5 SUMMARY Context: Acute methanol poisoning is a life-threatening condition. Methanol is metabolized in the organism to formaldehyde and than to formic acid, which inhibits cytochrome c oxidase in mitochondria and thus contributes to the development of oxidative stress. Aim: To study the role of oxidative stress in the pathogenesis of acute neuronal damage to the central nervous system (CNS), in the development of long-term sequelae of methanol poisoning and chronic neurodegenerative processes in the years following acute methanol exposure. Material and Methods: Methanol intoxication was confirmed analytically in 55 patients included in he d ; hei age a he ime f i ning a 46.7 3.6 ea (9 female and 46 male ). All a ien , together with 41 control subjects, were examined in a prospective longitudinal cohort study. At admission, during hospitalisation, and at regular intervals after discharge during the follow-up, the patients were sampled for serum concentrations of lipid oxidative damage markers 4-hydroxy-trans-2- hexenal (HHE), 4-hydroxynonenal (HNE), malondialdehyde (MDA), and 8-isoprostane, for nucleic acids oxidative damage markers 8-hydroxy-2 -deoxyguanosine (8-OHdG), 8-hydroxyguanosine (8- OHG), 5- (hydroxymethyl) uracil (5-OHMU), for proteins oxidative damage markers ortho-tyrosine (o- Tyr),...

No / Potential protective effects of the flavonoids quercetin and luteolin have been examined against the oxidative stress of MC3T3-E1 osteoblast-like cells. Although hydrogen peroxide and menadione reduced cell viability, the toxicity was prevented by desferrioxamine or catalase but not superoxide dismutase, suggesting the involvement of hydrogen peroxide in both cases. Quercetin and luteolin reduced the oxidative damage, especially that caused by hydrogen peroxide. When cultures were pre-incubated with quercetin or luteolin, protection was reduced or lost. Protection was also reduced when a 24 h pre-incubation with the flavonoids was followed by exposure to menadione alone. Pretreating cultures with luteolin impaired protection by quercetin, whereas quercetin pretreatment did not affect protection by luteolin. It is concluded that quercetin and luteolin suppress oxidative damage to MC3T3-E1 cells, especially caused by peroxide. The reduction in protection by pretreatment implies a down-regulation of part of the toxic transduction pathway.

Doctor of Philosophy / Department of Biochemistry / Dolores J. Takemoto / Exposure to oxidative stress leads to accumulation of reactive oxygen species and this stimulates protective cellular functions as a compensatory response to prevent the spread of apoptotic signal and prevent cell death. The purpose of this dissertation is to understand the importance of PKCγ activation and regulation of the retinal gap junction protein Cx50, and what role PKCγ plays in this neuro-protective effect. Through electron microscopy we were able to show that PKCγ knockout mice retinas had incomplete cellular organization in the outer plexiform layer (OPL) of the retina, the layer of retina where Cx50 plays an important role in retinal cellular synapses. Electroretinograms confirmed that this structural disorganization also led to loss of functional response to light stimuli in PKCγ knockout mice retinas. In vivo exposure to 100% hyperbaric oxygen (HBO) caused significant degradation of the retina in knockout mice compared to control mice. Thicknesses of the inner and nuclear and ganglion cell layers were increased, with complete disruption of OPL in PKCγ KO mice retinas. Damage to the outer segments of the photoreceptor layer and ganglion cell layer was significantly more apparent in the central retinas of HBO-treated knockout mice. Cx50 immunolabeling showed significant reduction to HBO treatment of PKCγ control mice retinas, HBO treatment failed to produce reduction of Cx50 immunolabeling in KO mice retinas. In the R28 retinal cell line, PKCγ enzyme was shown to be activated by phorbol ester (TPA) and hydrogen peroxide. This resulted in translocation to the cellular membrane as confirmed by western blot and confocal microscopy. Suppression of PKCγ by siRNA rendered R28 cells more sensitive to oxidative stress-induced cell apoptosis, the process of apoptosis started earlier, and this resulted in cell death. R28 treatment with phorbol esters and hydrogen peroxide led to reduction in gap junction activity and Cx50 gap junction cell disassembly. This dissertation shows that PKCγ plays an important role in structural organization of retina and has a neuro-protective effect in response to oxidative stress, in part because of its control of Cx50.

Oxidative stress is a pathophysiological condition defined by an increased production of reactive oxygen species (ROS), which can result in the growth arrest of cells followed by cell disintegration or necrosis. A number of small molecule antioxidants (e.g. curcumin, quercetin and resveratrol) are capable of directly scavenging ROS, thereby short-circuiting the self-propagating oxidative stress state. However, poor solubility and rapid 1st pass metabolism results in overall low bioavailability and acts as a barrier for its use as a drug to suppress oxidative stress efficiently. To overcome this limitation, these small molecule antioxidants were covalently conjugated into poly(β-amino ester) (PβAE) cross-linked networks to formulate prodrug gel microparticles and nanoparticles (nanogels). Being hydrolytically degradable in nature, these PβAE crosslinked systems released antioxidants in their original structural form in a sustained controlled fashion. Both quercetin and curcumin-PβAE nanogels showed prolonged suppression of cellular oxidative stress induced by H2O2. Curcumin PβAE nanogels also demonstrated protection against mitochondrial oxidative stress induced by H2O2 and polychlorinated biphenyls. Curcumin-PβAE gel microparticles were also developed as a platform to treat oral mucositis through a local antioxidant delivery route. The same synthesis chemistry was transferred to formulate resveratrol PβAE gel microparticles for topical applications, to treat UV radiation induced oxidative stress. Both formulations showed suppression of induced oxidative stress. An in vivo trial with curcumin-PβAE microparticles further showed relatively reduced the severity of induced oral mucositis (OM) in hamster check pouch as compared to placebo.

Nitric oxide (NO), a vasoprotective and ubiquitous signaling molecule generated from the endothelial cells (EC) by the enzyme endothelial nitric oxide synthase (eNOS) have a vital role in regulation of vascular function and integrity. However, a significant attenuation of eNOS and NO leads to endothelial dysfunction (ED) and increased risk of cardiovascular disease (CVD) in diabetes. Lipoproteins particularly LDL, undergo glycation in diabetic patients and turns it into pro-atherogenic glycated LDL (glyLDL). However, the impact of glyLDL on eNOS, the transmembrane signalling events, involvement of mitochondrial and endoplasmic reticulum (ER) stress in EC remains unclear. Also, literatures reveal impaired platelet mitochondrial function in diabetes patients; however, the impact of family history of diabetes on platelet mitochondrial bioenergetics still remains unknown. In the present study, we had provided the evidence for diabetes-associated metabolic stress involving glyLDL can attenuate eNOS protein, gene and activity in EC, as well as glyLDL and high glucose attenuates eNOS content in EC. Receptor of advanced glycation end products (RAGE) and H-Ras pathway are implicated in the upstream signalling events in the downregulation of eNOS in EC. In addition, ER stress, impaired mitochondrial function due to significant reduction of complex-specific oxygen consumption and bioenergetics were identified in glyLDL-treated EC. Further, we have also detected significant impairment in platelet mitochondrial bioenergetics in healthy individuals with familial history of diabetes. Identifying the mechanisms involved in diabetes associated metabolic stress induced signaling in EC and early detection of mitochondrial impairment in healthy individuals will help to find new targets for the prevention and treatment of diabetic cardiovascular complications and improve quality of life in diabetic patients. / May 2015

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

The rise of nanotechnology industries has led to the design and production of new nano-scaled materials such as quantum dots, nano-metals, carbon nanotubes, fullerenes and a myriad of functionalised derivatives. Extensive work concerning well characterised pathogenic fibres has led to the development of a fibre paradigm that suggests respirable fibres vary in their ability to cause disease based on length and pulmonary bio-persistence. Induction of oxidative stress is also a central plank of the mechanism used to explain inflammatory, fibrotic and carcinogenic effects of fibres. The toxicity of different particle types has consistently been shown to depend upon particle size and surface area, reactive surface molecular groups, metal content, organic content and the presence of endotoxins. A growing body of work has begun to examine the potential pathogenicity of carbon nanotubes to the pulmonary system as a consequence of superficial similarities to known pathogenic particle and fibres. The aim of this thesis was to investigate the potential toxicity of two commercially manufactured multi-walled carbon nanotubes (MWCNT) compared to a panel of low and high toxicity particles and fibres. The pro-inflammatory nature of MWCNT was examined in vitro and in vivo to determine the effects they may exert in the pulmonary system. In aqueous solutions of phosphate buffered saline, saline and cell culture medium (with or without foetal calf serum supplementation) MWCNT were found to exist as tight aggregates even after sonication. Analysis of metal content of MWCNT by ICP-AES revealed the presence of a low percentage of non extractable residual iron. From analysis of MWCNT by electron spin resonance (ESR) the CNT were found to be ready producers of a free radical species, despite this MWCNT were not able to cleave plasmid DNA. Upon incubation with the alveolar epithelial cell line A549 MWCNTs did not cause noticeable toxicity but did dose dependently deplete total glutathione levels. No increase in production of the pro-inflammatory cytokine IL-8 could be detected at the level of protein or at the level of mRNA. Analysis of the levels (protein and mRNA) of the pro-fibrotic mediator TGF-β did not indicate induction of a fibrotic response to MWCNT. Neither were MWCNTs found to consistently activate the pro-inflammatory associated transcription factor nuclear factor kappa B (NF-κB). Upon instillation into the peritoneal cavity of mice MWCNT failed to induce a pro-inflammatory response in contrast to long amosite asbestos that induced an extensive inflammatory reaction. Analysis of the diaphragms of exposed animals revealed the induction by MWCNT of an apparent foreign body type reaction. Overall with limited processing and dispersion MWCNT were morphologically more akin to particles than fibres. Although apparently able to spontaneously generate ROS in aqueous solution this did not translate into a capacity to cause toxicity or a capacity to induce inflammation either in vitro or in vivo.

Background: Physically injured elder adults present challenges in the emergent injury phase. Oxidative stress contributes to cellular deterioration, resulting in decreases in physiological reserve. Imbalance of oxidative stress pathways lead to damage and drive the aging process and frailty. Goals of this study were to determine if a new plasma biomarker of oxidative stress is related to: 1) oxidation reduction status in patients who have experienced traumatic injury as well as healthy community dwellers, 2) outcomes of patients who have experienced trauma, 3) frailty measured by established frailty scales in healthy community dwellers. Methods: Prospective study included 1) trauma patients ≥65 admitted to Level I trauma center 2) age, gender matched healthy, community-dwelling participants. Plasma samples tested in duplicate for capacity oxidative reductive potential (cORP, μC; antioxidant reserve), and static oxidative reductive potential (sORP, mV; the current state of oxidative stress). Frailty assessments were performed in healthy participants using established frailty scales. ORP measurements were analyzed using correlation analyses. Univariate analysis analyzed cORP and sORP for differences by the variables gender, age, smoking, diabetes, statin use, vitamin use and any alcohol use in both the injured and healthy populations. Results: 186 subjects included in study (N=93 for both groups). Trauma groups's cORP values were significantly lower in patients with diabetes (p<0.05) and patients that smoked (p<0.01). Similarly the healthy group's cORP was significantly lower for those who smoked and those with diabetes (p<0.05). Non-vitamin use in the healthy group was related to lower cORP values (p<0.05). Trauma patients who smoked and those with diabetes exhibited higher sORP values (p<0.05). In the healthy group, sORP did not differ when considering the variables. No12significant differences were found based on gender, statin or alcohol use for either group. Significant correlation was found for both sORP and cORP with CSHA Clinical Frailty Scale in the healthy group. Conclusion: Findings suggest that the variables of smoking and diabetes are contributory to frailty trajectory. Data suggest the capacity to tolerate oxidative stress, measured by cORP, is lower in aged individuals that smoke or are diabetic and contributes to frailty as a result of oxidative damage.

INTRODUCTION: Airway inflammatory response is widely believed to be a central mechanism in the development of adverse health effects related to air pollution exposure. Increased ventilation and inspiratory flow rates due to physical activity in the presence of air pollution will increase the inhaled dose of air pollutants. However, physical activity can also affect lung function and may moderate the relationship between air pollution and lung function. The mechanisms that underpin the complex interplay between air pollution, physical activity, and lung function may be more sensitive to the inhaled dose of air pollution than to ambient air pollution exposure alone. Despite this, the majority of literature on the topic measures only the ambient concentration of air pollution. AIM: This study aims to characterize the relationship between inhaled air pollution dose, physical activity, and respiratory response markers of lung function, oxidative stress and inflammation among healthy adolescents. Respiratory response measures include exhaled nitric oxide (eNO), percent oxidized exhaled breath condensate glutathione (%GSSG), percent oxidized exhaled breath condensate cysteine (%CYSS), the percentage of total oxidized compounds (%Oxidized), and changes in pulmonary function, namely, forced vital capacity (FVC), forced expiratory volume (FEV1), and forced expiratory flow (FEF25-75). Air pollution measures include cumulative inhaled doses of fine particulate matter (PM2.5), ozone (O3), black carbon (BC), and particle number total (PNT). METHODS: Using a non-probability sample of high school athletes, outcomes were measured prior to and after participation in extracurricular sports practice. The inhaled dose of air pollutants during the sports practice was estimated for each participant using a novel method developed by Dr. Roby Greenwald. This observational study estimates the association between air pollution dose and outcome measures using general linear mixed models with an unstructured covariance structure and a random intercept for subject to account for repeated measures within subjects. All data analysis was completed using SAS. RESULTS: A one IQR (i.e. 345.64 µg) increase in O3 inhaled dose is associated with a 29.16% average decrease from baseline in %Oxidized. A one IQR (i.e. 2.368E+10 particle) increase in PNT inhaled dose is associated with an average decrease in FEF25-75 of 0.168 L/second from baseline. The relationship between PNT inhaled dose and eNO is moderated by activity level, with increasing activity levels attenuating the relationship. Similarly, the relationship between O3 inhaled dose and %CYSS is attenuated by activity level, with increasing activity levels corresponding to smaller changes from baseline for a constant O3 inhaled dose. DISCUSSION: Someone who inhales a high cumulative dose despite a low activity level is likely breathing in a higher concentration of air pollution in a shorter period of time than a person who receives the same dose with a high activity level. The moderating effects of activity level suggest that peaks of high concentration doses of air pollution may overwhelm cells’ endogenous redox balance resulting in increased airway inflammation. Further research that examines the relationships between dose peaks over time and inflammation could help to determine whether a high concentration dose over a short period of time has a different effect than a lower concentration dose over a longer period of time.

Thesis (PhD (Science) (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Carnitine plays an essential role in eukaryotic metabolism by mediating the shuttling of activated acyl residues between intracellular compartments. This function of carnitine, referred to as the carnitine shuttle, is supported by the activities of carnitine acyltransferases and carnitine/acylcarnitine transporters, and is reasonably well studied and understood. While this function remains the only metabolically well established role of carnitine, several studies have been reporting beneficial effects associated with dietary carnitine supplementation, and some of those beneficial impacts appear not to be directly linked to shuttle activity. This study makes use of the yeast Saccharomyces cerevisiae as a cellular model system in order to study the impact of carnitine and of the carnitine shuttle on cellular physiology, and also investigates the eukaryotic carnitine biosynthesis pathway. The carnitine shuttle of S. cerevisiae relies on the activity of three carnitine acetyltransferases (CATs), namely Cat2p (located in the peroxisome and mitochondria), Yat1p (on the outer mitochondrial membrane) and Yat2p (in the cytosol), which catalyze the reversible transfer of activated acetyl units between CoA and carnitine. The acetylcarnitine moieties can be transferred across the intracellular membranes of the peroxisomes and mitochondria by the activity of the carnitine/acetylcarnitine translocases. The activated acetyl groups can be transferred back to free CoA-SH and further metabolised. In addition to the carnitine shuttle, yeast can also utilize the glyoxylate cycle for further metabolisation of in particular peroxisomally generated acetyl-CoA. This cycle results in the net production of succinate from two molecules of acetyl-CoA. This dicarboxylic acid can then enter the mitochondria for further metabolism. Partial disruption of the glyoxylate cycle, by deletion of the citrate synthase 2 (CIT2) gene, generates a yeast strain that is completely dependent on the activity of the carnitine shuttle and, as a consequence, on carnitine supplementation for growth on fatty acids and other non-fermentable carbon sources. In this study, we show that all three CATs are required for the function of the carnitine shuttle. Furthermore, overexpression of any of the three enzymes is unable to crosscomplement deletion of any one of the remaining two, suggesting a highly specific role for each CAT in the function of the shuttle. In addition, a role for carnitine that is independent of the carnitine shuttle is described. The data show that carnitine can influence the cellular response to oxidative stresses. Interestingly, carnitine supplementation has a protective effect against certain ROS generating oxidants, but detrimentally impacts cellular survival when combined with thiol modifying agents. Although carnitine is shown to behave like an antioxidant within a cellular context, the molecule is unable to scavenge free radicals. The protective and detrimental impacts are dependent on the general regulators of the cells protection against oxidative stress such as Yap1p and Skn7p. Furthermore, from the results of a microarray based screen, a role for the cytochrome c heme lyase (Cyc3p) in both the protective and detrimental effects of carnitine is described. The requirement of cytochrome c is suggestive of an involvement in apoptotic processes, a hypothesis that is supported by the analysis of the impact of carnitine on genome wide transcription levels. A separate aim of this project involved the cloning and expression in S. cerevisiae of the four genes encoding the enzymes from the eukaryotic carnitine biosynthesis pathway. The cloned genes, expressed from the constitutive PGK1 promoter, were sequentially integrated into the yeast genome, thereby reconstituting the pathway. The results of a plate based screen for carnitine production indicate that the engineered laboratory strains of S. cerevisiae are able to convert trimethyllysine to L-carnitine. This work forms the basis for a larger study that aims to generate carnitine producing industrial yeast strains, which could be used in commercial applications. / AFRIKAANSE OPSOMMING: Karnitien vervul ‘n noodsaaklike rol in eukariotiese metabolisme deur die pendel van asiel residue tussen intersellulêre kompartemente te medieer. Hierdie funksie van karnitien heet “die karnitien-pendel“ en word ondersteun deur verskeie karnitien asieltransferases en karnitine/asielkarnitien oordragsprotiëne. Die rol van die karnitien-pendel is redelik goed gekarakteriseer en is tot op hede die enigste bevestigde rol van karnitien in eukariotiese metabolisme. Verskeie onlangse studies dui egter op voordele geasosieer met karnitien aanvulling, wat in sommige gevalle blyk om onafhanklik te wees van die pendel aktiwiteit van karnitien. Hierdie studie maak gebruik van die gis, Saccharomyces cerevisiae, as ‘n sellulêre model sisteem om die impak van karnitien op sel fisiologie asook die eukariotiese karnitien biosintese pad te bestudeer. Die karnitien-pendel van S. Cerevisiae is afhanklik van die aktiwiteite van drie afsonderlike karnitien asetieltransferases (CATs), naamlik Cat2p (gelokaliseer in die peroksisoom en die mitochondria), Yat1p (op die buitenste membraan van die mitochondria) en Yat2p (in die sitosol). Die drie ensieme kataliseer die omkeerbare oordrag van asetielgroepe tussen CoA en karnitien. Die terugwaartse reaksie stel CoA-SH vry om sodoende verbruik te word in verdere metaboliese reaksies. Gis is in staat om, afsonderlik van die karnitien-pendel, gebruik te maak van die glioksilaat siklus vir verdere metabolisme van asetiel-CoA wat gevorm word in die peroksisoom. Gedeeltelike onderbreking van hierdie siklus deur uitwissing van die sitraat sintase (CIT2) geen, genereer ’n gisras wat afhanklik is van die funksie van die karnitienpendel en ook van karnitien aanvulling vir groei op vetsure en nie-fermenteerbare koolstofbronne. Hierdie studie dui daarop dat al drie CATs noodsaaklik is vir die funksionering van die karnitien-pendel. Ooruitdrukking van enige van die drie ensieme lei slegs tot selfkomplementasie en nie tot kruis-komplementasie van die ander twee CATs nie. Hieruit word ’n hoogs spesifieke rol vir elk van die drie ensieme afgelei. ’n Pendel-onafhanklike rol vir karnitien word ook in hierdie werk uitgewys in die bevordering van weerstand teen oksidatiewe stres. Dit is noemenswaardig dat karnitien ’n beskermende effek het in kombinasie met oksidante wat ROS genereer en ’n nadelige effek in kombinasie met sulfhidriel modifiserende agente. Dit word aangedui dat karnitien antioksidant funksie naboots in die konteks van ’n gis sel terwyl die molekuul nie in staat is om vry radikale te deaktiveer nie. Beide die beskermende asook die nadelige inwerking van karnitien is afhanklik van Yap1p en Skn7p, wat reguleerders is in die algemene beskerming teen oksidatiewe stres. Die resultate van ’n “microarray“ gebaseerde studie dui op ’n rol vir die sitokroom c heem liase (Cyc3p) in beide die beskermende en nadelige gevolge van karnitien aanvulling. Die vereiste vir sitochroom c dui op ’n moontlike rol vir apoptotiese prosesse. Hierdie hipotese word verder versterk deur ‘n analise van die impak van karnitien op genoomwye transkripsievlakke. ’n Afsonderlike doelwit van hierdie studie was toegespits op die klonering en uitdrukking van die vier ensieme betrokke in eukariotiese karnitien biosintese in S. cerevisiae. Die gekloneerde gene, uitgedruk vanaf die konstitutiewe PGK1 promotor, was geïntigreer in die gisgenoom om die pad op te bou. Die resultate van ’n plaat gebaseerde karnitien produksie toets dui aan dat die geneties gemanipuleerde gisrasse wel in staat is om trimetiellisien oor te skakel in Lkarnitien. Hierdie werk vorm die hoeksteen van ’n studie wat die ontwikkeling van karnitien produserende kommersiële gisrasse as doelwit stel.