La cytokinèse est un processus dont le but est une séparation de deux cellules soeurs en deux entités suite à une mitose. La cytokinèse nécessite la formation d’un anneau contractile (AC) qui va conduire un sillon de clivage vers une ingression à l’équateur de la cellule. L’une des étapes critiques de ce processus est la transition d’un AC dynamique vers une structure stable surnommée l’anneau du midbody (AM), organelle qui va guider la cellule vers l’abscision. La compréhension des mécanismes moléculaires impliqués dans cette transition nous permettrait de mieux comprendre les complexes protéiques impliqués autant au niveau de l’initiation qu’à la terminaison de la cytokinèse. Des défauts ayant lieu lors de cette transition mènent à la formation de cellules binucléées tétraploïdes qui sont observées dans plusieurs pathologies comme le cancer. Afin d’approfondir nos connaissances à ce sujet j’ai utilisé un modèle d’imagerie optique en temps réel dans un modèle cellulaire de Drosophila melanogaster : les cellules S2 de Schneider. Ces études ont mis l’emphase sur un nouveau mécanisme de maturation de la transition AC/AM. Nous avons pu démontrer que la kinase Citron, Sticky, et la septine, Peanut, agissent de manière opposée sur la protéine Anillin pour retenir ou éliminer, respectivement, la membrane plasmique lors de la transition AC/AM. En effet, la diminution d’expression de Sticky par ARNi engendre une perte de contrôle de rétention membranaire de l’AM. À l’inverse, la diminution d’expression de Peanut inhibe la maturation par excrétion membranaire de l’AM. La diminution d’expression simultanée de Sticky et de Peanut conduit l’AC vers des mouvements oscillatoires typiques d’une instabilité de l’AC suite à la perte de fonction de l’Anillin. Sticky est une protéine corticale lors de la cytokinèse dont le rôle et les partenaires d’interaction restent controversés. Pour approfondie nos connaissance de ce sujet, nous avons effectué une étude structurelle et fonctionnelle de Sticky. Cette étude démontre que Sticky possède deux mécanismes de localisation corticale. Le premier dépend de l’Anillin et le deuxième dépend de la petite GTPase Rho1, le régulateur maître de la cytokinèse. Sticky est capable de se localiser à l’AC en présence de l’un ou l’autre de ces deux mécanismes, mais chacun semble être essentiel pour la réussite de la cytokinèse. Le domaine minimal d’interaction entre la Sticky et l’Anillin a été identifié. Une version d’Anillin qui manque le site de liaison à la Sticky est incapable de supporter l’achèvement de la cytokinèse, et les cellules échouent la cytokinèse d’une manière semblable aux cellules dont l’expression de Sticky est diminuée. Similairement, les cellules exprimant une protéine Sticky mutée au site d’interaction avec Rho1-GTP, sont incapables de compléter la cytokinèse lorsque les niveaux endogènes de Sticky sont diminués par ARNi. Ceci suggère que Sticky agit avec Anillin et Rho1 au niveau du cortex pour guider la transition d’un AC dynamique vers un AM stable. Par la mise en évidence et la caractérisation d’un nouveau mécanisme moléculaire essentiel à la cytokinèse, cette thèse constitue des avancements importants au niveau de la cytokinèse. / Cytokinesis is a multistep process that allows two sister cells to undergo complete separation following mitosis. Cytokinesis requires the formation of a contractile ring (CR) that will drive cleavage furrow ingression at the equator of the cell. One of the crucial steps in this process is the transition from a dynamic CR to a more stable structure named the midbody ring (MR), which directs the final separation or abscission. Our knowledge of the molecular mechanisms involved in the CR-to-MR transition would presumably improve our understanding of the molecular complexes involved throughout cytokinesis from initiation to abscission. Defects that occur during this transition can lead to the formation of bi-nucleate tetraploid cells that are often observed in pathological conditions such as cancer. I have used Drosophila melanogaster Schneider’s S2 cells to study the CR-to-MR transition. My findings have highlighted a previously uncharacterized maturation process essential for the transition. More specifically, I demonstrate that the Citron Kinase, Sticky, and the Septin, Peanut, have opposing actions on the scaffold protein Anillin to either retain or extrude, respectively, membrane-positive proteins during the CR-to-MR transition. Indeed, Sticky depletion by RNAi led to uncontrolled loss of membrane-associated Anillin at the MR. Conversely, Peanut depletion led to inhibition of MR maturation by membrane extrusion. Co-depletion of Sticky and Peanut led to oscillatory movements of the CR, typical of Anillin depletion. Sticky is a cortical protein during cytokinesis whose role and interacting partners are controversial. I have performed a structure/function analysis of Sticky to better define its role and regulation during cytokinesis. My work shows that Sticky has two mechanisms of cortical localization. The first is through an Anillin interaction and the second is through the small GTPase Rho1, a master regulator of cytokinesis. Sticky can localize to the cortex in the absence of either one of these mechanisms. However, loss of both inhibits its localization. Following the identification of the minimal interaction sites of Anillin and Sticky, I expressed an Anillin mutant that lacked part of this site and found that cells failed cytokinesis in a similar manner to cells depleted of Sticky. Mutation of the Rho1 binding site on Sticky produced similar cytokinesis failures. Altogether, the results suggest that Sticky interacts with Anillin and Rho1 at the cortex to guide the transition from dynamic CR to stable MR. This thesis advances our understanding of cytokinesis by highlighting a previously uncharacterized process of MR maturation and by defining the importance and regulation of Citron Kinase during this process.

No description available.

The cytoplasm of eukaryotic cells contains several membrane-delimited compartments of specific molecular compositions and functions. Among those, the vacuole of fungal cells is often described as an organelle equivalent to the lysosomes of animal cells and the vacuoles of plant cells. These compartments indeed share two similar features: they contain a wide variety of hydrolases and are the most acidic compartments of the cell, which accounts for their key role in the intracellular degradation of macromolecules. In humans, dysfunctions of the lysosomes often give rise to lysosomal related diseases, such as lysosomal storage disorders. These are a class of metabolic disorders caused by the accumulation of non-degraded macromolecules or impaired export of hydrolytic degradation products. Cystinosis is an autosomal recessive disorder (1/200 000 incidence) generally associated with renal dysfunctions. It is caused by the accumulation and crystallization of cystine, the disulfide of cysteine, into the lumen of lysosomes. Cystinosin, the causative gene product of cystinosis, is present at the lysosomal membrane and catalyses the export of cystine from this compartment. The human cystinosin is a member of the Lysosomal Cystine Transporter (LCT) family. LCT proteins are conserved in all eukaryotic species and are defined by the presence of highly conserved PQ-loop motifs. <p>During this thesis work, we have studied three LCT proteins of the yeast Saccharomyces cerevisiae, named Ypq1, Ypq2 and Ypq3 (Yeast PQ-loop proteins 1, 2 and 3). We first showed that these proteins localize to the vacuolar membrane. We next studied the roles of these proteins, the regulation of their genes and the mechanisms and signals implicated in their delivery to the vacuolar membrane. We also contributed to the functional characterization of a mammalian homologue of yeast Ypq proteins, named rPqlc2. <p>In the first part of this work, we report that the Ypq proteins are most probably implicated in the export of basic amino acids from the vacuole to the cytosol. More precisely, Ypq2 and Ypq3 behave like vacuolar arginine and lysine exporters, respectively. Interestingly, the mammalian rPqlc2 protein expressed in yeast reaches the vacuolar membrane and functions as an orthologue of the Ypq proteins. Our results also reveal that the expression of the YPQ3 gene is regulated by the Lys14 transcription factor, responsible for the transcriptional activation of the LYS genes encoding enzymes implicated in the biosynthesis of lysine. We have also noted that, in general, the expression of the expression of the YPQ genes is regulated according to the quality of the nitrogen source available in the extracellular medium, eg. YPQ3 is sensitive to the nitrogen catabolite repression regulatory mechanism. <p>In the last part of this thesis work, we investigated the intracellular trafficking of the Ypq proteins and show that these predominantly reach the vacuolar membrane via the ALP (alkaline phosphatase) pathway due to the presence of a dileucine-based sorting signal in their sequences. Interestingly, a similar mechanism seems responsible for targeting to the yeast vacuole of the mammalian rPqlc2 protein.<p><p><p>Une caractéristique des cellules eucaryotes est leur organisation en compartiment internes délimité par une membrane lipidique, appelé organelles. Ces compartiments intracellulaires présentent une composition lipidique et protéique particulaire conforme à leur identité et fonction. Les lysosomes de cellules de mammifères et la vacuole fongique jouent un rôle clé dans la digestion intracellulaire de macromolécules et de ce fait leurs lumières sont enrichis d’enzymes hydrolytiques nécessaires à cette action. Des disfonctionnements du lysosome peuvent être la conséquence de pathologie chez l’homme, regroupé sous le nom de maladie lysosomale, lié à un à une accumulation de macromolécules non digéré ou un default d’export des produits d’hydrolysé depuis la lumière du lysosome. La cystinose est une maladie autosomale récessive avec une faible fréquence d’incidence (1/200 000) qui regroupe trois formes cliniques :deux formes rénales graves et une forme extra-rénale. Cette maladie est due à une accumulation et cristallisation de cystine dans la lumière du lysosome qui est corrélé à des mutations ponctuelles dans le gène CTNS qui code pour l’exporteur de cystine, la cystinosine. Cette protéine est un membre de la famille LCT (Lysosomal Cystine Transporter) qui possède des représentants chez les cellules animales, végétales et fongiques. Les protéines de la famille possèdent une taille et une topologie prédite similaire (7 segments transmembranaires) et on retrouve aussi au sein de ces protéines deux exemplaires de motifs PQ. Lors de ce travail de thèse nous nous sommes intéressés à trois membres de la famille LCT chez Saccharomyces cerevisiae que nous avons nommé Ypq1, Ypq2 et Ypq3 pour Yeast PQ-loop proteins. Ces protéines n’ayant pas fait l’objet de nombreuses études, nous nous sommes orientés vers une analyse fonctionnelle et transcriptionnelle. De plus, nous avons également étudié les mécanismes et signaux impliqué dans leur adressage vers la vacuole. Finalement, nous avons également inclus dans notre étude un homologue mammalien de ces protéines, rPqlc2. <p>\ / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Introduction Diabetes constitutes a major health challenge. Since cardiovascular complications are common in diabetic patients this will further increase the overall burden of disease. Furthermore, stress-induced hyperglycemia in non-diabetic patients with acute myocardial infarction is associated with higher inhospital mortality. Hyperglycemia-induced oxidative stress results in DNA damage and subsequent activation of poly-ADP-ribose polymerase (PARP) as a restorative mechanism. However, PARP attenuates glyceraldehyde–3-phosphate dehydrogenase (GAPDH) activity, thereby diverting upstream glycolytic metabolites into damaging non-oxidative glucose pathways (NOGP). For example, hyperglycemia-induced stimulation of four NOGP, i.e. the polyol pathway, hexosamine biosynthetic pathway (HBP), advanced glycation end products (AGE), and PKC activation elicit cardiovascular complications. The current thesis examined the regulation of NOGP in the setting of ischemia and reperfusion under hyperglycemic conditions. Here we hypothesized that administration of two unique therapeutic interventions, i.e. oleanolic acid (OA; clove extract) and benfotiamine (BFT; vitamin B1 derivative), can blunt oxidative stress and NOGP-induced cardiac dysfunction under hyperglycemic conditions following ischemia and reperfusion. Our choice for these agents was based on the principle that OA possesses antioxidant properties; and BFT stimulates transketolase (pentose phosphate pathway [PPP] enzyme) thereby shunting flux away from the NOGP pathways. Additionally, hyperglycemia-induced oxidative stress can also result in dysregulation of the ubiquitin-proteasome system (UPS) that removes misfolded proteins. There are conflicting data whether increased/decreased UPS is detrimental with hyperglycemia and/or in response to ischemia and reperfusion. In light of this, we also hypothesized that BFT and OA act as novel cardio-protective agents by diminishing myocardial UPS activity in response to ischemia and reperfusion under acute hyperglycemic conditions. Materials and Methods For the first part of the study, we employed several experimental systems: 1) H9c2 cardiac myoblasts were exposed to 33 mM glucose for 48 hr vs. controls (5 mM glucose); and subsequently treated with two OA doses (20 and 50 μM) for 6 and 24 hr, respectively; 2) Isolated rat hearts were perfused ex vivo with Krebs-Henseleit buffer containing 33 mM glucose vs. controls (11 mM glucose) for 60 min, followed by 20 min global ischemia and 60 min reperfusion ± OA treatment; 3) Infarct size was determined using Evans Blue dye and 1% 2,3,5-triphenyl tetrazolium chloride (TTC) staining with 20 min regional ischemia and 2 hr reperfusion 4) In vivo coronary ligations were performed on streptozotocin-diabetic rats ± 0.45 mg/kg OA administration within the first two minutes of reperfusion; and 5) Effects of long-term OA treatment (2 weeks) on heart function were assessed in streptozotocin (STZ)-diabetic rats. Here, STZ was dissolved in citrate buffer (p.H 6.3) and diabetes was induced by administering 60 mg/kg i.p Tissues were collected at the end of the global ischemia experiments and analyzed for oxidative stress, apoptosis, UPS activity and HBP activation. For the second part of the study we employed several experimental systems: 1) Isolated rat hearts were perfused ex vivo with Krebs-Henseleit buffer containing 33 mM glucose vs. controls (11 mM glucose) for 90 min, followed by 30 min global ischemia and 60 min reperfusion ± 25, 50 and 100 μM BFT treatment, respectively, added during the first 20 min of reperfusion; 2) Infarct size determination as in #3 above but with 30 min regional ischemia and 2 hr reperfusion ± 100 μM BFT treatment; and 3) In vivo coronary ligations performed on streptozotocin-diabetic rats ± 0.50 mg/kg BFT treatment within the first two min of reperfusion. In parallel experiments, NOGP inhibitors were added during the first 20 min of reperfusion. The following inhibitors were individually employed: AGE pathway (100 μM aminoguanidine); PKC (5 μM chelerythrine chloride); HBP (40 μM 6-diazo-5-oxo-L-norleucine); and polyol pathway (1 μM zopolrestat); Infarct size determination as in #2) with 30 min regional ischemia and 120 min reperfusion ± similar treatments. Results Our data show decreased cardiac contractile function in response to ischemia and reperfusion under hyperglycemic conditions. This was linked to increased PARP and attenuated GAPDH activities, together with higher activation of the NOGP. Moreover, we found elevated myocardial oxidative stress, UPS and cell death under these conditions. OA treatment resulted in cardio-protection, i.e. for ex vivo and in vivo rat hearts exposed to ischemia and reperfusion under hyperglycemic conditions. In parallel, OA decreased oxidative stress, apoptosis, HBP flux and UPS activity following ischemia and reperfusion. Long-term OA treatment also improved heart function in streptozotocin-diabetic rats. Our data also reveal that acute BFT treatment significantly decreased myocardial oxidative stress and apoptosis, and provided cardio-protection in response to ischemia and reperfusion under hyperglycemic conditions. In parallel, BFT blunted hyperglycemia-induced activation of four NOGP in the rat heart. Acute administration of each of the NOGP inhibitors decreased PARP and enhanced GAPDH activities, while diminishing oxidative stress and myocardial apoptosis. Moreover, each of the NOGP inhibitors (individually) employed blunted activation of the other three pathways here examined. Hearts treated with NOGP inhibitors also displayed improved functional recovery and smaller infarct sizes following ischemia and reperfusion. Interestingly, NOGP inhibitors resulted in the same degree of change (for all above-mentioned parameters evaluated) when compared to each other. Conclusions This study shows that acute and chronic hyperglycemia trigger myocardial oxidative stress that eventually results in NOGP activation and contractile dysfunction following ischemia and reperfusion. Moreover, our findings establish - for the first time as far as we are aware - that there is a convergence of downstream NOGP effects in our model, i.e. increased myocardial oxidative stress, further NOGP pathway activation, apoptosis, and impaired contractile function. Thus a vicious metabolic cycle is established whereby hyperglycemia-induced NOGP further fuels its own activation by generating even more oxidative stress, thereby exacerbating damaging effects on the heart under these conditions. We also found that both OA and BFT treatment blunted high glucose-induced detrimental effects and provided robust cardio-protection in response to ischemia and reperfusion under hyperglycemic conditions (acute and chronic). These findings suggest that the UPS may be a unique therapeutic target to treat ischemic heart disease in individuals that present with stress-induced, acute hyperglycemia. Moreover, BFT exhibited its cardio-protective effects by NOGP inhibition after ischemia and reperfusion under acute and chronic high glucose conditions. A similar effect was observed at baseline although the underlying mechanisms driving this process still need to be elucidated. In summary, the findings of this thesis are highly promising since it may eventually result in novel, cost-effective therapeutic interventions to treat acute hyperglycemia (in non-diabetic patients) and diabetic patients with associated cardiovascular complications. / AFRIKAANSE OPSOMMING: Inleiding Diabetes skep ‘n groot gesondheidsuitdaging. Omrede kardiovaskulêre komplikaseis algemeen onder diabetiese pasiënte is, sal dit oorkoepelend die las van hierdie siekte verder laat toeneem. Verder word stresgeïnduseerde hiperglukemie in nie-diabetiese pasiënte met akute miokardiale infarksie geassosieër met ‘n hoër binne-hospitaalmortaliteit. Hiperglukemies-geïnduseerde oksidatiewe stres veroorsaak DNA skade, en gevolglike aktivering van poli-ADF-ribose polimerase (PARP), as ‘n herstelmeganisme. Nietemin, PARP verminder gliseraldehied–3-fosfaatdehidrogenase (GAPDH) aktiwiteit om sodoende die opstroom glikolitiese metaboliete te herlei na skadelike nie-oksidatiewe glukose weë (NOGW). Byvoorbeeld, hiperglukemie-geïnduseerde stimulasie van vier NOGW, i.e. die poliolweg, heksosamienbiosintetiese weg, (HBW), gevorderde glukasie eindprodukte (GGE), en PKC aktivering, lei tot kardiovaskulêre komplikasies. Die huidige tesis ondersoek die regulering van NOGW in ‘n isgemiese-reperfussie onder hiperglukemiese toestande. Ons hipotetiseer dat die toediening van twee unieke terapeutises intervensies, i.e. oleanoliese suur (OS, naaltjie ekstrak), en benfotiamien (BFT, vitamien B1 derivaat) oksidatiewe stress kan versag, en NOGW geinduseerde kardiale disfunksie onder hiperglukemiese toestande na ischemie en reperfussie. Ons keuse vir hierdie middels is gebaseer op die beginsel dat OS antioksidanteienskappe bevat, en dat BFT transketolase (pentosefosfaat weg (PFW) ensiem) stimuleer en sodoende die fluks weg van die NOGW weg veroorsaak. Addisioneel kan hiperglukemiegeïnduseerde oksidatiewe stres ook tot wanregulering van die ubikwitien-proteosoomsisteem (UPS) wat wangevoude protïene verwyder, aanleiding gee. Daar bestaan kontrasterende data oor ‘n verhoogde/verlaagde UPS, tesame met hiperglukemie en/of in reaksie tot isgemie-reperfussie. In die lig hiervan, hipotetiseer ons dat BFT en OS as ‘n nuwe kardiobeskermingsmiddel kan optree deur miokardiale oksidatiewe stres en UPS aktiwiteit in reaksie op isgemie-reperfussie tydens akute hiperglukemiese toestande kan verlaag. Materiale en Metodes Vir die eerste deel van die studie het ons van verskeie eksperimentele sisteme gebruik gemaak: 1) H9c2 kardiale mioblaste is aan 33 mM glukose vir 48 uur vs. kontrole (5 mM glukose) blootgestel; en gevolglik met twee OS dosisse (20 en 50 μM) vir 6 en 24 hr, onderskeidelik behandel; 2) geïsoleerde rotharte is ex vivo met Krebs-Henseleit buffer, wat, 33 mM glukose vs. kontrole (11 mM glukose) bevat, vir 60 min geperfuseer, daarna is dit deur 20 min globale isgemie gevolg en 60 min reperfussie ± OS behandeling; 3) Infarkgrootte is bepaal deur Evans bou kleursel en 1% 2. 3-5 tripfeniel tetrazoloimcholierd (TTC) kleuring met 20 minute regionale ischemie, en 2 uur reprefussie 4) In vivo koronêre liggasies is op streptozotosien-diabetiese rotte uitgevoer ± 0.45 mg/kg OS toedienning binne die eerste twee minute van reperfussie; en 5) effekte van langtermyn OS behandeling (2 weke) op hartfunskie is in hierdie streptozotosien-diabetiese rotte ondersoek. Hier is STZ opgelos in ‘n sitraatbuffer (pH 6.3), en diabetes is geinduseer deur 60mg/kg i.p. toe te dien. Weefsels is aan die einde van die globale isgemie eksperimente versamel, en vir oksidatewe stres, apoptose, UPS aktiwiteit en HBW aktivering, ontleed. Vir die tweede deel van die studie het ons van verskeie eksperimentele sisteme gebruik gemaak: 1) geïsoleerde rotharte is ex vivo met Krebs-Henseleit buffer, wat 33 mM glukose vs. kontrole (11 mM glukose) bevat, vir 90 min geperfuseer. Daarna is dit gevolg met 30 min globale isgemie en 60 min reperfussie ± 25, 50 en 100 μM BFT behandeling onderskeidelik, gevolg, bykomend, gedurende die eerste 20 min reperfussie; 2) Infarkgrootte is bepaal soos in #3 hierbo, maar met 30 minute regionale ischemie en 2 uur reperfussie ± 100 μM BFT behandeling; en 3) In vivo koronêre liggasies is op streptozotosien-diabetiese rotte uitgevoer ± 0.50 mg/kg BFT behandeling binne die eerste twee minute van reperfussie. Met parallele eksperimente is NOGW inhibeerders bygevoeg binne die eerste 20 min van reperfussie. Die volgende inhibeerders is individueel ontplooi: GGE weg (100 μM aminoguanidien); PKC (5 μM chelleritrienchloried); HBW (40 μM 6-diazo-5-oxo-L-nor-leusien); en poliolweg (1 μM zopolrestaat); 2) Infarkgrootte is bepaal soos in #2) met die uitsondering van 30 min regionale isgemie en 120 min reperfussie ± identiese behandelings. Resultate Ons data toon aan dat kardiale kontraktiele funksie, in reaksie op isgemie-reperfussie onder hiperglukemiese toestande, verlaag. Dit is verwant aan verhoogde PARP en verminderde GAPDH aktiwiteit, tesame met ‘n hoër aktivering van die NOGW. Verder het ons bevind dat verhoogde miokardiale oksidatiewe stres, UPS en seldood onder die toestande voorkom. OS behandeling lei tot kardiale beskerming, i.e. vir ex vivo en in vivo rotharte wat aan isgemie-reperfussie onder hiperglukemiese toestande blootgestel is. Parallel hiermee het OS oksidatiewe stres, apoptose, HBW invloed, en UPS aktiwiteit na isgemie-reperfussie, verlaag. Langtermyn OS behandeling het ook hartfunksie in streptozotosien-diabetiese rotte verbeter. Ons data vertoon verder dat akute BFT behandeling, miokardiale oksidatiewe stres en apoptose, betekenisvol verlaag het in reaksie op isgemie-reperfussie onder hiperglukemiese toestande. Parallel hiermee het BFT hiperglukemiegeïnduseerde aktivering van vier NOGWë in die rothart, verminder. Akute toediening van die elk van die NOGW inhibeerders het PARP verlaag, en GAPDH aktiwiteite verhoog, terwyl oksidatiewe stres, en miokardiale apoptose verminder. Verder het elk van die NOGW inhibeerders wat (individueel) toegedien is, aktivering van die ander drie weë, hier ondersoek, verlaag. Die harte wat met NOGW inhibeerders behandel is het ook ‘n verbeterde herstel en kleiner infarkgrootte na isgemie-reperfussie getoon. Interessant is hoe die NOGW inhibeerders tot dieselfde graad verandering (vir al die bogemelde parameters wat geevalueer is) indien dit vergelyk word teen mekaar, gelei het. Gevolgtrekking Hierdie studie het bevind dat akute en chroniese hiperglukemie, miokardiale oksidatiewe stres ontlok, en dat dit geleidelik tot NOGW aktivering en kontraktiele wanfunksionering na isgemie-reperfussie lei. Verder het ons bevindinge vir die eerste keer, volgens ons wete, bewys dat daar ‘n ineenloping is van afstroom NOGW effekte in ons model, i.e. verhoogde miokardiale oksidatiewe stres, verdere NOGW weg aktivering, apoptose, en ingeperkte kontraktiele funksie. Dus, ‘n gebrekkige metaboliese siklus word verkry waarby hiperglukemies-geïnduseerde NOGW verder sy eie aktivering aanvuur deur meer oksidatiewe stres, en sodoende die skadelike effekte op die hart onder hierdie toestande verder versleg. Ons het verder bevind dat beide OS en BFT behandeling, hoë glukose-geïnduseerde skadelike effekte onderdruk, en kragtige kardiale-beskerming in reaksie op isgemie-reperfussie onder hiperglukemiese toestande (akuut en chronies), teweeg bring. Hierdie bevindinge dui moontlik daarop dat die UPS ‘n unieke terapeutiese teiken kan wees vir die behandeling van isgemiese hartsiekte in individue wat presenteer met stres-geïnduseerde, akute hiperglukemie. BFT het ook sy kardiale beskermende effekte getoon deur NOGW inhibering na isgemie-geïnduseerde reperfussie onder aktute en chroniese hoë glukose toestande. ‘n Soorgelyke effek is tydens die basislyn waargeneem, alhoewel die onderliggende meganisme wat hierdie proses dryf verder ondersoek moet word. Opsommend is ons bevindinge baie belowend omrede dit daartoe kan aanleiding gee tot ‘n nuwe, meer koste-effektiewe terapeutiese intervensie vir die behandeling van akute hiperglukemie (in niediabetiese pasiënte) en diabetiese pasiënte met geassosieërde kardiovaskulêre komplikasies. / Oppenheimer, Beit Trust and Harry Crossley

Angiotensin Converting Enzyme (ACE) IID, and a actinin 3 (AC1N3) R577X polymorphisms have been linked to the strength and power performance of elite athletes and suggested to influence skeletal muscle function in the general popUlation. This research investigated the association of these two candidate gene polymorphisms with the muscle function of young and older men, and the distribution of these genotypes in frequent fallers compared to controls. Muscle function measurements of young and older men included isometric strength, absolute and relative isokinetic strength at high velocity (ratio of torque at 2400 ·s"; torque at 30°·s") and the time course of an evoked twitch. Additionally body composition was measured by skinfold thickness (young men) and DXA scanning (old men) to estimate fat-free mass, an index of muscularity, and fat mass. ACE and AC1N3 genotypes were determined from whole blood samples using polymerase chain reaction, and serum ACE activity using spectrophotometry. The gemtypes of frequent fallers referred to a Falls Clinic were compared to a control group of healthy men. ACE genotype was not associated with any measure of muscle function, including the time course of an evoked twitch or absolute and relative high velocity torque, or body composition in these populations (ANOVA, 0.12<P<0.97). Serum ACE activity appeared to be weakly associated with knee extensor (R = 0.19, P = 0.07) and elbow flexor (R = 0.20, P = 0.06) isometric strength in older men, and was negatively correlated with the relative torque at high velocity (R = -0.23, P = 0.03). AC1N3 genotype was associated with fat mass in older men (P = 0.04), but was not associated with any measure of muscle function or muscularity (KruskalWaIIis, 0.26<P<0.95). Finally there was no apparent difference in the distribution of ACE IID (r: = 0.54, P = 0.77) and AC1N3 RIX (r: = 0.76, P = 0.68) genotypes between frequent fallers and controls. Any influence of these individual polymorphisms seems unlikely to be of sufficient magnitude to produce genotype related differences in muscle function in young or older free living UK Caucasian men. Serum ACE activity may have a small association with the isometric and dynamic strength of older men. However, AC1N3 genotype was associated with increased fat mass in XX individuals, that suggests this polymorphism may have an association with the accumulation of body fat over the life span of older men.

La stabilité structurale et l'intégrité mécanique sont des éléments clés pour le bon fonctionnement et la préservation des systèmes vivants complexes. Étant en interaction constante avec leur environnement et en ce qui concerne les intrants externes, de tels systèmes doivent pouvoir faire face aux changements afin de prospérer. Ces entrées peuvent affecter le système dans son ensemble. Toute perturbation qui ne peut pas être supportée mécaniquement par le système vivant entraînera un dysfonctionnement crucial ou, en fin de compte, sa mort. Le mécanisme responsable du maintien des conditions physiologiques du système à l'état correct, malgré les variations environnementales, est identifié comme étant l'homéostasie. Plus précisément, le processus connu en mécanobiologie pour préserver l'équilibre mécanique approprié d'un système vivant est appelé homéostasie tensionnelle.Il est important de noter que tout ce qui précède est vrai à la fois à l'échelle du comportement collectif des organismes complexes et jusqu'au niveau de la cellule unique. En fait, c'est en fait cette dernière petite échelle qui nous intéresse. Les cellules font face à des perturbations mécaniques constantes de leur environnement et sont capables de répondre au maintien d'un état mécanique interne relativement stable. L'existence de cet équilibre tensionnel interne est liée à un processus très dynamique avec des boucles de rétroaction constantes entre les machines contractiles biochimiques internes et les forces actives externes générées.Notre intérêt est de comprendre ce mécanisme dynamique en perturbant dynamiquement le système homéostatique tensionnel en étudiant son retour à l'équilibre. / The structural stability and mechanical integrity are key elements for the proper functioning and preservation of complex living systems. Being in constant interaction with their surroundings and subjected to external inputs, such systems need to be able to face changes in order to thrive. These inputs can affect the system both in a localized way or disturb it as a whole. Any perturbations that cannot be mechanically withstand by the living system will result in a crucial malfunctioning or, ultimately, in its death. The mechanism responsible for maintaining the system’s physiological conditions at the proper state, despite environmental variations, is identified as homeostasis. More specifically, the process known in mechanobiology to preserve the appropriate mechanical equilibrium of a living system is called tensional homeostasis.It is important to note that all of the above stated holds true both at the scale of collective behaviour of complex organisms, and all the way down to the single cell level. In fact, it is actually this last small scale which draws our interest. Cells face constant mechanical perturbations from their surrounding and are able to respond accordingly maintaining a relatively stable internal mechanical state. The existence of this internal tensional equilibrium relies on a very dynamic process with constant feedback loops between the internal biochemical contractile machinery and the external active generated forces.Our interest is to understand better this active mechanism by dynamically perturbing the tensional homeostatic system while studying its return to equilibrium.

The primary purpose of this thesis was to assess the effect of knee-joint angle on the neuromechanics of maximal and explosive contractions, specifically torque and neuromuscular activation, as well as the influence of isometric resistance training (RT) on these variables and thus joint angle specificity of training adaptations. It was found that electrode location had a pronounced effect on surface electromyography (sEMG) amplitude during maximum isometric voluntary contractions (MVCs) and moderate relationship between subcutaneous tissue thickness and sEMG amplitude (R2=0.31 up to 0.38) was reduced but not consistently removed by maximal M-Wave (MMAX) normalization [up to R2= 0.16 (peak-to-peak) and R2= 0.23 (Area)]. Thus, MMAX peak-to-peak was the better normalization parameter that removed the influence of electrode location and substantially reduced the influence of subcutaneous tissue thickness. Maximal torque-angle relationship presented an inverted U shape with both, agonist (measure by two different techniques) and antagonist neuromuscular activation both differing with knee-joint angle and thus, both likely contributing to the torque-angle relationship. Absolute explosive torque-angle relationship exhibited higher torques at mid-range knee joint angles in a similar manner to maximal strength, whilst the ability to explosively express the available torque (i.e. relative to maximal strength) revealed only subtle differences between joint angles. Agonist neuromuscular activation showed increases from extended to flexed positions during both maximum and explosive contractions (at all time points; ~6% to ~34%) and evoked contractile properties presented opposite patterns with twitch torque increasing (~5% to ~30%) and octet torque decreasing (~2% to ~14%) with knee flexion. Finally, after 4 weeks of RT at a 65° knee-joint angle evidence of joint angle specificity was provided from both within-group (greater gains at 3 angles than others) and between-group evidence (greater gains at 2 angles than others) for maximal strength but not for explosive strength and neuromuscular activation. In summary, this thesis demonstrated: (1) higher strength values at middle knee-joint positions than more flexed and/or extended positions during maximal and explosive contractions; (2) how agonist neuromuscular activation contributes to the beforementioned changes in strength; (3) how muscle contractile properties contribute to the explosive strength across knee-joint angles; and finally (4) that joint angle specificity has a neural basis.

A previous study in chickens revealed that myosin light chain kinase (MLCK), f actin, and myosin are found on the crystalline lens. Their polygonal arrangement at the posterior surface resembles a muscle tissue, which suggests that these proteins may have a contractile role in accommodation. The ciliary muscle in chickens is skeletal in nature and, therefore, chickens were used to test the hypothesis that contractile microfilaments play a role in accommodation. Ciliary nerve-induced accommodation was measured in the presence of an MLCK inhibitor 1-(5-Iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7). Eyes of 6-day old white Leghorn chickens (gallus gallus domesticus) were enucleated in Tyrode’s saline solution while keeping the ciliary nerve intact. One eye was treated with ML-7 and the other eye was treated with vehicle only. Three concentrations of ML-7 were used: 1 µM, 10 µM, and 100 µM. Two experiments were carried out, one including a (3×10 min) wash and one without. Focal lengths of the vehicle- and ML-7-treated eyes were measured before, during and after accommodation. Immunoblots were used to detect the amount of phosphorylated myosin with and without the inhibitor. Focal lengths for accommodation were shorter than those at rest (p<0.001). In the wash experiment, vehicle-treated eyes had higher accommodative amplitudes compared to ML-7-treated eyes for all three dosage groups. In the no-wash experiment, only the 1 µM group demonstrated the same trend as the wash experiment. For the 10 µM and 100 µM groups, ML-7-treated eyes had higher accommodative amplitudes compared to vehicle-treated eyes. Immunoblots revealed varying amounts of inhibition within pairs of eyes as well as between birds for both experiments. Results from this experiment indicate that ML-7 was not effective at determining whether contractile microfilaments found on the lens contribute to accommodation.

A previous study in chickens revealed that myosin light chain kinase (MLCK), f actin, and myosin are found on the crystalline lens. Their polygonal arrangement at the posterior surface resembles a muscle tissue, which suggests that these proteins may have a contractile role in accommodation. The ciliary muscle in chickens is skeletal in nature and, therefore, chickens were used to test the hypothesis that contractile microfilaments play a role in accommodation. Ciliary nerve-induced accommodation was measured in the presence of an MLCK inhibitor 1-(5-Iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7). Eyes of 6-day old white Leghorn chickens (gallus gallus domesticus) were enucleated in Tyrode’s saline solution while keeping the ciliary nerve intact. One eye was treated with ML-7 and the other eye was treated with vehicle only. Three concentrations of ML-7 were used: 1 µM, 10 µM, and 100 µM. Two experiments were carried out, one including a (3×10 min) wash and one without. Focal lengths of the vehicle- and ML-7-treated eyes were measured before, during and after accommodation. Immunoblots were used to detect the amount of phosphorylated myosin with and without the inhibitor. Focal lengths for accommodation were shorter than those at rest (p<0.001). In the wash experiment, vehicle-treated eyes had higher accommodative amplitudes compared to ML-7-treated eyes for all three dosage groups. In the no-wash experiment, only the 1 µM group demonstrated the same trend as the wash experiment. For the 10 µM and 100 µM groups, ML-7-treated eyes had higher accommodative amplitudes compared to vehicle-treated eyes. Immunoblots revealed varying amounts of inhibition within pairs of eyes as well as between birds for both experiments. Results from this experiment indicate that ML-7 was not effective at determining whether contractile microfilaments found on the lens contribute to accommodation.

<p>Transplantation of a functional engineered skeletal muscle substitute is a promising therapeutic option to repair irreversible muscle damage, and, on the other hand, functional muscle tissue constructs can serve as in vitro 3D tissue models that complement the conventional 2D cell cultures and animal models to advance our limited understanding of intrinsic myogenesis and muscle regeneration process. However, the engineering of skeletal muscle constructs with comparable contractile function to the native muscle is hampered by the lack of 1) effective and reproducible methods to form relatively large muscle constructs composed of viable, dense, aligned and matured myofibers, and 2) beneficial microenvironmental cues as well as physiological stimulations that favor the growth, differentiation and maturation of myogenic cells. Thus, in this thesis, I have developed a mesoscopic hydrogel molding approach to fabricate relatively large engineered muscle tissue networks with controllable thickness, pore dimensions, overall myofiber alignment and regional myofiber orientation. I then investigated the effect of variation in pore length on the force generation and passive properties of engineered muscle networks and the potential to improve the contractile function of engineered muscle networks with the treatment of a soluble neurotrophic factor, agrin.</p><p>Specifically, high aspect-ratio soft lithography was utilized to precisely fabricate elastomeric molds containing an array of staggered hexagonal posts which created elliptical pores in muscle tissue sheets made from a mixture of primary skeletal myoblasts, fibrin and Matrigel. The improved oxygen and nutrient access through the pores increased the viability of the embedded muscle cells and prevented the formation of an acellular core. The differentiated myofibers were locally aligned in tissue bundles surrounding the elliptical pores. The length and direction of the microfabricate posts arbitrarily determined the length of elliptical pores and the mean orientation of myofibers formed around the pores, which enables the control of pore dimensions and regional myofiber orientation. Contractile force analysis revealed that engineered muscle networks with more elongated pores generated larger contractile force due to the increased myonuclear density and degree of overall myofiber alignment, despite the larger porosity and reduced tissue volume. Furthermore, the introduction of elliptical pores resulted in distinct deformational changes in tissue bundles and node regions that connect the ends of bundles with the applied unaxial macroscopic stretch, but such spatial alteration of local strain field resulted in no significant change in macroscopic length- tension relationships among engineered muscle networks with different pore length. </p><p>In addition, supplementing culture medium with soluble recombinant agrin significantly increased contractile force production of engineered muscle networks in the absence of nerve-muscle interaction, primarily or partially due to the agrin-induced upregulation of dystrophin. As expected, alteration in the levels endogenous ACh or ACh-like compound affected the agrin-induced AChR aggregation. Furthermore, increased autocrine AChR stimulation, a novel mechanism underlying survival and maturation of aneural myotubes, attenuated the agrin-induced force increase, while suppressed autocrine AChR stimulation severely comproised the overall force production of engineered muscle networks, of which the underlying mechanisms remains to be elucidated in the future studies. </p><p>In summary, a novel tissue engineering methodology that enables the fabrication of relative large muscle tissue constructs with controllable structure and function has been developed in this thesis. Future studies, such as optimizing cell-matrix interaction, incorporating beneficial regulatory proteins in the fibrin-based matrix, and applying specific patterns of electro-mechanical stimulations are expected to further augment the contractile function of engineered muscle networks. The potential application of this versatile tissue fabrication approach to engineer different types of soft tissue might further advance the development of tissue regeneration therapies as well as deepen our understanding of intrinsic tissue morphogenesis and regeneration process.</p> / Dissertation