An online, semi-continuous instrument to measure both total and gas phase atmospheric reactive oxygen species (ROS) and determine the concentration of ROS in the particle phase (ROS(p)) was developed. This instrument was based on a fluorescent probe for quantifying ambient ROS, specifically 2'7'-dichlorodihydrofluorescin, or DCFH probe. This probe was analyzed for sensitivity to a variety of offline and online parameters for efficient use in a field instrument. The ROS(p) instrument measures the peak light intensity at 530 nm to determine ambient ROS concentrations. ROS particles and gases are collected in a mist chamber in a nebulized mist. The instrument alternates measurements of ROS(p+g), or ROS(tot) by means of an inline filter. Fine (PM₂.₅) (ROS(p) is determined by subtraction of the ROS(g) concentration from the ROS(tot), as the ROS(g) signal could not be excluded. This instrument was tested during the summer (May-July) of 2012 at urban and rural sites in the metropolitan Atlanta and surrounding region. Concentrations of ROS(p) determined from this instrument were often below limit of detection. Average concentrations of ROS(p) were found to be 0.25 nmol/m³ in urban Atlanta (Jefferson St. and Georgia Tech), and 0.15 nmol/m³ in Yorkville, a rural site. A side by side comparison of this method with a filter collection method was made in July. The average ROS(p) offline concentrations were 0.15 nmol/m³. These concentrations were comparable to the online average concentrations of 0.21 nmol/m³ for the same period of time. This average and the majority of the measurements comprising it is dominated by the high limit of detection. The ROS instrument as constructed and operated is an efficient way to conduct ROS(p) measurements at the level of a filter study while reducing the labor intensive filter collection and extraction. In order for this instrument to be successful at measuring ambient ROS in the particle phase, the removal of the gas phase from the current sampling scheme is critical as the ROS(g) concentrations are over 90% of the measured ROS. The system as currently operable is best suited for source measurements, including biomass burning plumes or fresh exhaust to capture immediate formation.

Thesis (Ph. D.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains xii, 216 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains xiv, 180 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains xiv, 197 p. : ill. Vita. Includes abstract. Includes bibliographical references.

Evolutionary life history theory predicts that tradeoffs among fitness-related phenotypes will occur as a result of resource limitations and/or physiological constraints. Such tradeoffs are defined as the cost(s) incurred on one component of fitness (e.g., reproduction) by the increased expression of another fitness-related trait (e.g., longevity). Only recently have researchers begun to investigate the mechanistic bases of life history tradeoffs. A recent proposal is that reactive oxygen species (ROS) have a central role in shaping life history traits and tradeoffs. Research on disparate animal taxa has highlighted strong correlations between oxidative stress resistance and fitness-related life history traits, for example. Here, I use the model organism Caenorhabditis elegans to test several hypotheses concerning the effects of ROS on life history traits and the manifestation of life history tradeoffs. Additionally, I use heat stress and an alternate food source to explore the responses of life history traits to other forms of physiological stress. Relative fitness and other traits related to reproduction were found to be affected in mostly negative ways by increasing oxidative insult. Lifespan was surprisingly unaffected by oxidative stress, but was modified by temperature. In vivo ROS levels as measured by fluorescent microscopy reveal a tradeoff between antioxidant production and reproduction in this species.

M. Tech. (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology. / The aim of the study is to synthesise, characterize and assess the antimicrobial activity of cobalt oxide, zinc oxide and cobalt-doped zinc oxide nanoparticles against selected waterborne pathogenic fungi (yeasts and moulds) and bacteria. Various types of oxide based nanomaterial are an attractive option for the disinfection of water due to its high chemical stability and non-toxicity towards human cells. Synthesis of Co -doped ZnO and Co3O4nanoparticles was done through mechanochemical synthesis and urea based synthesis and microwave heating was employed for the preparation of ZnO nanoparticles. The ZnO nanoparticles were produced in short reaction and it was white color. Cobalt oxide (Co3O4) nanoparticles appeared as a pink precipitate but was turned black after being calcined. The synthesis of Co- ZnO nanoparticles was successfully prepared and blue solid was obtained from pink cobalt ion solution. The nanoparticles were characterised by X- Ray Diffraction (XRD), Fourier Infrared Spectroscopy (FTIR), UV–visible spectroscopy, Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) (Yang et al. 2003). In this research project, the antibacterial activities of NPs were carried out by well diffusion method and minimum inhibitory concentration (MIC). MIC is the lowest concentration of a chemical, usually a drug, which prevents visible growth of bacterium. Bacterial strains used in the study are: Salmonella enterica, Escherichia coli, Shigella sonnei and Staphylococcus aureus, yeast and mould is: Candida albicans and Aspergillus niger. The antimicrobial results obtained showed that ZnO nanoparticles are more effective than Co- ZnO and Co3O4 nanoprticles against all the microorganisms used. The toxicity studies were performed using DAPHTOXKIT F and the 24h EC50 and 48h EC50 were calculated according to the manufactures’ instructions. The results showed that Co- ZnO nanoparticles is less toxic to Daphnia magna compared to ZnO and Co3O4 NPs.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The human body has developed an integrated antioxidant defence system to protect against free radical damage. Acute exercise may result in the increased generation of free radicals, including reactive oxygen species, and this may overwhelm antioxidant defence systems resulting in oxidative stress. However, it has been shown that individuals who undergo regular exercise training may have improved antioxidant capacity when compared to sedentary controls. Results from research regarding the association between antioxidant capacity and exercise training are however not conclusive and further investigation is required. Therefore, the aim of this study was to investigate the association between the total plasma antioxidant status and selected plasma indicators of antioxidant status and the MnSOD Ala-9Val (-28C®T) polymorphism in trained male athletes (rugby players) and sedentary male students while controlling for dietary intake of the major antioxidants using a validated dietary assessment method. In order to address the potential confounding effect of dietary antioxidant intake on antioxidant status in the main study, a FFQ that measures vitamin C, vitamin E, carotenoid and flavonoid intake was developed. The reproducibility was assessed by the repeat administration of the FFQ (n = 38), while the va lidity was assessed using a 28-day closeended dietary record and repeated plasma vitamin C values (n = 18). Several statistical tests were conducted to compare the values obtained from the FFQ with values obtained from the various reference methods. While results from Bland-Altman plots suggested that the reproducibility and validity of FFQ was not completely satisfactory, similar mean values, moderate to strong correlation coefficients, and a high percentage of individuals classified correctly according to quartiles of intake indicated satisfactory reproducibility and validity of the FFQ in assessing antioxidant intake. Furthermore, moderate to strong validity coefficients obtained from the method of triads also indicated satisfactory validity for the FFQ. The main study involved a cross-sectional study that compared plasma vitamin C and carotenoid levels as well as total plasma antioxidant status in trained rugby players (n = 76) and sedentary male subjects (n = 39) with different MnSOD genotypes, while controlling for dietary antioxidant intake. Rugby players had significantly higher plasma vitamin C and carotenoid levels compared to sedentary students, which indicated more satisfactory plasma antioxidant status. This was also reflected in the tendency for total plasma antioxidant status (ORAC assay) to be higher in rugby players than sedentary students. MnSOD genotype did not influence plasma vitamin C and carotenoid levels or plasma total antioxidant status, with or without control for dietary antioxidant intake. Dietary vitamin C, vitamin E, carotenoid an flavonoid intake (from foods + supplements) was similar for rugby players and sedentary students and was adequate for both groups. Thus the association between antioxidant status and MnSOD genotype in rugby players and sedentary students seemed not to be influenced by dietary antioxidant intake. In conclusion therefore, rugby players undergoing regular exercise training had a more satisfactory antioxidant status compared to sedentary students. Based on this conclusion, the widespread use of antioxidant supplements by athletes is questioned. / AFRIKAANSE OPSOMMING: Die menslike liggaam beskik oor ‘n geintegreerde antioksidantmeganisme om dit teen vryradikaalskade te beskerm. Akute oefening kan bydra tot ‘n verhoogde produksie van vry radikale, insluitend reaktiewe suurstofspesies, wat kan veroorsaak dat die antioksidantbeskermingsmeganisme oorlaai word, wat dan kan aanleiding gee tot die ontstaan van oksidatiewe stress. Dit is aangetoon dat persone wat gereeld oefening doen verbeterde antioksidantkapasiteit toon in vergelyking met persone wat geen oefening doen nie. Die resultate van navorsingstudies wat die verband tussen antioksidantkapasiteit en oefening ondersoek is egter teenstrydig en verdere navorsing op hierdie gebied is essensieël om uitsluitsel te kry oor kontensieuse vraagstukke. Die doel van hierdie studie was dus om ondersoek in te stel na die verband tussen plasma antioksidant status, die MnSOD Ala-9Val (-28C T) polimorfisme en geselekteerde plasma antioksidantmerkers in geoefende manlike atlete (rugby spelers) en ‘n onaktiewe manlike kontrolegroep terwyl gekontroleer word vir die dieetinname van die vernaamste antioksidante. Om vir die potensiële invloed van dieetantioksidantinname op die antioksidantstatus van proefpersone in die hoofstudie te kontroleer, is ‘n voedsel frekwensievraelys wat vitamien C-, vitamien E-, karotenoïed- en flavinoïedinname meet, ontwikkel. Die herhaalbaarheid (betroubaarheid) van die vraelys is getoets deur herhaalde voltooiing daarvan deur ‘n toetsgroep (n=38), terwyl die geldighied getoets is deur gebruik te maak van ‘n 28-dag geslote dieetrekord en herhaalde plasma vitamien C bepalings as verwysingswaardes (n=18). Verskeie statistiese toetse is uitgevoer om die frekwensievraelys waardes met die verskillende verwysingswaardes te vergelyk. Alhoewel die Bland -Altman grafieke nie dui op bevredigende herhaalbaarheid en geldigheid van die voedselfrekwensie vraelys nie, dui gelyke gemiddelde waardes, matig tot sterk en betekenisvolle korrelasiekoeffisiënte en ‘n hoë persentasie individue korrek geklassifiseer volgens kwartiele van inname, wel op bevredigende herhaalbaarheid en geldigheid. Matige tot sterk geldigheidskoeffisiënte is ook verkry met die toepassing van “The method of Triads”, wat verdere steun bied vir bevredigende geldigheid. In die hoofstudie is plasma vitamien C, karotenoïedvlakke en totale plasma antioksidantstatus in manlike rugby spelers (n=76) vergelyk met dié van onaktiewe manlike kontroles (n=39). Vergelykings tussen MnSOD genotipes binne die aktiwiteitsgroepe is ook getref. Al genoemde analises is gekontroleer vir dieet antioksidantinname. Resultate dui daarop dat die plasma vitamien C en karotenoïedvlakke van rugby spelers betekenisvol hoër was as dié van die kontrolegroep, wat dui op ‘n meer bevredigende antioksidantstatus. Hierdie resultaat is ook weerspieël in die feit dat totale plasma antioksidantstatus (ORAC) in die rugby spelers oog geneig was om hoër te wees as dié van die kontrole groep. Dit het ook geblyk dat MnSOD genotipe nie ‘n effek gehad het op plasma vitamien C-, karotenoïed- of totale antioksidantstatus nie, met of sonder kontrole vir dieet antioksidantinname. Die dieet vitamien C-, vitamien E-, karotenoïed- en flavinoïedinname (vanaf voedsel en supplemente) was dieselfde vir rugby spelers en kontrole en was toereikend vir beide groepe. Dit blyk dus dat dat die verband tussen antioksidantstatus en MnSOD genotipe in die twee groepe nie beinvloed is deur antioksidantinname nie. Ten slotte kan die gevolgtrekking gemaak word dat manlike rugby spelers ‘n meer bevredigende antioksidant status het as onaktiwe manlike kontroles. Op grond van hierdie gevolgtrekking word die algemene gebruik van antioksidant supplemente deur atlete bevraagteken.

Law, Sau Kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 111-117). / Abstract also in Chinese. / Thesis Committee --- p.i / Acknowledgements --- p.ii / Contents --- p.iii / Abstract --- p.vii / 論文摘要 --- p.x / Abbreviations --- p.xi / List of Figures --- p.xiii / List of Tables --- p.xxiii / Chapter CHAPTER ONE --- INTRODUCTION / Chapter 1.1 --- Embryonic Stem (ES) Cells / Chapter 1.1.1 --- Characteristics of ES Cells l / Chapter 1.1.2 --- Therapeutic Potential of ES Cells --- p.3 / Chapter 1.1.3 --- Myocardial Infarction and ES cells-derived Cardiomyocytes --- p.4 / Chapter 1.1.4 --- Current Hurdles of Using ES cells-derived Cardiomyocytes for Research and Therapeutic Purposes --- p.6 / Chapter 1.2 --- Transcription Factors for Cardiac Development / Chapter 1.2.1 --- GATA-binding Protein 4 (GATA-4) --- p.8 / Chapter 1.2.2 --- Myocyte Enhancer Factor 2C (MEF2C) --- p.10 / Chapter 1.2.3 --- "NK2 Transcription Factor Related, Locus 5 (Nkx2.5)" --- p.11 / Chapter 1.2.4 --- Heart and Neural Crest Derivatives Expressed 1 /2 (HANDI/2) --- p.11 / Chapter 1.2.5 --- T-box Protein 5 (Tbx5) --- p.13 / Chapter 1.2.6 --- Serum Response Factor (SRF) --- p.14 / Chapter 1.2.7 --- Specificity Protein 1 (Spl) --- p.15 / Chapter 1.2.8 --- Activator Protein 1 (AP-1) --- p.16 / Chapter 1.3 --- Reactive Oxygen Species (ROS) / Chapter 1.3.1 --- Cellular Production of ROS --- p.18 / Chapter 1.3.2 --- Maintenance of Redox balance --- p.18 / Chapter 1.3.3 --- Redox Signaling --- p.19 / Chapter 1.4 --- Nitric Oxide (NO) and NO Signaling --- p.20 / Chapter 1.5 --- Aims of the Study --- p.22 / Chapter CHAPTER TWO --- MATERIALS AND METHODS / Chapter 2.1 --- Mouse Embryonic Fibroblast (MEF) Culture / Chapter 2.1.1 --- Derivation of MEF --- p.23 / Chapter 2.1.2 --- Maintenance of MEF Culture --- p.24 / Chapter 2.1.3 --- Irradiation of MEF --- p.25 / Chapter 2.2 --- Mouse ES Cell Culture / Chapter 2.2.1 --- Maintenance of Undifferentiated Mouse ES Cell Culture --- p.26 / Chapter 2.2.2 --- Differentiation of Mouse ES Cells --- p.26 / Chapter 2.2.3 --- Exogenous addition of hydrogen peroxide (H2O2) and NO --- p.27 / Chapter 2.3 --- ROS Localization Study / Chapter 2.3.1 --- Frozen Sectioning --- p.28 / Chapter 2.3.2 --- Confocal microscopy for ROS detection --- p.28 / Chapter 2.4 --- Intracellular ROS Measurement / Chapter 2.4.1 --- "Chemistry of 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA)" --- p.29 / Chapter 2.4.2 --- Flow Cytometry for ROS Measurement --- p.29 / Chapter 2.5 --- Gene Expression Study / Chapter 2.5.1 --- Primer Design --- p.30 / Chapter 2.5.2 --- RNA Extraction --- p.31 / Chapter 2.5.3 --- DNase Treatment --- p.32 / Chapter 2.5.4 --- Reverse Transcription --- p.32 / Chapter 2.5.5 --- Quantitative Real Time PCR --- p.33 / Chapter 2.5.6 --- Quantification of mRNA Expression --- p.34 / Chapter 2.6 --- Protein Expression Study / Chapter 2.6.1 --- Total Protein Extraction --- p.34 / Chapter 2.6.2 --- Nuclear and Cytosolic Protein Extraction --- p.35 / Chapter 2.6.3 --- Measurement of Protein Concentration --- p.36 / Chapter 2.6.4 --- De-sumoylation Assay --- p.36 / Chapter 2.6.5 --- De-phosphorylation Assay --- p.37 / Chapter 2.6.6 --- De-glycosylation Assay --- p.38 / Chapter 2.6.7 --- Western Blot --- p.39 / Chapter 2.7 --- Statistical Analysis --- p.41 / Chapter CHAPTER THREE --- RESULTS / Chapter 3.1 --- Study of Endogenous ROS / Chapter 3.1.1 --- Level and Distribution of Endogenous ROS --- p.47 / Chapter 3.1.2 --- Quantification of intracellular ROS --- p.48 / Chapter 3.2 --- Effect of Exogenous Addition of Nitric Oxide (NO) on Cardiac Differentiation / Chapter 3.2.1 --- Beating Profile of NO-treated Embryoid Bodies (EBs) --- p.50 / Chapter 3.3 --- Effect of Exogenous Addition of H2O2 on Cardiac Differentiation / Chapter 3.3.1 --- Beating Profile of H2O2-treated EBs --- p.51 / Chapter 3.3.2 --- mRNA Expression of Cardiac Structural Genes --- p.52 / Chapter 3.3.3 --- Protein Expression of Cardiac Structural Genes --- p.54 / Chapter 3.3.4 --- mRNA Expression of Cardiac Transcription Factors --- p.58 / Chapter 3.3.5 --- Protein Expression of Cardiac Transcription Factors --- p.67 / Chapter 3.3.6 --- Post-translational Modifications of Cardiac Transcription Factors --- p.74 / Chapter 3.3.7 --- Translocation of Cardiac Transcription Factors --- p.89 / Chapter CHAPTER FOUR --- DISCUSSION / Chapter 4.1 --- Changes in the Level of Endogenous ROS During Cardiac Differentiation of Mouse ES Cells --- p.96 / Chapter 4.2 --- H2O2 and NO Have Opposite Effects Towards Cardiac Differentiation --- p.97 / Chapter 4.3 --- Exogenous Addition of H2O2 Advances Differentiation of Mouse ES Cells into Cardiac Lineage --- p.99 / Chapter 4.4 --- Possible Role of H2O2 in Mediating Cardiac Differentiation of Mouse ES Cells --- p.103 / Chapter 4.5 --- Future Directions --- p.108 / Conclusions --- p.110 / References --- p.111

Yung, Lai Hang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 93-115). / Abstract also in Chinese. / Declaration --- p.i / Acknowledgements --- p.ii / Abbreviations --- p.iii / Abstract in English --- p.v / Abstract in Chinese --- p.ix / Contents --- p.xi / Chapter Chapter I - --- Introduction / Chapter 1.1) --- Endothelial cells function --- p.1 / Chapter 1.2) --- Oxidative stress in the vascular wall --- p.2 / Chapter 1.2.1) --- Sources of ROS --- p.3 / Chapter 1.2.2) --- Actions of ROS --- p.3 / Chapter 1.2.2.1) --- Impaired endothelium-dependent vasodilatation --- p.3 / Chapter 1.2.2.2) --- VSMC migration --- p.4 / Chapter 1.2.2.3) --- Programmed cell death (cell apoptosis) --- p.4 / Chapter 1.3) --- Endothelial cell apoptosis --- p.7 / Chapter 1.3.1) --- Apoptosis and cardiovascular diseases --- p.7 / Chapter 1.3.2) --- Mechanisms of endothelial cells apoptosis --- p.7 / Chapter 1.3.2.1) --- What are caspases? --- p.8 / Chapter 1.3.2.2) --- Death receptor-mediated apoptosis --- p.9 / Chapter 1.3.2.3) --- Mitochondria-dependent pathway --- p.9 / Chapter 1.3.3) --- Regulations of endothelial cells apoptosis --- p.10 / Chapter 1.3.3.1) --- Oxidative stress --- p.10 / Chapter 1.3.3.2) --- Shear Stress --- p.11 / Chapter 1.3.3.3) --- Growth factors --- p.12 / Chapter 1.3.3.4) --- NO --- p.12 / Chapter 1.3.3.5) --- Inflammatory mediators --- p.13 / Chapter 1.4) --- Mitogen activated kinases signaling in apoptosis --- p.15 / Chapter 1.5) --- Bone morphogenic proteins (BMPs) --- p.17 / Chapter 1.5.1) --- BMPs functions and cardiovascular system --- p.17 / Chapter 1.5.2) --- BMPs signaling pathways --- p.18 / Chapter 1.5.2.1) --- Smad-dependent pathway --- p.18 / Chapter 1.5.2.2) --- MAPKs and SAPKs pathways --- p.19 / Chapter 1.5.2.3) --- Antagonists of BMPs signaling --- p.20 / Chapter 1.5.3) --- BMP4 and cardiovascular diseases --- p.20 / Chapter 1.6) --- "Justification, long-term significance and objectives of the present project" --- p.23 / Chapter Chapter II - --- Methods and Materials / Chapter 2.1) --- Animal handling --- p.24 / Chapter 2.2) --- Endothelial cell isolation and culture --- p.24 / Chapter 2.2.1) --- Primary culture of rat endothelial cells --- p.24 / Chapter 2.2.2) --- Culture of human umbilical cord vein endothelial cells… --- p.25 / Chapter 2.3) --- Apoptosis assessment --- p.25 / Chapter 2.3.1) --- Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay --- p.25 / Chapter 2.3.2) --- Cell death detection ELISA kit --- p.26 / Chapter 2.3.3) --- Flow cytometry --- p.27 / Chapter 2.4) --- Western blot analysis --- p.28 / Chapter 2.4.1) --- Sample preparation --- p.28 / Chapter 2.4.2) --- SDS-PAGE and transfer --- p.28 / Chapter 2.5) --- DHE fluorescence --- p.29 / Chapter 2.6) --- "Drugs, chemicals and other reagents" --- p.30 / Chapter 2.6.1) --- Drugs and chemicals used in the present experiments --- p.30 / Chapter 2.6.2) --- Reagents for Western blot analysis --- p.30 / Chapter 2.6.3) --- Primary antibodies --- p.33 / Chapter 2.7) --- Small interfering RNA experiment --- p.34 / Chapter 2.8) --- Statistical analysis --- p.34 / Chapter Chapter III - --- BMP4 induces endothelial cell apoptosis in ROS related p38 MAPK and JNK mediated caspase-3 dependent pathway / Chapter 3.1) --- Introduction --- p.35 / Chapter 3.2) --- Methods and materials --- p.39 / Chapter 3.2.1) --- Isolation and culture of endothelial cells --- p.39 / Chapter 3.2.2) --- Drugs treatment --- p.39 / Chapter 3.2.3) --- Assay for cell apoptosis --- p.40 / Chapter 3.2.3.1) --- Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay --- p.40 / Chapter 3.2.3.2) --- Cell death detection ELISA kit --- p.41 / Chapter 3.2.3.3) --- Flow cytometric analysis --- p.41 / Chapter 3.2.4) --- Western blot analysis --- p.41 / Chapter 3.2.5) --- Dihydroethidium (DHE) staining --- p.42 / Chapter 3.2.6) --- Statistical analysis --- p.42 / Chapter 3.3) --- Results --- p.43 / Chapter 3.3.1) --- Dose- and time-dependent effect of BMP4 --- p.43 / Chapter 3.3.2) --- Role of caspases in apoptosis of RAECs and HUVECs --- p.43 / Chapter 3.3.3) --- Roles of BMP4 and ROS in endothelial cell apoptosis --- p.44 / Chapter 3.3.3.1) --- Noggin antagonism of BMP4-induced effect --- p.44 / Chapter 3.3.3.2) --- NAD(P)H oxidase-mediated ROS production --- p.44 / Chapter 3.3.3.3) --- Inhibition of endothelial cell apoptosis by ROS scavengers --- p.45 / Chapter 3.3.4) --- Roles of MAPKs/SAPKs in BMP4-induced endothelial cell apoptosis --- p.45 / Chapter 3.3.5) --- Relationship between ROS and MAPKs/SAPKs --- p.46 / Chapter 3.3.6) --- Relationship between p38 MAPK and JNK --- p.46 / Chapter 3.4) --- Discussion --- p.82 / Chapter 3.4.1) --- Caspase-dependent pathways --- p.82 / Chapter 3.4.2) --- Oxidative stress --- p.85 / Chapter 3.4.3) --- Role of MAPKs activation in BMP4-induced endothelial cell apoptosis --- p.87 / Chapter 3.4.4) --- ROS mediates BMP4-induced activation of MAPKs --- p.88 / Chapter 3.4.5) --- Role of p38 MAPK in the activation of JNK 1 --- p.89 / Chapter 3.5) --- Concluding remarks --- p.91 / References --- p.93 / Publications and Awards --- p.116

胰臟內分泌細胞分化的調控事件的研究揭示了胰島素分泌細胞的形成。這一原理既有利於體外誘導用於移植的胰島素分泌細胞，又可應用于糖尿病病人自體胰島素分泌細胞的再生。正在發育的組織和器官中，發現了腎素血管緊張素（RAS）成員，揭示了他們在發育過程中的潛在調控作用。另外，對 RAS 信號系統做出應答的活性氧化物質（ROS），被認為是第二信使，通過對轉錄調控因子的氧化還原的修飾促進分化。作為 ROS 的主要來源，NADPH 已被證實在各類細胞和組織中參與了祖細胞的分化。儘管如此，依賴於 NADPH 氧化酶的 ROS對于胰腺內分泌細胞分化的調控作用仍不清楚。基於這個背景，本研究致力於揭示 RAS 和 NADPH 氧化酶依賴性 ROS 在胰腺內分泌細胞分化中的作用。本實驗將在小鼠胰臟原基培養物和尿鏈黴素（STZ）誘導的新生大鼠上進行。 結果顯示，經典 RAS 成員中的血管緊張素 2 型受體（AT₂R）分佈於內分泌祖細胞的細胞核，之後穿梭定位於胰島素分泌細胞的細胞質。阻斷 AT₂R 功能抑制了Ngn3，胰島素的表達以及 β 細胞的增值。在不同的胚胎期 ROS 的水平發生了改變。對于培養的胰臟原基施加適當的外源 ROS，刺激了內分泌細胞的分化。同時，ROS 清除劑減弱了胰島細胞分化和成熟的標記基因的表達。NOX4 以及其相關的亞基 p22phox 是 NADPH 氧化酶成員，其在胰臟發育過程中的變化同 ROS 水平的變化相似，並且持續表達與內分泌細胞系統。在 NGN3 高表達的胚胎期15.5 天，它們定位于表達 NGN3 的細胞；在 NGN3 表達下調，且胰島素表達升高的胚胎期 17.5 天，它們分佈於胰島素表達細胞。而且，NADPH 氧化酶的抑製劑 DPI 削弱了胰臟培養物中的內分泌祖細胞的分化, 外源 H₂O₂ 的加入扭轉了這一現象。 / 另一方面，在 STZ 誘導的新生大鼠的研究中，DPI 負調節 β 細胞的再生。血糖失調，胰島結構毀壞以及血清胰島素匱乏的現象發生在了 DPI 處理組。另外，DPI 減弱了 NGN3 的表達而並非 Ki67, 顯示 β 細胞的分化而並非增值對於 ROS 的刺激進行了應答。在體內和體外的實驗中，DPI 也抑制了 NGN3 的轉綠調控因子 SOX9 在胰腺祖細胞中的表達。有趣的是，過表達 SOX9 可以恢復 DPI 引發的對於 NGN3 的抑制 。結合以上數據，本研究顯示 NADPH 氧化酶依賴性ROS 誘導的信號通路參與了胰腺祖細胞到胰島素分泌細胞的分化。 / Investigations into the regulatory events that modulate pancreatic endocrine cell differentiation shed light on the generation of sufficient insulin-producing cells in vitro for transplantation or regeneration of β cells in patients with diabetes. The expression of renin-angiotensin system (RAS) components has been detected in development tissue and organs, implicating their regulatory role in developmental processes. On the other hand, reactive oxygen species (ROS) are responsive to RAS signaling pathways and act as second messengers to promote differentiation through redox modification of transcriptional factors essential for differentiation. As a major source of ROS, NADPH oxidase has been shown to participate in the progenitor differentiation in a variety of cells and tissues. Despite this finding, the role of NADPH oxidase-dependent ROS in regulating pancreatic endocrine cell differentiation remains ambiguous. Against this background, the study was aimed at elucidating the roles of RAS components and NADPH oxidase-derived ROS during differentiation of pancreatic endocrine cells using mouse pancreatic rudiments and streptozotocin-treated neonatal rats. / Results showed that angiotensin II type 2 receptor (AT₂R), a major component of the classical RAS, was localized within the nuclei of endocrine progenitors in the cultured pancreatic rudiments; following the differentiation of endocrine progenitors into insulin producing cells, it translocated to cytoplasm. Blockade of AT₂R impeded the expression of Ngn3 and insulin as well as proliferation of β-cells. In addition, the dynamic changes of ROS levels were found in mouse pancreata at different embryonic days, concomitant with induction of endocrine cell differentiation induced by modest exogenous ROS in pancreatic rudiment cultures. Moreover, scavenger of ROS diminished the expression of islet cell markers for differentiation and maturation. NOX4 and its associated subunit p22phox, which are the member of NADPH oxidase, exhibited similar changes of expression to that of ROS levels during pancreas development and persisted in the endocrine lineage; they were located in NGN3⁺ cells at E15.5 during the burst of NGN3 expression and then distributed in insulin⁺ cell at E17.5, the latter being the phase that has a decline in NGN3 expression with an increase of insulin. Furthermore, administration of NADPH oxidase inhibitor, diphenylene iodonium (DPI) attenuated the differentiation of endocrine progenitors in rudiment cultures, while exogenous ROS reversed this effect. / On the other hand, studies performed in streptozotocin-induced neonatal rats showed that β cell regeneration was negatively affected by DPI treatments; consistently, impaired blood glucose control, disturbed islet architecture and deficient serum insulin were observed in DPI-treated groups. In addition, DPI treatments blunted NGN3 expression, but not Ki67-labeling beta-cells, suggesting that differentiation beyond proliferation of β-cells was accountable in response to ROS stimulation. Administration of DPI also suppressed the levels of SOX9, a transcriptional regulator of NGN3, in pancreatic progenitor cells, as evidenced by both in vivo and in vitro studies. Interestingly, over-expression of SOX9 could restore the repression of NGN3 induced by DPI. Taken all these data together, our results indicate that NADPH oxidase-dependent ROS-induced signaling pathway is involved in the differentiation of pancreatic endocrine progenitors into insulin-producing β cells. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liang, Juan. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 171-205). / Abstracts also in Chinese.