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The aquaculture sector is currently thriving, and has expanded to meet the demand for fish and shellfish as an alternative protein source to meat. This is especially true for high value products such as Atlantic salmon, where in Scotland salmon farming is reported to be worth> £1 billion to the national economy. Currently around 40% of farmed fish and shellfish destined for human consumption are derived from aquaculture. Therefore, a great deal of attention is paid to problems that the industry faces, with fish diseases of paramount importance. A variety of species of bacteria, viruses and parasites are common in the aquatic environment, which can result in serious diseases amongst fish stocks. As a result, ways to improve disease resistance have been the focus of much attention, with the use of vaccines considered a desirable way forward. However, other approaches are also followed, such as the use of immunostimulants to improve fish health in a more limited, non-specific way, or the use of genetic markers to allow selective breeding of important disease resistance traits. For all of these approaches more information is needed on the pathways that give rise to disease resistance in fish in different situations, to allow their manipulation or monitoring, and the studies in this thesis are directed towards this goal. Fish has been used as a model to study the evolution of vertebrate immunolity for some deacades, especially work on humoral immune responses where knowledge on antibody production has dominated much of the literature on fish immunology. In contrast, little known about specific cell-mediated immunity in fish, even though it also likely plays an important role in the immune system and disease resistance. Therefore, this thesis has been focused on analysing such responses, taking advantage of the recently discovered cytokines of adaptive immune responses in fish, which allow transcriptomic studies in particular to look at the molecules turned on during infection and after vaccination. Thus the goal of this thesis was to take advantage of some successful vaccines that exist for rainbow trout, and examine the gene expression changes that occur in vaccinated trout post-challenge with the homologus pathogen, and to try to dissect pathways that may correlate with disease resistance in this species.

African trypanosomiasis remains a major health problem to both humans and animals due to lack of effective treatment or vaccine to control the disease. Animal trypanosomiasis is considered one of the most important diseases affecting livestock production and agricultural development in sub-Saharan Africa. Although the use of trypanocides remain the most important method for controlling the disease in animals, the mechanisms of action of these compounds are not completely known. The overall aim of this thesis is to decipher the molecular mechanisms involved in Trypanosoma congolense-induced cytokine production and how this is modulated by the trypanocide, Diminazene aceturate (Berenil). First, I investigated the molecular and biochemical mechanisms of action of Berenil to determine whether in addition to trypanolytic effect, it exerts a modulatory effect on the host immune system. Although it is known that T. congolense infection in mice is associated with increased production of pro-inflammatory cytokines by macrophages, the intracellular signaling pathways leading to the production of these cytokines remain unknown. Therefore, I investigated the innate receptors and intracellular signaling pathways that are involved in T. congolense-induced pro-inflammatory cytokine production in macrophages. Next I further determined whether the inhibitory effect of Berenil on proinflammatory cytokine production in macrophages is specific to T. congolense. I found that Berenil treatment significantly reduced the immune activation and proinflammatory cytokine production in infected mice suggesting that in addition to its direct trypanolytic effect, Berenil also modulates the host immune response to the parasite. Next, I show that T. congolense induced pro-inflammatory cytokine production in macrophages is dependent on phosphorylation of mitogen-activated protein kinase (MAPK) and signal transducer and activation of transcription (STAT) proteins in a TLR2-dependent manner. I further show that Berenil treatment downregulates T. congolense as well as LPS induced cytokine production by affecting the phosphorylation of MAPK and STAT proteins. Collectively, the results from this thesis provide novel insights into T. congolense-induced activation of the innate immune system and modulation of host immune response by Berenil. These findings are significant and could help in developing newer and better therapeutic strategies against the disease, in particular, and inflammatory responses in general. / October 2016

Chronic pain is a major health problem worldwide. We have recently demonstrated the analgesic effect of the nitroxyl donor, Angeli's salt (AS) in models of inflammatory pain. In the present study, the acute and chronic analgesic effects of AS was investigated in chronic constriction injury of the sciatic nerve (CCI)-induced neuropathic pain in mice. Acute (7th day after CCI) AS treatment (1 and 3 mg/kg; s.c.) reduced CCI-induced mechanical, but not thermal hyperalgesia. The acute analgesic effect of AS was prevented by treatment with 1H-[1,2, 4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, a soluble guanylate cyclase inhibitor), KT5823 (an inhibitor of protein kinase G [PKG]) or glibenclamide (GLB, an ATP-sensitive potassium channel blocker). Chronic (7-14 days after CCI) treatment with AS (3 mg/kg, s.c.) promoted a sustained reduction of CCI-induced mechanical and thermal hyperalgesia. Acute AS treatment reduced CCI-induced spinal cord allograft inflammatory factor 1 (known as Iba-1), interleukin-1β (IL-1β), and ST2 receptor mRNA expression. Chronic AS treatment reduced CCI-induced spinal cord glial fibrillary acidic protein (GFAP), Iba-1, IL-1β, tumor necrosis factor-α (TNF-α), interleukin-33 (IL-33) and ST2 mRNA expression. Chronic treatment with AS (3 mg/kg, s.c.) did not alter aspartate aminotransferase, alanine aminotransferase, urea or creatinine plasma levels. Together, these results suggest that the acute analgesic effect of AS depends on activating the cGMP/PKG/ATP-sensitive potassium channel signaling pathway. Moreover, chronic AS diminishes CCI-induced mechanical and thermal hyperalgesia by reducing the activation of spinal cord microglia and astrocytes, decreasing TNF-α, IL-1β and IL-33 cytokines expression. This spinal cord immune modulation was more prominent in the chronic treatment with AS. Thus, nitroxyl limits CCI-induced neuropathic pain by reducing spinal cord glial cells activation.