Meagre and the greater amberjack are promising emerging species in aquaculture. This is due to their fast growth rate, large size, high processing yield and high quality fillet. However, little is known about the immune system of these fish and several potential pathogenic threats to sustainable culture of these fish have been identified. Therefore, this thesis has begun to characterise the immune response of these species in order to better combat pathogenic threats. The focus of this thesis is the identification, modulation and monitoring of key innate and adaptive immune genes. This is achieved by identifying conserved regions of target gene sequence and designing consensus primers to these in order to generate a partial sequence, which could then be used as a base from which RACE PCR could be performed to obtain full sequence. The identified sequence was then used to produce qPCR primers, anti-meagre IgM and anti-meagre IgT monoclonal antibodies and a functional synthetic greater amberjack piscidin active peptide. Using the developed qPCR primers the constitutive expression of target genes was determined in the gills, gut, head kidney and spleen. Changes in transcript expression were then monitored in these tissues in response to PAMP stimulation in vivo and in vitro in order to better understand how these genes are modulated by viral, bacterial and fungal stimuli. Furthermore, the meagre development of the immune system was monitored from 1 day post hatch (dph) until 120 dph, providing insights into when immune maturity is achieved. Monoclonal antibodies were also produced and their viability tested in a variety of assays including Western blot, Immunohistochemistry and ELISA. Finally, A synthetic greater amberjack piscidin was produced and the bacteriostatic capabilities of this peptide were tested and showed promising results against known bacterial fish pathogens, indicating a potential for future uses within and outwith aquaculture.

Plants have evolved a plethora of effective mechanisms to protect themselves from biotic stresses. Jasmonates (JAs) are employed as vital defence signals against both insect and pathogen attack. Jasmonic acid (JA) signalling plays a central role in plant defence and development. S-nitrosylation, a redox-based post-translational modification plays an important role in plant disease resistance. S-nitrosoglutathione (GSNO) is formed by the reaction of antioxidant glutathione (GSH) and nitric oxide (NO) and acts as a mobile reservoir of NO bioactivity. The Arabidopsis thaliana S-NITROSOGLUTATHIONE REDUCTASE (AtGSNOR1) controls multiple modes of disease resistance via S-nitrosylation. In this context, the Arabidopsis lossof- function mutant atgsnor1-3 exhibits higher susceptibility to Botrytis cinerea a necrotrophic pathogens and Pieris rapae insect attack. Accumulation of JA was reduced in atgsnor1-3 after mechanical wounding. JA marker genes were also downregulated in atgsnor1-3 compared to Col-0 after Methyl Jasmonate (Me-JA) treatment. The relative gene expression of Vegetative Storage Protein (VSP) was reduced in atgsnor1-3 compared to wild type. Further, protein-protein interaction experiments in yeast two hybrid assays revealed an inhibition of Coronatine-insensitive 1 (COI1) and Jasmonate ZIM domain (JAZ1) interactions upon NO donor application. Interestingly it was also shown that Nitric oxide donor may inhibited the degradation of JAZ1-β-glucoronidase (GUS) fusion protein driven by a CaMV35s:: JAZ1-GUS transgene in GUS histochemical analysis but not in flurometric assay. A biotin switch assay of recombinant JAZ1-Maltose-binding protein (MBP) has shown that JAZ1-MBP was S-nitrosylated and mass spectrometry suggested Cysteine229 (Cys229) was the site of this modification. Further, CaMV35S::JAZ1-Flag transgene expressed in either a wild-type or atgsnor1-3 genetic background, suggested that JAZ1 was S-nitrosylated in vivo. Collectively, our data imply that JA-signalling engaged in response to either insect predation or attempted B. cinerea infection is under redox control as high SNO in atgsnor1-3 has disrupted the JA signalling pathway. Furthermore, our data suggest that S-nitrosylation of Cys-229 of JAZ1 may control JA-mediated signalling by blocking the interaction of this protein with COI1, thus reducing the turnover of JAZ1 by the 26S proteasome and consequently enabling continued JAZ1-mediated repression of JA-dependent gene expression in the presence of Me-JA. Thus our findings highlight the importance of NO and associated S-nitrosylation in JA signalling during plant immune response.

The immune system’s vitality and function is of the upmost importance in the human body. The ingenuity and performance of this defence mechanism also plays a role in the prevention of mutated, transformed cells becoming malig- nant tumours (cancers). More recently, the subject of cancer immunology has been concerned with examining the local effects the immune system has on a pre-angiogenic tumour site. A recent immunological review article - “The Three Es of Cancer Immunoediting” - discusses the way the immune system interacts with cancer cells: elimination, equilibrium, and escape. This bio- logical explanation underpins the mathematical modelling in this PhD thesis where mathematical models of pre-angiogenic immune-tumour interactions are presented and analysed. Chapter two develops an individual-based model of immune-cancer cell interactions using the computational simulation plat- form, CompuCell3D, to extend an earlier spatio-temporal model of tumour dormancy (Matzavinos et al. [2004]). Chapter three investigates and analyses an ODE model of the interaction of two immune cells and two tumour cells. This model is extended to include spatial movement terms for the tumour and immune cells (in both one and two dimensions) and investigates the rich heterogeneous spatio-temporal dynamics of the system in the presence of a limit cycle in the reaction kinetics. Finally, chapter four extends the models in the previous two chapters by examining an individual based model of two immune cell populations interacting with two tumour cell populations.

In this thesis mathematical models describing the growth of a solid tumour in the presence of an immune response are presented. Specifically, attention is focused on the interactions between cytotoxic T-lymphocytes (CTLs) and tumour cells in a small, avascular multicellular tumour. At this stage of the disease the CTLs and the tumour cells are considered to be in a state of dynamic equilibrium or cancer dormancy. The precise biochemical and cellular mechanisms by which CTLs can control a cancer and keep it in a dormant state are still not completely understood from a biological and immunological point of view. The mathematical models focus on the spatio-temporal dynamics of tumour cells, immune cells, chemokines and “chemo-repellors” in an immunogenic tumour. The CTLs and tumour cells are assumed to migrate and interact with each other in such a way that lymphocyte-tumour cell complexes are formed. These complexes result in either the death of the tumour cells (the normal situation) or the inactivation of the lymphocytes and consequently the survival of the tumour cells. In the latter case, we assume that each tumour cell which survives its “brief encounter” with the CTLs undergoes certain beneficial phenotypic changes. We explore the dynamics of the model under these assumptions and show that the process of the immuno-evasion can arise as a consequence of these encounters.Our computational simulations suggest that the proposed mechanism is able to mimic various dynamics of immunoevasion during the lifespan of a mouse. We also highlight the differential spatiotemporal contributions to evasion due, respectively, to: i) a decrease in the probability pi of being lethally hit; ii) a decrease in the probability, embedded in k+ i , that a tumour cell is recognized by a CTL. In particular, our model suggests that a decrease in the parameters pi is needed to produce evasion, which does not occur in the case where pi remains constant at its baseline level inferred from the experimental data. However, the role of the parameters k+ i is important since it can greatly accelerate the simulated process. Moreover, our computational simulations also show that the proposed mechanism can also deeply affect the spatial patterning of the tumour. In particular, our model suggests that to have a uniform invasion profile for the tumour cells necessitates also having a decrease in the recognition rate, embedded in the parameters k+ i . These parameters also differentially shape the spatial distribution of the various classes of tumour cells. Also in this thesis, we discuss mathematical models of the interactions between a tumour and both the innate and the cellular part of the adaptive immune system. We have developed and formulated spatiotemporal models of the interactions between macrophages, natural killer cells, cytotoxic T lymphocytes and tumour cells. In addition to presenting computational simulations of our ODE and PDE models, we investigate the linear stability analysis of steady states of the model and the effect of the initial conditions on the behaviour of the ODE solution. We show that limit cycle behaviour could be obtained by making some changes in the parameter values, which gave us oscillations in the solution of the ODE and PDE systems. We observe that there is a slowly damped oscillation in the behaviour of the tumour, natural killer and CTL cells. Also we note that the solution converges to the second steady state where the tumour size is small (dormant state).A model of cancer invasion and metastasis is also discussed in this thesis. This model attempts to describe the interactions between cancer cells, urokinase plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), plasmin, extracellular matrix (ECM) and the immune response. The mathematical model focuses on the effect of the immune response on cancer invasion by assuming that there is some form of limit cycle behaviour between the cancer cells and the effector cells. The work we present in this chapter develops a mathematical model for tumour invasion with an immune response using a continuum model in 1 and 2 space dimensions. This model consists of a system of nonlinear partial differential equations and examines the effector cell response the tumour invasion. This model consists of effector cells, tumour cells, ECM, uPA, PAI-1, and plasmin. First, we set all spatial components of the model to zero and consider only the reaction kinetics in order to compare between the behaviour of our model and the original Chaplain and Lolas model (Chaplain and Lolas, 2005). The spatially homogeneous simulation shows the behaviour of solutions have regular oscillations because there is a closed orbit. Second, we present the computational results of the spatio-temporal model, and we note from these simulations that the tumour size of our model is smaller than the tumour size of the Chaplain and Lolas model because the immune cells are interacting with the tumour cells, and also the degradation of ECM is less than that in the Chaplain and Lolas model. In addition, the number of tumour cell clusters in our model is less than those in the Chaplain and Lolas model. Also we found the tumour clusters of the mathematical model which was discussed in this chapter to have the same range than the tumour clusters of the Chaplain and Lolas model. The final model presented in this thesis is a mathematical model of cancer cells and effector cells which exhibit standing-wave behaviour between them. We show tha the wave of invading cancer cells can be stopped by the wave of effector cells or ECM.This model also focuses on the effect of the mutation of cancer cells to another subpopulation which is more malignant and which has the ability to invade the ECM or the effector cells to occupy space. The numerical simulations discussed in this chapter are essentially associated with an initial model of two equations representing the effector cells and tumour cells, such that there is a standing wave between these species. We note that the solution of the mathematical model is a travelling wave and also has a standing wave solution (i.e. the wave of effector cells stops the wave of tumour cells when they meet). This phenomenon occurs when the two diffusion coefficients are the same. We calculate the wave speed to illustrate that the speed tends to zero when the two waves meet - a positive speed of tumour cells refers to an invading tumour, and a negative speed refers to the decreasing of effector cells. After this we modify the model by adding an equation for a second cancer cell population T2, which is a sub-population 2 of tumour cells. This is to reflect the fact that cancer is a progressive disease, and as such it becomes more malignant as the cancer cells undergo successive mutations. We show in this case how the new type of cancer cells start to invade the effector cells after the failure of the first type. The third model discussed in this chapter is arrived at by adding an ECM equation to the second model, and it explains how the standing wave arise from two types of equations - the first one contains diffusion, and the second one has no diffusion.All the mathematical models in this thesis use numerical analysis of nonlinear partial differential equations and computational simulations to obtain insight into the underlying biological systems. The systems of nonlinear partial differential equations were numerically solved by a PDE solver in MATLAB for 1D and COMSOL for 2D. We used the MATLAB PDE solver pdepe which uses the method described in Skeel and Berzins (1990) for the spatial discretisation and the MATLAB routine ode15s for the time integration.The numerical simulations demonstrate the existence of cell distributions that are quasi-stationary in time and heterogeneous in space.

Marsupials and eutherians are the two principal groups of modern mammals. Mammalian immunological studies, to date, have focused on eutherian systems with little or no comprehensive work having been carried out on marsupials. This project investigates the functional and developmental aspects of T-cell responses in the marsupial, Macropus eugenii (Tammar wallaby) in both adults and pouch young at various stages of development. Determination of the age at which the Tammar wallaby immune system becomes competent has been examined through the use of cellular and molecular studies carried out on developing pouch young tissue. The capacity for generating an immunological response in adult and pouch young marsupials has been studied by following cellular proliferation in response to mitogens or mixed lymphocyte culture (MLC). After examining adult responses to mitogens and allogenic lymphocytes, optimised conditions were then used to examine the development of responsiveness in pouch young. Several further tests were conducted and findings shown. The study has shown that the earliest age at which Macropus eugenii is capable of mounting a T-cell mediated immune response is between 5 to 13 days post-partum / Doctor of Philosophy (PhD)

An animal model of respiratory infection was used to determine the effect of various factors, thought to influence the ability of the host to clear bacteria, on the host?s innate response to an NTHi lung infection. Mucosal immunisation with NTHi has previously been shown to enhance the clearance of NTHi from the lung in an animal model of infection through the increased recruitment of phagocytes. Comparisons of cytokine and chemokine kinetic profiles were made in order to determine differences between innate and acquired immune response and the way in which mucosal immunisation controls the innate immune response to NTHi. Increased production of proinflammatory cytokines and chemokines in the early stages of NTHi lung infection enhanced the ability to clear bacteria from the rat lung in the immune animals through the increased recruitment of phagocytes to the site. Mucosal immunisation was found to alter the cytokine and chemokine mRNA profiles of CD4+ and CD8+ cells, with increased levels of MCP-1 protein being detected in both types of immune cells. An antecedent viral infection has been shown to increase the chance of developing a respiratory bacterial infection. The NTHi model of respiratory infection was used to characterise the effect that a viral infection had on the host response to the host?s innate response to a bacterial infection and the ability to clear the bacteria. The host?s ability to clear NTHi from the rat lung was enhanced by an antecedent viral infection through alterations to the innate immune response and the cytokine and chemokine kinetic profiles. The use of a mutant strain of NTHi deficient in a component of Lipooligosaccharide (LOS), Phosphorylcholine (ChoP), was utilised as a tool to characterise the innate immune response to LOS. Animals challenged with the LOS mutant strain had a reduced inflammatory response to NTHi through the decreased production of pro-inflammatory cytokines and chemokines and the reduced recruitment of phagocytes to the site of infection. This thesis has contributed valuable information to enable a better understanding of the host?s innate immune response to respiratory infection. This study has identified the role of cytokines and chemokines in the innate response to a respiratory bacterial infection and the enhanced ability of the host to clear NTHi from the lung.

Typescript (photocopy) / 199 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (M.D.)--Dept. of Medicine, University of Adelaide, 1981

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

Typescript (photocopy)

"February 1977." Includes bibliographical references (leaves [105]-[120]) The malignant transformation, and the relevance of developmental antigens to the cancer process, is broadly reviewed. The two developmental antigens - foetal and placental - are then examined in experimental mouse and rat tumour systems.