Ischemic stroke is the leading cause of death and disability in human diseases all around the world. As effective treatment for ischemic stroke is still absent, seeking for new therapy is of great interest. Currently, several key pathological cascades following cerebral ischemia have been explored to develop further therapies. Among them, reactive nitrogen species (RNS) has been indicated to play a critical role in cerebral ischemia reperfusion injury. As one of the RNS, peroxynitrite contributes to the neural cell death and subsequent brain dysfunction in the process. Thus, development of antioxidants targeting on peroxynitrite could be an important strategy for the treatment of cerebral ischemia-reperfusion injury. Baicalin is a polyphenolic compound isolated from roots of Scutellaria baicalensis. Baicalin exerted protective effects against cerebral ischemia-reperfusion injury but the mechanisms are not clear yet. In this study, we investigated the free radical scavenging ability and neuroprotective effects of baicalin. According to our results, baicalin neutralized DPPH radicals effectively. By using electron paramagnetic resonance (EPR) spin trapping technology and fluorescent probe DAF-2DA, we found that baicalin dose-dependently scavenged superoxide, but had very low effect on elimination of nitric oxide. The immunofluoresent results revealed that baicalin at the concentration of 50 M completely suppressed the nitrotyrosine formation induced by 3-morpholinylsydnoneimine chloride (SIN-1, a peroxynitrite donor) in neuroblastoma SH-SY5Y cells. Mass spetrum provided direct evidence of the peroxynitrite scavenging ability of baicalin. Using MTT assays, we found that baicalin totally reversed peroxynitrite-induced cytotoxicity in SH-SY5Y cells and protected SH-SY5Y cells in oxygen glucose deprivation (OGD) and following reoxygenation injury. Furthermore, in vivo experiments revealed that intravenous injection of baicalin exerted better neuroprotective effect than intraperitoneal administration in rats underwent middle cerebral artery occlusion (MCAO). After cerebral ischemia reperfusion, rats treated with 3 mg/kg of peroxynitrite decomposition catalyst (FeTMPyP) or 25 mg/kg of baicalin revealed a smaller size of infarction volume, suppressed neural cell death and reduced nitrotyrosine formation than MCAO rats. However, baicalin did not alter the expression of tight junction proteins, claudin-5 and ZO-1, in brain endothelial bEnd3 cell line treated with OGD following reoxygenation. In cerebral ischemia reperfusion rats, administration of FeTMPyP at the dosage of 3 mg/kg diminished the Evans blue leakage caused by blood brain barrier disruption, whereas treatment of baicalin did not show significant effect. In conclusion, this study suggests that baicalin can scavenge peroxynitrite and protect neural cells from peroxynitrite-induced injury. Furthermore, baicalin could prevent brains from cerebral ischemia-reperfusion injury and the neuroprotective mechanisms are associated with the scavenging effects on peroxynitrite. These findings provide new insights into the antioxidant and neuroprotective properties of baicalin and indicate the potential application of baicalin for the treatment of ischemic stroke. / published_or_final_version / Chinese Medicine / Master / Master of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

In nature bacteria predominantly live on surfaces, in matrix-encased communities called biofilms. Biofilm formation displays dynamic developmental patterns resembling those of multicellular organisms. Using cooperative traits such as cell-cell signaling, bacteria in biofilms form complex architectures, known as microcolonies, in which cells become highly differentiated from their planktonic counterparts. Microcolonies are generally highly tolerant to bactericides, rendering biofilms extremely difficult to eradicate. The aim of this study was to investigate the last, and least understood stage of biofilm development, which involves the coordinated dispersal of single cells that revert to a free-swimming planktonic phenotype and escape from the biofilm. Strategies to induce biofilm dispersal are of interest due to their potential to prevent biofilms and biofilm-related infections. In the model organism Pseudomonas aeruginosa, reproducible patterns of cell death and dispersal can occur within biofilm structures, leaving behind empty or hollow microcolonies. These events were previously linked with the appearance of oxidative and/or nitrosative stress in mature microcolonies. Here, the involvement of reactive oxygen and nitrogen intermediates in biofilm development and dispersal processes was investigated in both mono- and mixed-species biofilms. By using specific fluorescent dyes and P. aeruginosa mutant strains, nitric oxide (NO), a by-product of anaerobic respiration and an important messenger molecule in biological systems, was found to play a major role in P. aeruginosa biofilm dispersal. Further, the results demonstrated that exposure to physiological, non-toxic concentrations of NO (in the low nanomolar range) causes biofilm dispersal in P. aeruginosa and restores its vulnerability to conventional antimicrobials. By using microarray techniques, NO was shown to induce global changes in genetic expression, including enhanced metabolic activity and motility and decreased adhesion and virulence in P. aeruginosa biofilms. The regulatory pathway implicated c-di-GMP, a newly discovered messenger molecule involved in the transition from sessility to motility in many bacterial species. NO-mediated dispersal was also observed in other single- and multi-species biofilms of clinically and industrially relevant organisms. Hence, the combined exposure to NO and bactericides was identified as a potential novel strategy for the removal of microbial communities, providing a low cost and environmentally safe solution to biofilm control.

Intervertebral disc (IVD) degeneration is a major cause of low back pain (LBP) worldwide which is expected to affect 80% of the world’s population. IVD degeneration (IDD) is a key player in the degenerative cascade associated with LBP. Pro-inflammatory cytokines and mediators, such as nitric oxide, have been shown to be triggers and mediators of IDD. Due to the avascular nature of the adult IVD, the disc is unable to heal or regenerate when damaged. The multi-components of the IVD, namely glycosaminoglycan (GAG)-rich nucleus pulposus (NP), a concentric collagen dense annulus fibrosis (AF), and cartilage endplates (CEPs), further complicate possible regenerative solutions. Cell therapies show promise. This is supported by studies that demonstrate the use of mesenchymal stem cells (MSCs) in animal models showing potential in mitigating inflammatory signaling as well as recovering proteoglycan content. Despite these promising findings, several gaps in knowledge remain. While the biochemical and mechanical properties of an injured disc (via physical or chemical stimulation) have been characterized, the resulting inflammatory signaling cascades remain undefined. A growing body of evidence suggests that TLR4 is involved in the pathogenesis of the IVD. However, it is unknown how TLR4 mediates injury responses of the IVD. Second, it is unknown how mechanical loading of IVDs can influence the transcriptome or secretome of the IVD. The IVD is normally exposed to multimodal loading (e.g., compression, tension, shear, hydrostatic pressure, and osmotic pressure). Both frequency and magnitude regulate whether loading is beneficial or detrimental to disc integrity, which will be explored. Furthermore, the secretome of the IVD, especially during loading, may be essential to creating therapies targeted for regeneration of the IVD. There may be key, distinct paracrine factors that are released in IVD conditioned loading media which can influence the regenerative and anti-inflammatory capabilities of cell-based therapies. To address these gaps, this thesis describes a series of experiments employing novel ex vivo organ culture model to study the response of the IVD to various injury modalities (inflammatory stimulation, puncture injury, compressive loading), and resulting changes in inflammatory, biomechanical, and biochemical responses. Through methods such as RNA sequencing and proteomics, we now have expanded the characterization to beyond candidate genes or proteins, and are more informed on (1) the IVD response to injury, (2) the role of TLR4 signaling in this ex vivo organ culture model, in addition to (3) the downstream effects of loading and how paracrine factors can be used to improve and develop potential cell and molecular therapies. Sex-based differences, in male and female rat caudal IVDs, were also identified and are analyzed in the context of response to injury.

In the studies about ovarian cancer cells, basal iNOS expression in the chemosensitive OV2008 cells was significantly higher than in the chemoresistant C13* cells. Cisplatin further increased iNOS expression in OV2008 cells, but had no effect in C13* cells. Furthermore, cisplatin dramatically reduced the expression levels of eNOS and nNOS, but again only in OV2008 cells. The data suggest that failure of cisplatin to upregulate iNOS and downregulate eNOS and nNOS in C13* cells could be an etiological factor in chemoresistance. Addition of exogenous NO at high levels, using SNAP, significantly increased p53 protein levels and caused apoptosis in both cell types. Specific iNOS inhibitor (1400W) partially blocked the pro-apoptotic effects of cisplatin in OV2008 cells, suggesting involvement of iNOS in cisplatin-induced apoptosis. However, blocking of all three isoforms of NOS with NG-amino-L-arginine in C13* cells dramatically changed these cells from chemoresistant to chemosensitive, greatly potentiating the pro-apoptotic effects of cisplatin. / Inhibition of Src-kinase activity reduces DNA synthesis in ovarian cancer cells. In an in vitro experiment, Src phosphorylated PKG on a tyrosine residue and PKG, presumable via serine-phosphorylation of Src, enhanced Src auto(tyrosine)phosphorylation. In ovarian cancer cells, inhibition of basal PKG activity with DT-2 decreased both basal and EGF-stimulated Src kinase activation and DNA synthesis. The data suggest that PKG at basal activity, is necessary for both basal and growth factor-stimulated Src kinase activation and enhanced DNA synthesis in human ovarian cancer cells. / The novel role of sGC/cGMP/PKG pathway on stimulating cell proliferation, potentially via interaction with the Src kinase pathway in human ovarian cancer cells, was demonstrated. ODQ dramatically reduced DNA synthesis rates, suggesting that basal sGC activity and basal cGMP levels are needed for ovarian cancer cell proliferation. DT-2 also reduced cell proliferation, suggesting the direct involvement of PKG. ANP and BNP had no effect on cell proliferation, suggesting that further activation of cGMP/PKG pathway above basal levels does not further enhance cell proliferation. / The present study also demonstrated that elevating cGMP slightly above the basal levels further protects pancreatic islet cells against spontaneous onset of apoptosis. The results showed that natriuretic peptides (both ANP and BNP) and low-level NO (i.e. physiological levels) as supply by NO donor, S-nitroso-N-acetylpenicilamine (SNAP) further prevented spontaneous apoptosis in pancreatic islets after isolation, whereas NO at high concentrations (i.e. pathological levels) promoted apoptosis in pancreatic islet cells. The commonly-used PKG inhibitor KT5823 and the newly-developed specific PKG inhibitor DT-2 completely prevented anti-apoptosic effect of ANP, suggesting the direct involvement of PKG in protection against spontaneous apoptosis. / The present study demonstrated that basal activity of sGC/cGMP/PKG signaling pathway is essential for partially limiting spontaneous apoptosis in pancreatic islet cells. The sGC inhibitor ODQ caused induction of apoptosis, which was completely blocked by co-treatment with ANP or BNP, agents that elevate cGMP via pGC, bypassing the ODQ block. Co-treatment with 8-Br-cGMP, a direct activator of PKG also completely prevented ODQ-induced apoptosis in islets. / Leung Lai-han. / "July 2006." / Adviser: Ronald Ray Fiscus. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1483. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 175-191). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Abstract not available

Zhang Rongzhen. / "July 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 130-176). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Many members of the Phylum Cnidaria are mutualistic with unicellular dinoflagellates belonging to the genus Symbiodinium. Corals are the most widely recognized example of these associations due to their key ecological importance in coral reef ecosystems where they serve as the structural and trophic foundation of these rich ecosystems. Coral reefs are severely threatened by human activities worldwide and are at great risk from global climate change, in particular the increase in seasurface temperatures. Detailed knowledge of how corals respond to stress is scarce. The most serious and immediate response of corals to environmental stress is a process referred to as coral bleaching (a.k.a. cnidarian bleaching). Nevertheless, the cellular and molecular processes by which elevated temperatures elicit the bleaching response are poorly understood. This dissertation deals with this important question by describing two mediators of cnidarian bleaching in the model symbiotic tropical sea anemone Aiptasia pallida (Verril), namely nitric oxide and cyclophilin. After an introduction to the topic of cnidarian-algal symbioses and cnidarian bleaching (Chapter 1), I present results from a study describing the involvement of nitric oxide (NO) in the anemone A. pallida (Chapter 2). Elevated temperature as well as oxidative stress induces production of NO and exposure of A. pallida to NO induces bleaching at non-stressful temperatures. Co-incubation with an NO scavenger suppresses bleaching. I propose that the host up-regulates NO production in response to elevated oxidative stress and that this situation leads to cytotoxicity and bleaching. Chapter 3 examines the role of cyclophilin from A. pallida in the regulation of the symbiosis. Cyclophilins belong to a highly conserved family peptydyl-prolyl cistrans isomerases (PPIases). Incubation of A. pallida with cyclosporin A (CsA), a potent inhibitor of cyclophilin resulted in bleaching and a decrease in tolerance to elevated temperatures. Protein extracts from A. pallida exhibited CsA-sensitive PPIase activity. Laser scanning confocal microscopy using superoxide and nitric oxide-sensitive fluorescent dyes on live A. pallida revealed that CsA strongly induced the production reactive oxygen species as well as NO. We tested weather the CsAsensitive isomerase activity is important for maintaining the activity of the antioxidant enzyme superoxide dismutase (SOD). SOD activity of protein extracts was not affected by pre-incubation with CsA in vitro. In Chapter 4 I review what is known about the molecular and cellular mechanisms of bleaching and describe a model of bleaching based on the results presented herein as well as studies of non-cnidarian models. / Graduation date: 2007

Abstract not available