Thesis (Ph.D.)--Brown University, 2008. / Vita. Advisor : Laurent Brossay. Includes bibliographical references.

Acquired immunity in vertebrates is a major mechanism of the immune system, which enables the vertebrates to discriminate self and non-self The most important feature of acquired immunity is the major histocompatibility complex (MHC) restricted-cellular immune response. This involves several important elements including MHC molecules and T cell antigen receptor (TCR) complex. MHC molecules are glycoproteins that recognise and present self and foreign peptides to T-cells via the TCR, causing the T cell to proliferate and secrete cytokines. There is increasing evidence that peripheral T cells and MHC restricted cellular immune responses occur in all vertebrates. However, the exact nature of the TCR and MHC in lower vertebrates has still to be established. The aim of this thesis was firstly, to use the polymerase chain reaction (PCR) in conjunction with degenerative primers to the TCR β chain variable region (TCRBV) sequences of the homed shark to allow the amplification of the TCR-like genes from dogfish, Scyliorhinus canicula; secondly, to employ a similar strategy using degenerative primers to the banded houndshark MHC class-I α3 DNA sequence, to amplify MHC genes from S. canicula. Using the degenerative primers, four clones from dogfish genomic DNA were obtained. These clones showed homology to TCR genes of other species. The amino acid sequence of clone 2C144 showed homology to the TCRBV region of several species. The highest similarities include 51.7% with the honed shark TCRBV, 45.1% with human TCRBV, 41% with monkey TCRBV, 40.7% with bovine TCRBV and 38.8 and 37.5% with chimpanzee and rat TCRBV, respectively. The clone 6C43 also showed homology to the TCRBV genes of several species (similarity is between 21.1% to 23.9%). Another two clones (6C53 and 6C54) were identified that showed high homology to the TCR δ chain variable region (TCRDV) of other vertebrates with 45.7% identity to the honed shark TCRDV region, 43.2% identity to mouse TCRDV, 32.2% identity to porcine TCRDV region, and 30.7% identity to human TCRDV. The highly conserved residues in the other vertebrates TCR are present in these four clones as well, such as WYRQ37 and YY(F)C92 motifs. Southern blotting analysis with the putative TCRBV (2Cl44) and TCRDV (6C53 and 6C54) suggested that polymorphism existed between different fish. Northern blotting analysis with the probe 6C53 identified a transcript of approximately 2 kb in the spleen., lymphocytes and brain as well, with the probe 2C144 identified a transcript of approximately 2 kb in the lymphocytes of dogfish. The sequences of nine clones obtained using cDNA as a template from dogfish together with degenerative primers showed high homology to the membrane-proximal domain of the MHC class II α chain in several species. These clones exhibited a high degree of homology to the nurse shark MHC class II α chain (74.1%), zebrafish MHC class II α chain (52.8%), mouse MHC class II I-A (49.6%), rat MHC class II α chain (49.9%), bovine MHC class II DYα chain and DQα chain (52.7% and 44.9%, respectively), human HLA- DP, - DQα and -DRα chain (44.4%) respectively. The cysteine residues of the membrane-proximal domain are conserved in the dogfish, suggesting that it may have a similar tertiary structure to mammalian MHC class II proteins. A highly conserved tryptophan residue at position 121 was found in the dogfish and an N-linked glycosylation site was present at position 133, whilst in higher mammals it is usually found at position 118. The Southern blotting analysis using probe DM9 showed that there may be more than four loci of MHC class IIA in dogfish. These results suggest that the MHC class II, TCRB and TCRD genes are present in dogfish. The dogfish may have distinct T-cells, expressing either αlβ or γ/δ heterodimers. Conserved key residues in both MHC class IIA. TCRB and TCRD suggest that these genes may encode functional MHC class II, TCRB and TCRD molecules. The MHC restricted cellular immune response may be present in dogfish. Dogfish TCRB, TCRD and MHC class II genes show high homology with several species including human, rat, bovine and other fish species, which implies that the immunoglobulin (Ig) superfamily has evolved from a common ancestor.

Alcohol abuse is an enormous problem causing death and disability to over 43 million people worldwide each year (WHO). Chronic alcohol consumption also contributes to abnormal brain morphology and significant brain volume loss indicative of neurodegeneration. Until there are effective treatments to alter maladaptive behavioral patterns in alcohol abuse, more research is needed to prevent alcohol-induced toxicity and degeneration. We used C. elegans as a model system to identify genetic modulators of alcohol toxicity and explored whether prolonged alcohol exposure damages the nervous system. In our study, we exposed L4-larval stage worms to varying concentrations of ethanol for three days and found a dose-dependent deficit in crawling. Furthermore, we evaluated degeneration by assessing the health of neurons using fluorescent reporters. Compared to the untreated group, we found that ethanol-exposed worms had a significant neurodegeneration. Previous findings using C. elegans have suggested that the innate immune pathway may protect against neurodegeneration caused by drug toxicity (Schreiber & McIntire, 2012). We find that deletion of either the innate immune gene nsy-1 (orthologous to the mammalian ASK-1 MAPKKK) or pmk-1 (orthologous to the mammalian p38 MAPK) caused hypersensitivity to ethanol toxicity. Conversely, boosting innate immune signaling via gain-of-function mutation in nsy-1 produced resistance to ethanol toxicity and ameliorated ethanol-induced cholinergic degeneration. Our findings indicate that prolonged exposure to ethanol leads to both behavioral impairments and neuronal degeneration in C. elegans and that the ASK1/p38 MAP kinase pathway may play a role in ethanol-induced damage to the nervous system. / text

No description available.

The peripheral blood of O.mossambicus was examined using light and electron microscopy and was found to contain four forms of leucocytes: lymphocytes, thrombocytes, monocytes and three types of granulocytes. The monocyte and two types of granulocyte were found to be phagocytic and ingest colloidal carbon and bacteria. The alimentary tract was found to contain a number of leucocytes, some showing a morphological similarity to those in the peripheral blood, while others were unique to the gut tissue. These intestinal leucocytes were found mainly as a diffuse cell population in the epithelium and lamina propria, and only occasionally as discrete lymphoid accumulations within the gut tissue. Ontogenic studies showed that a limited number of leucocytes were found in the gut tissue after hatching, however, there was a gradual increase in these numbers once exogenous feeding began. The intestinal enterocytes of both the anterior and posterior intestine were found to take up intubated macromolecules. An electron microscopical investigation revealed that these macromolecules were absorbed by pinocytosis and were found within large intraepithelial macrophages. These macromolecules were also absorbed and transported into the systemic circulation. In juvenile fish macromolecules were detected in the plasma following both oral and anal intubation, however, in adult fish they were detected in the plasma only after anal intubation, and in smaller quantities. Macromolecular absorption in O.mossambicus was compared to that in two other fish species, Cyprinus carpio and Sa1mo gairdneri, and it was found that higher levels of absorbed macromolecules were found in the plasma of O.mossambicus. Bovine serum albumin absorption by the gut of the three species revealed that both the 'intact' macromolecule and smaller antigenic fragments, probably resulting from enzymatic modification, were ansorbed and transported into the plasma.

No description available.

No description available.

Nod1 and Nod2 are intracellular pattern recognition receptors that detect specific moieties of peptidoglycan, a critical component of the bacterial cell wall, to initiate host innate immune responses. Importantly, mutations in the human NOD2 gene have been associated with increased risk to develop mucosal auto-inflammatory disorders such as Crohn’s Disease. However, how Nod1 and Nod2 mediate mucosal homeostasis still remains unclear. In Chapter 2, I determined that mice deficient for both Nod1 and Nod2 (Nod1-/-Nod2-/-) exhibited delayed induction of intestinal inflammation at early timepoints after infection with Citrobacter rodentium compared to wild-type mice, which correlated with compromised control of the pathogen at later timepoints. Notably, I determined that induction of the cytokines IL-17 and IL-22 in the cecal lamina propria (LP) was blunted in Nod1-/-Nod2-/- mice after infection with either C. rodentium or Salmonella enterica serovar Typhimurium. Importantly, I found that Th17 cells were the principal producers of IL-17 and IL-22 after infection. Due to the rapid kinetics of activation and the regulation by Nod1 and Nod2, I termed this early mucosal response the innate Th17 (iTh17) response. The iTh17 cells exhibited an effector memory phenotype and required priming from the enteric microbiota for full induction. Therefore, in Chapter 3, I next determined that major histocompatibility complex (MHC) class II expression in hematopoietic cells was required for the induction of LP Th17 responses after infection. Interestingly, I found that the percentage IL-17+CD8+ T cells was strongly upregulated when MHCII signaling was ablated, suggesting a dynamic compensatory mechanism of IL-17-producing T cell responses in the mucosa. In Chapter 4, I identified MDP(D-Glu2)-OCH3 as a synthetic Nod2 agonist that exhibited increased stimulatory ability of Nod2-dependent NF-B activation compared to MDP in an unbiased screen. Moreover, I determined that MDP(D-Glu2)-OCH3 induced more potent inflammatory responses both in vitro and in vivo and was a better adjuvant than MDP. Together, the data presented in this thesis expand our current understanding of the roles of Nod1 and Nod2 in the intestinal LP, the regulation of IL-17 producing T cells in the gut and the therapeutic potential of novel Nod2 agonists.

Nod1 and Nod2 are intracellular pattern recognition receptors that detect specific moieties of peptidoglycan, a critical component of the bacterial cell wall, to initiate host innate immune responses. Importantly, mutations in the human NOD2 gene have been associated with increased risk to develop mucosal auto-inflammatory disorders such as Crohn’s Disease. However, how Nod1 and Nod2 mediate mucosal homeostasis still remains unclear. In Chapter 2, I determined that mice deficient for both Nod1 and Nod2 (Nod1-/-Nod2-/-) exhibited delayed induction of intestinal inflammation at early timepoints after infection with Citrobacter rodentium compared to wild-type mice, which correlated with compromised control of the pathogen at later timepoints. Notably, I determined that induction of the cytokines IL-17 and IL-22 in the cecal lamina propria (LP) was blunted in Nod1-/-Nod2-/- mice after infection with either C. rodentium or Salmonella enterica serovar Typhimurium. Importantly, I found that Th17 cells were the principal producers of IL-17 and IL-22 after infection. Due to the rapid kinetics of activation and the regulation by Nod1 and Nod2, I termed this early mucosal response the innate Th17 (iTh17) response. The iTh17 cells exhibited an effector memory phenotype and required priming from the enteric microbiota for full induction. Therefore, in Chapter 3, I next determined that major histocompatibility complex (MHC) class II expression in hematopoietic cells was required for the induction of LP Th17 responses after infection. Interestingly, I found that the percentage IL-17+CD8+ T cells was strongly upregulated when MHCII signaling was ablated, suggesting a dynamic compensatory mechanism of IL-17-producing T cell responses in the mucosa. In Chapter 4, I identified MDP(D-Glu2)-OCH3 as a synthetic Nod2 agonist that exhibited increased stimulatory ability of Nod2-dependent NF-B activation compared to MDP in an unbiased screen. Moreover, I determined that MDP(D-Glu2)-OCH3 induced more potent inflammatory responses both in vitro and in vivo and was a better adjuvant than MDP. Together, the data presented in this thesis expand our current understanding of the roles of Nod1 and Nod2 in the intestinal LP, the regulation of IL-17 producing T cells in the gut and the therapeutic potential of novel Nod2 agonists.

The essay discusses judicial jurisdiction over workplace injury claims against a state in the former Soviet Union Countries. Claiming that such cases should be dismissed in foreign jurisdiction, the paper seeks explanation to different approach and different outcomes of workplace injury cases in the courts of the same countries. The essay begins with background information on particularities of unusual workplace injury cases which emerged in connection with important political event - collapse of the USSR. Relevant provisions of domestic and . international law on judicial jurisdiction, their interpretation and application in Commonwealth of Independent States are discussed in this paper. Analyzing provisions and reasons of different decisions, the essay infers the implications from analysis in support of its main claim.