This study reports the localization and partial characterization of thioredoxin from the parasitic trematode Fasciola hepatica. Snails (Pseudosuccinia columella) were raised in culture and infected with F. hepatica so that Western blotting and immunohistochemical techniques could be utilized to determine the presence of thioredoxin in different stages of the parasite’s development. The results of these experiments showed that thioredoxin was present in the tegument, gut epithelium, excretory canal epithelium and sperm, of the adult parasite as well as in the tegument and gut of the redia and cercaria intermediate stages. In situ hybridization was used to determine the localization and possible differential mRNA expression of two different F. hepatica thioredoxin isotypes (Fh2020.A and Fh2020.SL) in the adult parasite. The in situ hybridization results showed that both isotypes are expressed in the tegument and gut epithelium. Fh2020.A stains with a greater intensity possibly demonstrating a difference in the amount of expression between the two isotypes. Recombinant F. hepatica thioredoxin expressed in bacteria using the pMAL™ Protein Fusion and Expression System was used to test its affects on the production of super oxide anion by murine peritoneal macrophages, bovine monocyte-derived macrophages and bovine whole blood neutrophils, and nitric oxide production by mouse peritoneal macrophages and bovine monocyte-derived macrophages. The results of the cellular assays were not definitive due to the fact that the maltose binding protein (MBP) moiety of the recombinant thioredoxin, when tested alone, increased production of nitric oxide by bovine monocyte-derived macrophages. Consequently, since the MBP could not be effectively separated from the thioredoxin portion of the recombinant, allowing the thioredoxin affects to be tested independently, no true conclusions regarding its affects on the host immune cells tested could be drawn. This is the first report of the localization of thioredoxin in both the adult F. hepatica as well as in specific intermediate stages of the parasite. These studies demonstrate the possible affects that a protein tag can have on experimental results and demonstrate how such data may be interpreted when a non-cleaved recombinant protein is used in cellular or other assays when compared to native or cleaved recombinant proteins.

<p>Biofilms are communities of sessile microorganisms attached to a surface and imbeddedin a matrix of extracellular polysaccharide substances. These communities can be foundin diverse aquatic environments, such as in industrial pipes and in humans. By formingmicrocolony structures, which are highly resistant to adverse physical conditions as wellas antimicrobial agents, biofilms are very problematic when associated with e.g.persistent infections. In order to find new ways of controlling biofilm growth, theprocesses involved in biofilm development must be investigated further. The maininterest of this study is the occurrence of void formation inside biofilms. Thisphenomenon has been observed in several studies and has been correlated to cell deathinside the microcolonies. The occurrence of cell death has recently been associated withthe presence of nitric oxide in the biofilm. In this study, the implications of nitric oxideaccumulation on biofilm development were investigated using an individual-basedmodel. Specifically, the role of nitric oxide in void formation was considered. A largenumber of simulations were run using different parameter settings in order to determine ifnitric oxide could account for the occurrence of void formation observed experimentally.The general predictions made by the model system showed agreement to someexperimental data, but not to others. Sloughing, the detachment of chunks of cells fromthe biofilm, was observed in the majority of simulations. In some cases, the model alsopredicted the presence of live cells inside the voids, which has been observedexperimentally.</p>

Tez (Doktora) - Süleyman Demirel Üniversitesi, Sağlık Bilimleri Enstitüsü, Histoloji ve Embriyoloji Anabilim Dalı, 2008. / Kaynakça var.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed on Feb. 4, 2010). Includes bibliographical references (p. 120-131).

Thesis (Ph. D.)--Ohio State University, 2003. / Title from first page of PDF file. Document formatted into pages; contains xii, 152 p. : ill., (some col.). Includes abstract and vita. Advisor: Anthony Young, Molecular, Cellular, and Developmental Biology Program. Includes bibliographical references (p. 137-150).

No description available.

While cellular levels of arginine greatly exceed the apparent Km for endothelial nitric oxide synthase (eNOS), nitric oxide (NO) production is limited by availability of arginine. Results from this work have provided a unique understanding of endothelial NO production, showing that arginine regeneration, that is the recycling of citrulline back to arginine by argininosuccinate synthase (AS) and argininosuccinate lyase (AL), defines the essential source of arginine for NO production. Using RNA interference analysis, selective reduction of AS expression was shown to directly correspond with a diminished capacity of endothelial cells to produce NO, despite saturating levels of arginine in the medium. In addition, the viability of AS siRNA-treated endothelial cells was compromised due to apoptotic cell death.AS expression was also investigated in response to two major vascular effectors. Tumor necrosis factor (TNF)-alpha; which is known to impair endothelial NO production, was shown to provoke a dose-dependent reduction of AS expression that corresponded to a decrease in NO production. Furthermore, TNF-alpha was shown to suppress AS expression through a NFkappaB mediated pathway, which involves three essential Sp1 elements in the proximal AS gene promoter. On the other hand, peroxisome proliferator-activated receptor gamma (PPARgamma) agonists, troglitazone and ciglitazone, which are known to elicit a vascular protective response against TNF-alpha effects, were shown to coordinately induce NO production and AS expression via a PPARgamma response element in the distal AS gene promoter. Importantly, these PPARgamma agonists were shown to restore AS expression and NO production following down-regulation by TNF-alpha, consistent with their vascular protective properties.

 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