen, die Morbidität und Mortalität für betroffene Patienten erhöht. Diese kardiale Dysfunktion bezeichnet man als myokardiales Stunning, welche durch die globale Ischämie ausgelöst wird. Das Phänomen der reversiblen kontraktilen Dysfunktion weißt eine hohe klinische Relevanz auf und ist somit in den vergangenen Jahrzehnten sowohl klinisch als auch experimentell intensiv erforscht worden. Dabei kamen unterschiedlichste Spezies, Methoden und Modelle zum Einsatz. Ziel der vorliegenden Arbeit ist die Etablierung eines neuartigen akut instrumentierten Tiermodells, anhand dessen Folgen des kardiopulmonalen Bypasses und Wirkung des endogenen Opioidsystems auf myokardiales Stunning untersucht werden können. Mit Hilfe des entwickelten Versuchsmodells können die Auswirkungen applizierter Opioidrezeptorantagonisten und die Effekte der kardiopulmonalen Zirkulation auf die kontraktile myokardiale Dysfunktion valide untersucht werden. Als Versuchstiere wurden 50 männlich kastrierte Schweine der Rassenkreuzung „Deutsche Landrasse“ und „Yorkshireschwein“ eingesetzt. In Allgemeinanästhesie wurden die Tiere über eine Thorakotomie instrumentiert und anschließend elektrisch Kammerflimmern induziert. Nach Erreichen einer stabilen extrakorporalen Zirkulation unter der Herz-Lungen-Maschine wurde nach Ablauf der ischämischen Phase eine standardisierte Reaninmation und Weaning durchgeführt. Alle 50 Tiere konnten den Versuch erfolgreich durchlaufen. Die Analyse und Auswertung sämtlicher archivierter Daten und Proben der Versuchstiere wurde zu einem späteren Zeitpunkt durchgeführt.

[ES] La cardiopatía isquémica, caracterizada por la falta de suministro adecuado de oxígeno al tejido cardíaco, es una afección grave que puede desencadenar un infarto agudo de miocardio y contribuir al desarrollo de la insuficiencia cardíaca (IC). A pesar de las terapias actuales, la IC sigue siendo una enfermedad con alta morbilidad y mortalidad, lo que destaca la necesidad de estrategias terapéuticas más efectivas. En este contexto, las células madre mesenquimales (MSCs) y, en especial sus vesículas extracelulares (EVs), han surgido como opciones prometedoras por sus propiedades regenerativas, pro-angiogénicas e inmunomoduladoras. Sin embargo, el reto actual se centra en mejorar la eficacia terapéutica de las EVs, ya sea mejorando su biodisponibilidad en el tejido cardíaco o potenciando sus capacidades intrínsecas, con el fin de hacer viable una terapia basada en las mismas. En este trabajo, nos hemos centrado en la mejora de las EVs mediante la modificación genética de las MSCs. Hemos seguido dos enfoques: la carga de oncostatina M (OSM) en la superficie de las EVs y la sobreexpresión inducible del dominio intracelular de Notch1 (N1ICD) junto con el factor inducible por hipoxia 1-alfa (HIF1A) en MSCs para enriquecer las EVs con factores terapéuticos, con la expectativa de mejorar su eficacia en el contexto de la isquemia cardíaca. Los resultados obtenidos muestran que las EVs cargadas con OSM poseen propiedades antifibroticas superiores a las EVs nativas, además de reducir el daño cardíaco provocado por la infusión de isoproterenol in vivo. Por otro lado, la sobreexpresión de N1ICD y HIF1A actúa a modo de precondicionamiento genético favoreciendo la carga de diferentes moléculas terapéuticas en las EVs. Estas EVs han demostrado ejercer efectos beneficiosos in vitro como la reducción de la fibrosis, la protección de los cardiomiocitos y reducción de la hipertrofia, la disminución de especies reactivas de oxígeno, y el aumento de la angiogénesis. En el estudio in vivo estas EVs redujeron el daño provocado por la infusión de isoproterenol. En conclusión, hemos generado dos tipos de EVs con un potencial terapéutico superior a las EVs nativas en el contexto de la patología cardíaca. Este trabajo abre la puerta al diseño de nuevas estrategias terapéuticas basadas en EVs, abordando de manera integral los diversos aspectos de la enfermedad cardíaca. / [CA] La cardiopatia isquèmica, caracteritzada per la falta de subministrament adequat d'oxigen al teixit cardíac, és una afecció greu que pot desencadenar un infart agut de miocardi i contribuir al desenvolupament de la insuficiència cardíaca (IC). Malgrat les teràpies actuals, la IC continua sent una malaltia amb alta morbiditat i mortalitat, la qual cosa destaca la necessitat d'estratègies terapèutiques més efectives. En este context, les cèl·lules mare mesenquimals (MSCs) i, especialment les seues vesícules extracelul·lars (EVs), han sorgit com a opcions prometedores per les seues propietats regeneratives, pro-angiogèniques i inmunomoduladores. No obstant això, el repte actual se centra en millorar l'eficàcia terapèutica de les EVs, ja siga millorant la seua biodisponibilitat en el teixit cardíac o potenciant les seues capacitats intrínseques, amb la finalitat de fer viable una teràpia basada en estes. En este treball, ens hem centrat en la millora de les EVs mitjançant la modificació genètica de les MSCs. Hem seguit dos enfocaments: la càrrega d'oncostatina M (OSM) en la superfície de les EVs i la sobreexpressió induïble del domini intracel·lular de Notch1 (N1ICD) juntament amb el factor induïble per hipòxia 1-alfa (HIF1A) en MSCs per a enriquir les EVs amb factors terapèutics, amb l'expectativa de millorar la seua eficàcia en el context de la isquèmia cardíaca. Els resultats obtinguts mostren que les EVs carregades amb OSM posseeixen propietats antifibròtiques superiors a les EVs natives, a més de reduir el dany cardíac provocat per la infusió d'isoproterenol in vivo. D'altra banda, la sobreexpressió de N1ICD i HIF1A actua a mode de precondicionament genètic afavorint la càrrega de diferents molècules terapèutiques en les EVs. Estes EVs han demostrat exercir efectes beneficiosos in vitro com la reducció de la fibrosi, la protecció dels cardiomiòcits i reducció de la hipertròfia, la disminució d'espècies reactives d'oxigen, i l'augment de l'angiogènesis. En l'estudi in vivo estes EVs van reduir el dany provocat per la infusió d'isoproterenol. En conclusió, hem generat dos tipus de EVs amb un potencial terapèutic superior a les EVs nadiues en el context de la patologia cardíaca. Este treball obri la porta al disseny de noves estratègies terapèutiques basades en EVs, abordant de manera integral els diversos aspectes de la malaltia cardíaca. / [EN] Ischemic heart disease, characterized by a lack of adequate oxygen delivery to the heart tissue, is a serious condition that can trigger acute myocardial infarction and contribute to the development of heart failure (HF). Despite current therapies, HF remains a disease with high morbidity and mortality, highlighting the need for more effective therapeutic strategies. In this context, mesenchymal stem cells (MSCs) and, especially their extracellular vesicles (EVs), have emerged as promising options due to their regenerative, pro-angiogenic and immunomodulatory properties. However, the current challenge focuses on improving the therapeutic efficacy of EVs, either by improving their bioavailability in cardiac tissue or by enhancing their intrinsic capabilities, in order to make a therapy based on them viable. In this work, we have focused on the improvement of EVs through genetic modification of MSCs. We have followed two approaches: loading of oncostatin M (OSM) on the surface of EVs and inducible overexpression of Notch1 intracellular domain (N1ICD) together with hypoxia-inducible factor 1-alpha (HIF1A) in MSCs to enrich EVs with therapeutic factors, with the expectation of improving its effectiveness in the context of cardiac ischemia. The results obtained show that OSM-loaded EVs have superior antifibrotic properties than native EVs, in addition to reducing cardiac damage caused by isoproterenol infusion in vivo. On the other hand, the overexpression of N1ICD and HIF1A acts as genetic preconditioning, favouring the loading of different therapeutic molecules in EVs. These EVs have been shown to exert beneficial effects in vitro such as reducing fibrosis, protecting cardiomyocytes and reducing hypertrophy, decreasing reactive oxygen species, and increasing angiogenesis. In the in vivo study, these EVs reduced the isoproterenol-induced myocardial damage. In conclusion, we have generated two types of EVs with a therapeutic potential superior to native EVs in the context of cardiac pathology. This work opens the door to the design of new therapeutic strategies based on EVs, comprehensively addressing the various aspects of heart disease. / Buigues Caravaca, M. (2024). Modificación genética de células estromales mesenquimales para potenciar la eficacia de las vesículas extracelulares en el ámbito de la terapia cardíaca [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/204408

Thesis (MScMedSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Introduction: Primary culture of isolated adult rat cardiomyocytes (ARCMs) is an important model for cardiovascular research, but successful maintenance of these cells in culture for their use in experiments remains challenging (Xu et al, 2009; Louch et al, 2011). Most studies are done on acutely isolated cardiomyocytes immediately after isolation, which is due to low survival of these cells in culture. Obstacles in culture are due to the type of medium and attachment factors (tissue culture adhesives) used to culture and grow these cells. Although we previously identified an optimum medium and adhesive for culture, an adhesive that permits cells to remain attached to the culture surface until after an ischemia/reperfusion insult was elusive. Aims: We therefore aimed to identify the best attachment factor and concentration that will allow adult rat cardiomyocytes to remain attached to the culture surfaces after ischemia/reperfusion experiments. Methods: Cardiomyocytes were isolated from adult Wistar rat hearts and cultured overnight on different concentrations (25 -200 μg/ml) of collagen 1, collagen 4, extracellular matrix (ECM), laminin/entactin (L/E) and laminin. Following overnight cultures, experiments were done in PBS and in PBS versus MMXCB to compare ARCM attachment and viability. Cardiomyocytes cultured on ECM, L/E and L (25−200μg/ml) were subjected to 1 hour of simulated ischemia using MMXCB that contained 3mM SDT and 10mM 2DG, followed by 15 minutes reperfusion. Cell viability was determined by staining cells with JC-1 and images of cells in a field view of 1.17μm/mm2 were captured using fluorescence microscopy. The cells were analysed according to morphology and fluorescence intensity. Results: Total and rod-shaped ARCMs attachment was improved when MMXCB was used as an experimental buffer instead of PBS. Regardless of the buffer used, morphological viability was poor on substrates of Col 1 and Col 4. In contrast to collagens, ARCMs attached efficiently and morphological viability was high on substrates of ECM, L/E and L in MMXCB, but this was greatly reduced in PBS. Mitochondrial viability was high in MMXCB compared to PBS on Col 1 and Col 4 at 75−175μg/ml and on ECM, L/E and L at all concentrations, except at 50 and 150μg/ml ECM, 175μg/ml L/E and 25μg/ml L. When cardiomyocytes cultured on ECM, L/E and L were subjected to simulated ischemia, total ARCMs, rod-shaped and R/G fluorescence (mitochondrial viability) was reduced at all concentrations compared to the control group. Hypercontracted cells were higher in the ischemic treated cells compared to the controls on ECM at 75−150μg/ml and 200μg/ml, L/E at 50,100μg/ml and 175μg/ml and on L at 125μg/ml. Total numbers of ARCMs attached on ECM, L/E and L in the ischemic group consisted of similar numbers of non-viable hypercontracted and viable rod-shaped cells. Conclusion: Cardiomyocytes should be cultured on ECM or L/E or L at concentrations from 25−200μg/ml in MMXCB. PBS is harmful to cultured ARCMs and should thus not be used as an experimental buffer. Ischemia/reperfusion can be simulated on ARCMs cultured on ECM, L/E or L from 25−200μg/ml, provided that a modified culture buffer is used as experimental buffer. / AFRIKAANSE OPSOMMING: Inleiding: Primêre selkulture van geïsoleerde volwasse rot kardiomiosiete (VRKMe) is ‘n belangrike model vir kardiovaskulêre navorsing, maar om hierdie selle suksesvol in kultuur te onderhou is ‘n groot uitdaging (Xu et al, 2009; Louch et al, 2011). Die meeste navorsingstudies maak gebruik van akuut geïsoleerde kardiomiosiete onmiddelik na isolasie omdat oorlewing van hierdie selle in kultuur baie laag is. Die struikelblokke in kultuur is as gevolg van die tipe medium en weefselkultuurgom wat gebruik word. Ons het voorheen 'n optimale medium en weefselkultuurgom geïdentifiseer vir VRKM kultuur oorlewing, maar die weefselkultuurgom was nie effektief genoeg om die selle aan die kultuuroppervlak te laat bly vaskleef, tot na die einde van 'n isgemie/herperfusie eksperiment nie. Doel: Die doel was dus om die beste weefselkultuurgom en konsentrasie te identifiseer, wat sal toelaat dat VRKMe verbonde bly aan die kultuuroppervlaktes tot na die einde van isgemie/herperfusie eksperimente. Metodes: Kardiomiosiete was geïsoleer vanaf volwasse Wistar rotharte en oornag in kultuur op verskillende konsentrasies (25 -200 μg/ml) van kollageen 1, kollageen 4, ekstrasellulêre matriks (ESM), laminin/entactin (L/E) en laminin onderhou. Die volgende dag was die VRKMe vir eksperimentasie in PBS en in PBS teenoor MMXCB gebruik, om selbehoud en oorlewing te vergelyk. Kardiomiosiete op ESM, L/E en L (25−200μg/ml) was aan 1 uur van gesimuleerde isgemie blootgestel, in MMXCB wat 3mM SDT en 10mM 2DG bevat het, gevolg deur 15 minute herperfusie. Sel oorlewing was bepaal deur selle te kleur met JC-1 en daarna was fluoressensiebeelde van die selle in ‘n veldgebied van 1.17μm/mm2 geneem. Die selle was volgens selmorfologie en fluoressensie intensiteit ontleed. Resultate: Met die gebruik van MMXCB as eksperimentele buffer in plaas van PBS, het die aantal totale en staafvormige VRKMe verbinding verbeter. Morfologiese onderhoud was sleg op kollageen 1 en 4, ongeag van watter buffer gebruik was. In kontras met die kollagene was die VRKM verbinding en morfologiese onderhoud op ESM, L/E en L in MMXCB effektief verbeter, maar in PBS aansienlik verminder. Mitochondriale lewensvatbaarheid in MMXCB teenoor PBS op kollageen 1 en 4 by 75−175μg/ml, sowel as op ECM, L/E en L by alle konsentrasies, was hoog, behalwe by 50 en 150μg/ml ESM, 175μg/ml L/E en 25μg/ml L. Isgemie blootstelling van kardiomiosiete gekultuur op alle konsentrasies van ESM, L/E en L, het ‘n afname in die totale, staafvormige en R/G fluoressensie (mitochondriale lewensvatbaarheid) teweeggebring. Meer hiperkontrakteerde kardiomiosiete was in die isgemie behandelde groepe as in die kontrole groepe teenwoordig, spesifiek op ESM by 75−150μg/ml en 200μg/ml, op L/E by 50,100μg/ml en 175μg/ml asook op L by 125μg/ml. In die isgemie groepe het die totale aantal VRKMe op ESM, L/E en L meestal uit ‘n gelyke hoeveelheid hiperkontrakteerde en staafvormige selle bestaan. Gevolgtrekking: Kardiomiosiete moet op ESM of L/E of L by konsentrasises van 25−200μg/ml in MMXCB gekultuur word. PBS is nadelig vir VRKMe in kultuur en moet dus nie gebruik word as eksperimentele buffer nie. Isgemie/herperfusie eksperimente kan gesimuleer word op VRKMe wat op 25−200μg/ml ESM, L/E of L gekultuur is, mits ‘n gemodifiseerde kultuur buffer gebruik word as eksperimentele buffer.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Ischaemic heart disease is a major contributor to global morbidity and mortality rates. Manoeuvres such as ischaemic preconditioning confer cardioprotection against ischaemia/reperfusion (I/R) injury by activating several intracellular signalling pathways. These pathways have been defined solely in terms of the kinases involved, despite the realization in recent years that protein phosphatase activity also contributes significantly to the attributes of the propagated signal. Protein phosphatase 2A (PP2A) is a heteromultimeric enzyme involved in an array of phosphatase reactions. We hypothesized that PP2A is an important participant in the myocardial response to I/R by regulating intracellular signalling. This project aimed to (i) characterize PP2A during myocardial I/R; (ii) determine the importance of its contribution to the cellular response to I/R; and (iii) investigate its role in the signalling pathways mediated by PKB/Akt, GSK-3β, ERK p42/p44 and p38 MAPK. Two models were used to characterize PP2A during I/R: (i) H9c2 cells exposed to simulated ischaemia (SI) buffer in conjunction with hypoxia (0.5% O2) for a maximum of 2 hours, followed by reoxygenation in standard growth medium for up to 30 minutes; and (ii) isolated working rat hearts exposed to a maximum of 20 minutes global ischaemia and 10 minutes reperfusion. In both models samples were collected at several time points during I/R for Western blotting analysis. PP2A-C (the catalytic subunit) accumulated in the nucleus during early ischaemia, but later redistributed to the cytosol. At the end of ischaemia there was an elevation of PP2A-C relative to PP2A-A in the unfractionated whole cell preparation concomitant with an increase in the inhibitory phosphorylation of PP2A-C. The impact of PP2A activity was evaluated by either inhibiting PP2A using okadaic acid (OA, 10 nM) or activating it by administering FTY720 (1 μM) in an isolated working rat heart model exposed to either 35 minutes of regional ischaemia (RI) with infarct size (IFS) as primary end-point, or 20 minutes global ischaemia (GI) with functional recovery as end-point. The results showed that the pre-ischaemic administration of OA or FTY720 reduced or exacerbated IFS respectively, indicating that PP2A activation during I/R favours cell death. OA and FTY720 were also employed to assess the contribution of PP2A to intracellular signalling in an isolated working rat heart exposed to I/R. Samples were collected at several timepoints and analyzed using Western Blotting. Pre-ischaemic administration of OA enhanced the phosphorylation of PKB/Akt, ERK p42/p44 and GSK-3β at the onset of reperfusion, while FTY720 given before ischaemia reduced the phosphorylation of GSK-3β, p38 MAPK and PKB/Akt at the end of ischaemia and onset of reperfusion. In summary, PP2A is part of an early nuclear-based response to ischaemia, while long-term ischaemia induces an increase in PP2A-C. A portion of this PP2A-C is stored in an inactive form, while an active portion acts as a regulator of the pro-survival signalling components PKB/Akt, GSK- 3β and ERK p42/p44 at the end of ischaemia and the onset of reperfusion. PP2A is therefore an important component of the myocardial response to I/R by regulating pro-survival signalling. / AFRIKAANSE OPSOMMING: Iskemiese hartsiekte is een van die belangrikste komponente wat bydra tot globale morbiditeit en mortaliteit. Ingrepe soos iskemiese prekondisionering aktiveer veelvoudige intrasellulêre seintransduksiepaaie om kardiobeskerming teen iskemie/herperfusie (I/H)-besering te ontlok. Die kinases betrokke in hierdie seintransduksiepaaie is reeds deeglik nagevors, terwyl die potensiële belang van die proteïenfosfatases in seintransduksie tot onlangs misken is. Ons hipotese was dat Proteïenfosfatase 2A (PP2A), wat in ‘n wye verskeidenheid fosfatase reaksies betrokke is, ‘n belangrike rolspeler in die miokardiale reaksie op I/H-besering is, deur deelname aan die regulering van intrasellulêre seintransduksie. Hierdie projek het ten doel gehad om (i) PP2A te karakteriseer tydens miokardiale I/H; (ii) die belang van PP2A in die sellulêre reaksie op I/H-besering te bepaal; en (iii) PP2A se rol in die seintransduksiepaaie, gemedieer deur PKB/Akt, GSK-3β, ERK p42/p44 en p38 MAPK, te evalueer. Twee modelle is aangewend om PP2A tydens I/H te karakteriseer: (i) H9c2-selle blootgestel aan ‘n simuleerde iskemiebuffer tesame met hipoksie (0.5% O2) vir ‘n maksimum van 2 uur gevolg deur heroksiginasie in standaardgroeimedium vir verskillende tydsperiodes tot ‘n maksimum van 30 minute; en (ii) geïsoleerde, werkende rotharte blootgestel aan ‘n maksimum van 20 minute globale iskemie en 10 minute herperfusie. In beide modelle is monsters op verskillende tye versamel vir Western-kladanalise. Tydens vroeë iskemie het PP2A-C in die kern toegeneem, waarna dit met verloop van tyd na die sitosol herversprei het. Teen die einde van iskemie was daar ‘n toename in die vlakke van PP2A-C relatief tot PP2A-A in ongefraksioneerde weefselhomogenate, tesame met ‘n toename in die inhibitoriese fosforilering van PP2A-C. Die belang van PP2A-aktiwiteit is ondersoek deur die effek te bepaal van die inhibisie of aktivering daarvan op infarktgrootte (IFS) en funksionele herstel in ‘n geïsoleerde werkende rothartmodel, blootgestel aan onderskeidelik 35 minute streeksiskemie (RI) of 20 minute globale iskemie. Preiskemiese toediening van die PP2A-inhibitor okadaïensuur (OA, 10 nM), of aktiveerder FTY720 (1 μm) het infarktgrootte respektiewelik beperk of vergroot. PP2A-aktivering tydens I/H is dus nadelig. OA en FTY720 is ook aangewend om die bydrae van PP2A tot I/H-verwante, intrasellulêre seintransduksie in die geïsoleerde, werkende rothart te bepaal. Monsters is op verskeie tydintervalle versamel en ontleed deur gebruik te maak van die Western-kladtegniek. Preiskemiese toediening van OA het die fosforilering van PKB/Akt, ERK p42/p44 en GSK-3β by die aanvang van herperfusie bevoordeel, terwyl pre-iskemiese toediening van FTY720, die fosforilering van GSK-3β, p38 MAPK en PKB/Akt aan die einde van iskemie en die begin van herperfusie verminder het. Ter opsomming: PP2A is deel van ‘n vroeë gelokaliseerde kerngebaseerde reaksie op iskemie, terwyl langdurige iskemie ‘n toename in PP2A-C relatief tot PP2A-A induseer. ‘n Deel van hierdie PP2A-C is onaktief, terwyl die res funksioneer in die regulering van die seintransduksiekomponente PKB/Akt, GSK-3β en ERK p42/p44 wat oorlewing fasiliteer met die aanvang van herperfusie. PP2A is dus ‘n belangrike komponent in die miokardiale reaksie op I/H deurdat dit tot die beheer van seintransduksiepaaie bydra.

Sudden cardiac death is one of the leading causes of mortality in the western world. One of the main factors is myocardial ischaemia, when there is a mismatch between blood demand and supply to the heart, which may lead to disturbed cardiac excitation patterns, known as arrhythmias. Ischaemia is a dynamic and complex process, which is characterised by many electrophysiological changes that vary through space and time. Ischaemia-induced arrhythmic mechanisms, and the safety and efficacy of certain therapies are still not fully understood. Most experimental studies are carried out in animal, due to the ethical and practical limitations of human experiments. Therefore, extrapolation of mechanisms from animal to human is challenging, but can be facilitated by in silico models. Since the first cardiac cell model was built over 50 years ago, computer simulations have provided a wealth of information and insight that is not possible to obtain through experiments alone. Therefore, mathematical models and computational simulations provide a powerful and complementary tool for the study of multi-scale problems. The aim of this thesis is to investigate pro-arrhythmic electrophysiological consequences of acute myocardial ischaemia, using a multi-scale computational modelling and simulation framework. Firstly, we present a novel method, combining computational simulations and optical mapping experiments, to characterise ischaemia-induced spatial differences modulating arrhythmic risk in rabbit hearts. Secondly, we use computer models to extend our investigation of acute ischaemia to human, by carrying out a thorough analysis of recent human action potential models under varied ischaemic conditions, to test their applicability to simulate ischaemia. Finally, we combine state-of-the-art knowledge and techniques to build a human whole ventricles model, in which we investigate how anti-arrhythmic drugs modulate arrhythmic mechanisms in the presence of ischaemia.

De manière générale, l'inhalation de particules entraîne des réactions inflammatoires et des réactions d'oxydo-réduction responsables de la dégradation des matrices biologiques qui exercent, de plus, un fort impact cardio-vasculaire. La combustion du bois est une source majeure de composés organiques semi-volatils, parmi lesquels les méthoxyphénols (MPs), tels que le coniféryl aldéhyde (CA), le syringaldéhyde (SR), ou l'acétosyringone (AS). Les MPs sont néanmoins peu étudiés dans la littérature alors que la toxicité d'autres composés également issus de la combustion de la biomasse, tels que le monoxyde de carbone, les suies et les hydrocarbures polyaromatiques est intensivement étudiée. Ce travail a montré par GC/MS que le vieillissement en atmosphère simulée (ozone, rayonnements lumineux) dégrade le CA en produits secondaires moins cytotoxiques, évalués sur des fibroblastes en culture, mais préserve le taux atmosphérique de SR et AS, de toxicité avérée. Un dispositif original de production de MPs particulaires (Ø~50nm, N~7E4particules/cm3, m~5µg/m3) en atmosphère contrôlée a été validé et permet la 1ièreétude in vivo des MPs. L'exposition chez le rat (1-3mois) montre une modification des défenses antioxydantes et des changements cardiaques principalement avec AS, puis CA et un peu moins pour SR. Des processus adaptatifs sont démontrés après 5mois d'exposition.Par ailleurs, il a été montré in vitro sur des adénocacinomes pulmonaires A549 en culture, que le CA induit une destructuration du tapis cellulaire et l'apoptose (caspase 3) mais pas d'effet pro-inflammatoire (IL8, Cox-2). En conclusion, ce travail contribue à étudier l'impact des MPs in vitro et in vivo. / In general, inhalation of particles is at the origin of inflammatory and oxidative reactions who are responsible of the degradation of biological cellular constituents, and could have a strong cardiovascular impact. The wood combustion is a major source of semivolatile organic compounds such as the methoxyphenols (MPs) including coniferyl aldehyde (CA), syringaldehyde (SR), or acetosyringone (AS). The MPs are however few studies into literature while toxicity of other compounds also from biomass combustion, as carbon monoxide, soot and polycyclic aromatic hydrocarbon are intensively studies.This work has shown by GC/MS that aging in simulated atmosphere (ozone + light rays) degraded CA in secondary products less cytotoxic, studies on fribroblastes culture but keep the atmospherical level of SR and AS which have a toxicity proved.A device of MPs particle production original by atomization, with a check system (height, composition, weight) and exposition flow continuous (Ø~50 nm, N~7E4 particles/cm3, m~5 µg/m3) adapted to little animals, was developed and validated, allowed the first study in vivo with these molecules. Between 1 and 3 month of exposition to rat Wistar, show modified antioxidant defences and cardiac modification (ischaemia/reperfusion) principally with AS, then CA and less SR. The adaptatives processes (remodeling) are demonstrated after 5 month of exposition.Furthermore, it is showed in vitro on lung adenocacinum cell lines (A549), CA induced a monolayer destructuration and apoptosis (caspase 3) but no effect proinflammatory (IL8, Cox-2 and iNOS).To conclude, this work contributes to study the impact of MPs in vitro and in vivo.

Après un traumatisme de la moelle épinière (TM), l’ischémieest un facteur d’aggravation des lésions. Cette ischémie peut être aggravée par l’augmentation depression du liquide cérébro-spinal (LCS) par le biais d’un effet tamponnade. Or chez l’homme,après un TM avec préservation de l’intégrité de la dure-mère, la pression de LCS augmentesignificativement. On suppose donc que le maintien d’une pression de LCS à des valeursphysiologique pourrait être une méthode de limitation de l’ischémie post-traumatique et doncd’amélioration du pronostic fonctionnel. Afin de pouvoir réaliser une étude expérimentale de cesphénomènes, nous avons consacré la première partie expérimentale de cette thèse à la mise au pointd’un modèle de TM à dure-mère intacte chez le rat permettant la mesure simultanée de la pressionde LCS et de la perfusion médullaire. Nous avons confirmé expérimentalement que la pression deLCS augmente après TM. Dans la seconde partie expérimentale, nous avons mis au point unetechnique expérimentale de quantification spatiale et temporelle de la perfusion médullaire grâce àl’échographie de contraste. Cette technique permettait aussi un suivi en temps réel de l’évolution dusaignement intra-parenchymateux induit par le TM. Dans la troisième partie expérimentale, nousavons utilisé notre modèle couplé avec l’échographie de contraste et le laser Doppler pour évaluerles effets de la noradrénaline injectée à la phase aigüe d’un TM sur la perfusion médullaire et lesaignement intra-parenchymateux. Nous avons montré que la noradrénaline augmentait trèslégèrement le flux sanguin superficiel mais pas le flux sanguin profond et qu’elle augmentait lataille du saignement. / After spinal cord injury (SCI), ischaemia aggravates lesions.Increase in cerebrospinal fluid (CSF) pressure can worsens ischaemia through a tamponnade effect.In humans, it has been shown that after SCI with intact dura mater, CSF pressure significantlyincreases. Therefore, preserving CSF pressure within a physiological range may limit post-traumaischaemia and improve neurological outcome. In order to experimentally study these phenomenon,we have dedicated the first part of that work to create a model of SCI in rats preserving dura’sintegrity and allowing simultaneous measurement of spinal cord blood flow (SCBF) and CSFpressure. We have confirmed that CSF pressure increases after SCI with intact dura. In the secondexperimental part, we have developed a technique allowing to perform spatial and temporalmeasurement of SCBF thanks to contrast enhanced ultrasonography (CEU). Moreover, thistechnique allows real-time measurement of the size of the parenchymal hemorrhage. In the thirdexperimental part, we have used our experimental model in association with CEU and LaserDoppler to assess the effects of early injection of norepinephrine on SCBF and parenchymalhemorrhage. We found that norepinephrine induces a slight increase in superficial SCBF while itdoesn’t modify deep SCBF and significantly increases the size of parenchymal hemorrhage.

Understanding and characterising the diffusive transport of capillary oxygen and nutrients in striated muscles is key to assessing angiogenesis and investigating the efficacy of experimental and therapeutic interventions for numerous pathological conditions, such as chronic ischaemia. In articular, the influence of both muscle tissue and microvascular heterogeneities on capillary oxygen supply is poorly understood. The objective of this thesis is to develop mathematical and computational modelling frameworks for the purpose of extending and generalising the current use of histology in estimating the regions of tissue supplied by individual capillaries to facilitate the exploration of functional capillary oxygen supply in striated muscles. In particular, we aim to investigate the balance between local capillary supply of oxygen and oxygen demand in the presence of various anatomical and functional heterogeneities, by capturing tissue details from histological imaging and estimating or predicting regions of capillary supply. Our computational method throughout is based on a finite element framework that captures the anatomical details of tissue cross sections. In Chapter 1 we introduce the problem. In Chapter 2 we develop a theoretical model to describe oxygen transport from capillaries to uniform muscle tissues (e.g. cardiac muscle). Transport is then explored in terms of oxygen levels and capillary supply regions. In Chapter 3 we extend this modelling framework to explore the influence of the surrounding tissue by accounting for the spatial anisotropies of fibre oxygen demand and diffusivity and the heterogeneity in fibre size and shape, as exemplified by mixed muscle tissues (e.g. skeletal muscle). We additionally explore the effects of diffusion through the interstitium, facilitated--diffusion by myoglobin, and Michaelis--Menten kinetics of tissue oxygen consumption. In Chapter 4, a further extension is pursued to account for intracellular heterogeneities in mitochondrial distribution and diffusive parameters. As a demonstration of the potential of the models derived in Chapters 2--4, in Chapter 5 we simulate oxygen transport in myocardial tissue biopsies from rats with either impaired angiogenesis or impaired arteriolar perfusion. Quantitative predictions are made to help explain and support experimental measurements of cardiac performance and metabolism. In the final chapter we summarize the main results and indicate directions for further work.