The goal of this study is to try to understand multi-strain diseases with the presence of cross-immunity by using mathematical models and other mathematical tools. Cross-immunity occurs when a host who is exposed to one disease, or one strain of a disease, develops resistance or partial resistance to related diseases or strains. It is an important factor in the epidemiology of diseases prone to mutation. This work includes modelling influenza in both presence and absence of controls. It also includes modelling malaria when cross-species immunity is present. In addition, vector-bias of mosquitoes to infected humans is also studied in the single-strain malaria model.

Endogenous retroviruses (ERVs) are fossils of ancient retroviral infection in the germline. In primates they represent around 5% of the genome sequence. During time spent in the genome, being transmitted in a Mendelian fashion, copies of ERVs have accumulated mutations, which rendered them inactive. However, some of them (the most recently integrated ones) are still able to transcribe and produce viral proteins, although few are capable of re-infection. In the past often considered as unharmful 'junk DNA', recent evidence link ERVs with cancer and several inflammatory diseases. For example, a few reports demonstrate that ERVs are involved in tumour development using shRNA knock-down and over-expression systems, and their overexpression tends to correlate with inflammation status, generating the hypothesis that they can act as pathogen-associated molecular patterns (PAMPs) and bind to innate sensors. Focusing on the Human (Homo sapiens) and the rhesus macaque (Macaca mulatta), the main aims of this thesis are to look for further evidence linking ERVs to tumour development, with possible implications for therapies, and test the hypothesis that ERVs are PAMPs by seeing if individuals with higher levels of ERV expression exhibit a higher innate immune response. The work on ERVs in cancer involved the human ERV type-K HML2 lineage (HERV-K (HML2)), an ERV lineage found in humans, in Merlin-deficient tumours. These are schwannomas that arise from Schwann cells and for which effective drug therapy is urgently needed. The work on ERVs in inflammation involved the Papio cynocephalus ERV (PcEV), in rhesus macaques infected with simian immunodeficiency virus (SIV) infection. The main outcomes are as follows: regarding HERV-K (HML2) in human schwannomas, (i) HERV-K (HML2) proteins are overexpressed in schwannoma compared to Schwann cells; (ii) these proteins are released from the tumour; (iii) regulation of HERV-K (HML2) expression in the tumour appears to involve the transcription factor TEAD; (iv) schwannomas are potentially treatable using anti-HERV-K (HML2) monoclonal antibodies and antiretroviral drugs since both decreased proliferation in vitro. Regarding PcEV in SIV-infected macaques: (i) PcEV is transcriptionally active; (ii) PcEV can be retrieved at low levels in the blood of some macaque animals; (iii) the levels of PcEV in cells correlates strongly with the strength of the innate response as measured by cellular levels of STAT1 transcripts - an interferon-stimulated gene (ISG). Other recent research has shown that human ERV lineages, namely HERV-W and HERV-H, have been co-opted and are involved in placentation and pluripotency during development, respectively. The present work suggests that ERVs are involved in a wide range of biological process and supports the need for further research into the biological significance of ERVs for their hosts.

Lee Ka Wai Sharon. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 227-260). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.I / ABBREVIATIONS --- p.III / ABSTRACT --- p.VI / 摘要 --- p.XI / CONFERENCE PUBLICATIONS --- p.XVII / TABLE OF CONTENTS --- p.XVIII / Chapter Part I - --- General Introduction / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1. --- The Search of Immunomodulatory Agents --- p.1 / Chapter 1.2. --- Cordyceps sinensis (Dong Cong Xia Cao) as an Immunomodulatory Agent --- p.2 / Chapter 1.2.1. --- General Aspects --- p.2 / Chapter 1.2.2. --- Evidence from the Traditional Chinese Medicine Concepts --- p.2 / Chapter 1.2.3. --- Evidence from the Traditional Chinese Medicine Classics --- p.4 / Chapter 1.2.4. --- Evidence from the Modern Research Literature --- p.4 / Chapter 1.2.4.1. --- lmmunomodulation --- p.4 / Chapter 1.2.4.2. --- Anti-tumor Effects --- p.7 / Chapter 1.2.4.3. --- Other Activities Related to the Immune System --- p.8 / Chapter 1.2.4.4. --- Potential Active Ingredients: Cordycepin and Polysaccharides --- p.8 / Chapter 1.2.5. --- Prescription and Usage: Single Vs Concocted --- p.11 / Chapter 1.2.5.1. --- Single Form as an Immunoactivating Agent --- p.11 / Chapter 1.2.5.2. --- Concocted as an Anti-asthmatic Agent --- p.12 / Chapter 1.3. --- Our Hypothesis and Rationale --- p.13 / Chapter Chapter 2: --- Experimental Design --- p.24 / Chapter 2.1. --- General Aspects of the Human Immune System --- p.24 / Chapter 2.2. --- Designing the In vitro Study on Cell-mediated Immunity --- p.24 / Chapter 2.2.1. --- T Cells --- p.25 / Chapter 2.2.2. --- Macrophages --- p.26 / Chapter 2.3. --- Designing the In vitro and In vivo Study of Anti-tumor Activities --- p.29 / Chapter 2.3.1. --- Tumor Biology --- p.29 / Chapter 2.3.2. --- Tumor and Immunity --- p.29 / Chapter 2.3.2.1. --- T-Cell-Mediated Cytolysis (Tc Cells) --- p.30 / Chapter 2.3.2.2. --- Delayed-Type Hypersensitivity (TDth Cells) --- p.30 / Chapter 2.3.2.3. --- Natural Killer (NK) Cells --- p.30 / Chapter 2.3.2.4. --- Lymphokine-Activated Killer (LAK) Cells --- p.31 / Chapter 2.3.2.5. --- Antibody-Dependent Cell-Mediated Cytotoxic (ADCC) Cells --- p.31 / Chapter 2.3.2.6. --- Activated Macrophages (AMΦ) --- p.31 / Chapter 2.3.3. --- Mechanism of Tumor Engulfment --- p.32 / Chapter 2.3.4. --- The Experimental Plan --- p.33 / Chapter 2.4. --- Designing the In vitro Study and Clinical Trials on Anti-asthmatic Activities --- p.36 / Chapter Part II - --- Methodology / Chapter Chapter 3: --- Materials and Methods / Chapter 3.1. --- List of Materials and Their Origin --- p.39 / Chapter 3.1.1. --- Traditional Chinese Medicine --- p.39 / Chapter 3.1.2. --- Cells for In vitro Experiments --- p.39 / Chapter 3.1.3. --- Mice for In vivo Experiments --- p.40 / Chapter 3.1.4. --- "Medium, Buffer, Supplements and Reagents for Cell Culture" --- p.40 / Chapter 3.1.5. --- Dye for Cellular Staining --- p.40 / Chapter 3.1.6. --- Cell Mitogens and Activator --- p.41 / Chapter 3.1.7. --- Reagents for Flow Cytometric Analysis --- p.41 / Chapter 3.1.8. --- Reagent Kits --- p.41 / Chapter 3.1.9. --- ELISA Kits --- p.42 / Chapter 3.1.10. --- Antibodies --- p.43 / Chapter 3.1.11. --- Reagents for RNA Extraction --- p.44 / Chapter 3.1.12. --- Reagents for Gel Electrophoresis --- p.44 / Chapter 3.1.13. --- Reagents for cDNA Expression Array --- p.44 / Chapter 3.1.14. --- Other Reagents --- p.45 / Chapter 3.1.15. --- Special Equipment and Apparatus --- p.45 / Chapter 3.2. --- Details of Materials --- p.46 / Chapter 3.2.1. --- Traditional Chinese Medicine --- p.46 / Chapter 3.2.1.1. --- Natural Cordyceps sinensis --- p.46 / Chapter 3.2.1.2. --- HERBSnSENSEŚёØ Cordyceps --- p.46 / Chapter 3.2.1.3. --- Wheeze-Relief Formula --- p.46 / Chapter 3.2.2. --- "Media, Supplements and Reagents for Cell Culture" --- p.47 / Chapter 3.2.2.1. --- Cell Culture Media --- p.47 / Chapter 3.2.2.2. --- Serum Supplements --- p.47 / Chapter 3.2.2.3. --- Anti-CD16 Magnetic Microbeads --- p.47 / Chapter 3.2.2.4. --- Fico´HёØ-Paque Plus Solution --- p.47 / Chapter 3.2.2.5. --- PercolĺёØ Solution --- p.48 / Chapter 3.2.2.6. --- Phosphate Buffered Saline (PBS) --- p.48 / Chapter 3.2.2.7. --- Water --- p.48 / Chapter 3.2.3. --- Dye for Cellular Staining --- p.48 / Chapter 3.2.3.1. --- HemacoloŕёØ for Microscopy --- p.48 / Chapter 3.2.3.2. --- Trypan Blue Dye --- p.49 / Chapter 3.2.4. --- Reagents for Flow Cytometry --- p.49 / Chapter 3.2.4.1. --- FACS Flow Sheath Fluid --- p.49 / Chapter 3.2.4.2. --- FACS Wash Medium --- p.49 / Chapter 3.2.4.3. --- Paraformaldehyde --- p.49 / Chapter 3.2.5. --- Special Equipments and Apparatus --- p.49 / Chapter 3.2.5.1. --- Magnetic Cell Sorting System (MACS) --- p.49 / Chapter 3.3. --- Human Subjects --- p.51 / Chapter 3.3.1. --- Inclusion Criteria --- p.51 / Chapter 3.3.2. --- Exclusion Crtieria --- p.51 / Chapter 3.3.3. --- Medication --- p.52 / Chapter 3.3.4. --- Informed Consent and Patient Information --- p.52 / Chapter 3.4. --- Animals --- p.53 / Chapter 3.4.1. --- Maintenance --- p.53 / Chapter 3.4.2. --- Survival Experiment Using Erhlich Ascites Tumor Bearing ICR Mice --- p.53 / Chapter 3.4.3. --- Experiments of Immunomodulatory activity in Sarcoma 180 Bearing BALB/c Mice --- p.54 / Chapter 3.5. --- Methodology --- p.55 / Chapter 3.5.1. --- Preparation of the Traditional Chinese Medicine --- p.55 / Chapter 3.5.1.1. --- Hot Water Extraction of Water Soluble Fraction of Natural Cordyceps sinensis --- p.55 / Chapter 3.5.1.2. --- Hot Water Extraction of Water Soluble Fraction of HERBSnSENSEŚёØ Corydceps and the Wheeze-relief Formula for In vitro Experiments --- p.55 / Chapter 3.5.1.3. --- HERBSnSENSEŚёØ Corydceps for the In Vivo Experiments --- p.56 / Chapter 3.5.1.4. --- Extraction Efficiency of the Hot Water Extracts --- p.56 / Chapter 3.5.2. --- Limulus Ameobocyte Lysate Test --- p.56 / Chapter 3.5.3. --- Cell Preparation --- p.57 / Chapter 3.5.3.1. --- "Isolation of Human Peripheral Blood Mononuclear Cells, Lymphocytes and Monocytes" --- p.57 / Chapter 3.5.3.2. --- Isolation of Eosinophils --- p.58 / Chapter 3.5.3.3. --- Isolation of Spleen Cells from BALB/c Mice --- p.58 / Chapter 3.5.3.4. --- "Murine Ehrlich Ascites Tumor (EAT), PU5-18, and Sarcoma 180 (SC-180) Cell Lines" --- p.59 / Chapter 3.5.3.5. --- Human Eosinophilic Leukemic Cell Line (EoL-1) --- p.59 / Chapter 3.5.3.6. --- Human Hepatocarcinoma Hep-3B Cell Line --- p.59 / Chapter 3.5.3.7. --- Human Leukemic Cell Line (HL-60) --- p.59 / Chapter 3.5.3.8. --- Human Mast Cell Line (HMC-1) --- p.60 / Chapter 3.5.4. --- Collection of Mouse Serum and Human Plasma --- p.60 / Chapter 3.5.5. --- Collection of Culture Supernatant --- p.60 / Chapter 3.5.6. --- The Trypan Blue Exclusion Assay --- p.61 / Chapter 3.5.7. --- Colorimetric 5-bromo-2'-deoxyuridine (BrdU) Cell Proliferation Enzyme Linked Immunosorbent Assay (ELISA) --- p.61 / Chapter 3.5.8. --- Immunophenotyping --- p.62 / Chapter 3.5.9. --- The Cytometric Bead Array (CBA) Kits --- p.62 / Chapter 3.5.10. --- Intracellular Florescence Staining for Reactive Oxygen Species --- p.63 / Chapter 3.5.11. --- The Intracellular Zymosan Florescence Assay --- p.64 / Chapter 3.5.12. --- Total Cellular RNA Extraction --- p.64 / Chapter 3.5.13. --- Gel Electrophoresis of RNA Integrity --- p.65 / Chapter 3.5.14. --- cDNA Expression Array --- p.65 / Chapter 3.5.15. --- Cell Staining Using Cytospin --- p.66 / Chapter 3.5.16. --- Annexin V-FITC/Propidium Iodide Apoptosis Detection --- p.66 / Chapter 3.5.17. --- Weighing the Spleen and Tumor --- p.67 / Chapter 3.5.18. --- Preparing Samples for the Eosinophilic Cationic Protein Fluoroenzymeimmunoassay --- p.67 / Chapter 3.5.19. --- Statistical Analysis --- p.67 / Chapter Part III - --- Results: Pre-functional Assays / Chapter Chapter 4: --- "Extraction, Endotoxin Measurement, In vitro Cytotoxicity Testing, and the Selection of Optimal Concentration" / Chapter 4.1. --- Extraction efficiency --- p.68 / Chapter 4.1.1. --- Introduction --- p.68 / Chapter 4.1.2. --- Results --- p.68 / Chapter 4.2. --- Endotoxin Level --- p.69 / Chapter 4.2.1. --- Introduction --- p.69 / Chapter 4.2.2. --- Results and Interpretation --- p.69 / Chapter 4.3. --- Cytotoxicity --- p.70 / Chapter 4.3.1. --- Introduction --- p.70 / Chapter 4.3.2. --- Results and Interpretation --- p.71 / Chapter 4.3.2.1. --- Peripheral Blood Mononuclear Cells (PBMC) --- p.71 / Chapter 4.3.2.2. --- Eosinophils --- p.72 / Chapter 4.4. --- The Optimal Concentration (OC) --- p.76 / Chapter 4.4.1. --- Introduction --- p.76 / Chapter 4.4.2. --- Results and Interpretation --- p.76 / Chapter Part IV- --- Results: Immunomodulatory Activities of Cordyceps sinensis as a Single Herb / Chapter Chapter 5: --- Mitogenic Activity --- p.80 / Chapter 5.1. --- Introduction --- p.80 / Chapter 5.2. --- Results --- p.80 / Chapter 5.3. --- Discussion --- p.81 / Chapter Chapter 6: --- Cytokines and Cytokine Receptors --- p.84 / Chapter 6.1. --- Introduction --- p.84 / Chapter 6.2. --- Results --- p.84 / Chapter 6.2.1. --- Effects of Natural Cordyceps sinensis and HERBSnSENSEŚёØ Cordyceps on the Induction of Cytokines from Lymphocytes --- p.84 / Chapter 6.2.1.1. --- TNFa --- p.84 / Chapter 6.2.1.2. --- IL-6 --- p.85 / Chapter 6.2.1.3. --- IL-10 --- p.85 / Chapter 6.2.2. --- Effects of Natural Cordyceps sinensis and HERBSnSENSEŚёØ Cordyceps on the Induction of Cytokines from Monocytes --- p.92 / Chapter 6.2.2.1. --- IL-1β --- p.92 / Chapter 6.2.2.2. --- IL-6 --- p.92 / Chapter 6.2.2.3. --- IL-10 --- p.97 / Chapter 6.2.2.4. --- TNFα --- p.97 / Chapter 6.2.3. --- Effects of Natural Cordyceps sinensis and HERBSnSENSEŚёØ Cordyceps on the Expression of Cytokine Receptor --- p.102 / Chapter 6.2.4. --- Effects of Natural Cordyceps sinensis and HERBSnSENSEŚёØ Cordyceps on the Gene Expression of Cytokines and Cytokine Receptors in Peripheral Blood Mononuclear Cells --- p.105 / Chapter 6.3. --- Discussion --- p.112 / Chapter Chapter 7: --- Macrophage Functions: Phagocytosis and Release of Reactive Oxygen Species (ROS) --- p.116 / Chapter 7.1 --- Introduction --- p.116 / Chapter 7.2. --- Results --- p.117 / Chapter 7.2.1. --- Phagocytosis --- p.117 / Chapter 7.2.2. --- Release of Reactive Oxygen Species (ROS) --- p.117 / Chapter 7.3. --- Discussion --- p.124 / Chapter Chapter 8: --- Apoptosis of Selected Cancer Cell Lines --- p.126 / Chapter 8.1. --- Introduction --- p.126 / Chapter 8.2. --- Results --- p.127 / Chapter 8.2.1. --- Differential Cytotoxic Effects of natural Cordyceps sinensis and HERBSnSENSEŚёØ Cordyceps on Various Cancer Cell Lines In vitro --- p.127 / Chapter 8.2.2. --- Differential Anti-Proliferative Effects of natural Cordyceps sinensis and HERBSnSENSEŚёØ Cordyceps on Various Cancer Cell Lines In vitro --- p.129 / Chapter 8.2.3. --- Differential Apoptotic Effects of Natural Cordyceps sinensis and HERBSnSENSEŚёØ Cordyceps on Various Cancer Cell Lines In vitro --- p.131 / Chapter 8.2.3.1. --- Peripheral Blood Mononuclear Cells --- p.131 / Chapter 8.2.3.2. --- Hepatocarcinoma Hep-3B --- p.131 / Chapter 8.2.3.3. --- Human Eosinophilic Leukemic Cell Line --- p.134 / Chapter 8.2.3.4. --- Human Mast Cell Line --- p.134 / Chapter 8.2.3.5. --- Human Leukemic Cell Line (HL-60) --- p.138 / Chapter 8.2.3.6. --- Murine Macrophages/Monocytes Cell Line PU5-18 --- p.138 / Chapter 8.2.3.7. --- Murine Erhlich Ascites Tumor (EAT) --- p.142 / Chapter 8.2.3.8. --- Murine Sarcoma 180 (SC-180) --- p.142 / Chapter 8.3. --- Discussion --- p.145 / Chapter Part V- --- Results: Immunomodulatory Activities of Cordyceps sinensis in Concoction / Chapter Chapter 9: --- The In vivo Animal Model --- p.147 / Chapter 9.1. --- introduction --- p.147 / Chapter 9.2. --- Results --- p.148 / Chapter 9.2.1. --- The ICR Mice Model --- p.148 / Chapter 9.2.1.1. --- In vivo Effects of Natural C. sinensis and HERBSnSENSEŚёØ Cordyceps on the Ascitic Fluid Production of ICR Mice --- p.148 / Chapter 9.2.1.2. --- Effects of Natural C. sinensis and HERBSnSENSEŚёØ Cordyceps on the Survival of Tumor-bearing ICR Mice --- p.149 / Chapter 9.3. --- The BALB/c Mice Model --- p.153 / Chapter 9.3.1. --- In vivo Effects of HERBSnSENSEŚёØ Cordyceps on Spleen and Tumor Weight --- p.153 / Chapter 9.3.2. --- Effects of HERBSnSENSEŚёØ Cordyceps on the Mitogenic Activities of Spleen Cells --- p.154 / Chapter 9.3.3. --- "In vivo Effects of HERBSnSENSEŚёØ Cordyceps on the Cell Surface Expression of CD3, CD4, and CD8" --- p.157 / Chapter 9.3.4. --- Effects of HERBSnSENSEŚёØ Cordyceps on the Cytokine Release from Cultured Spleen Cells --- p.161 / Chapter 9.3.4.1. --- TNFα --- p.161 / Chapter 9.3.4.2. --- IFNγ --- p.163 / Chapter 9.3.4.3. --- IL-2 --- p.163 / Chapter 9.3.4.4. --- IL-4 --- p.163 / Chapter 9.3.4.5. --- IL-6 --- p.167 / Chapter 9.3.4.6. --- IL-10 --- p.167 / Chapter 9.3.4.7. --- IL-12p70 --- p.167 / Chapter 9.3.4.8. --- Monocyte Chemoattractant Protein(MCP)-1 --- p.167 / Chapter 9.3.5. --- In vivo Effects of HERBSnSENSEŚёØ Cordyceps on the Cytokine Synthesis --- p.172 / Chapter 9.4. --- Discussion --- p.174 / Chapter Chapter 10: --- In vitro Studies on Eosinophils and Peripheral Blood Mononuclear Cells --- p.178 / Chapter 10.1. --- Introduction --- p.178 / Chapter 10.2. --- Results --- p.180 / Chapter 10.2.1. --- In vitro Effects of Wheeze-Relief Formula on the Survival of IL-5 Enhanced Eosinophils --- p.180 / Chapter 10.2.2. --- In vitro Effects of Wheeze-Relief Formula on the Degranulation of Eosinophils --- p.180 / Chapter 10.2.3. --- In vitro Effects of Wheeze-Relief Formula on the Surface Expression of Adhesion Molecules and Chemokine Receptors on Eosinophils --- p.183 / Chapter 10.2.4. --- In vitro Effects of Wheeze-Relief Formula on the Surface Expression of Adhesion Molecules on Eosinophils --- p.183 / Chapter 10.2.5. --- In vitro Effects of Wheeze-Relief Formula on the Cytokine Release from Peripheral Blood Mononuclear Cells --- p.187 / Chapter 10.2.6. --- In vitro Effects of Wheeze-Relief Formula on the Gene Expression Profile of Cytokines and Cytokine Receptors of Peripheral Blood Mononuclear Cells --- p.187 / Chapter 10.3. --- Discussion --- p.196 / Chapter Chapter 11: --- The Clinical Trial: Analysis of Serological Markers --- p.200 / Chapter 11.1. --- Introduction --- p.200 / Chapter 11.2. --- Results --- p.202 / Chapter 11.2.1. --- Demographic Data and Drop-out Cases --- p.202 / Chapter 11.2.2. --- Lung Function Test --- p.202 / Chapter 11.2.3. --- Steroid Dosage --- p.202 / Chapter 11.2.4. --- Serological Markers --- p.205 / Chapter 11.3. --- Discussion --- p.215 / Chapter Part VI - --- Conclusion / Chapter Chapter 12: --- Concluding Remarks and Future Perspectives --- p.217 / Chapter Part VII- --- Appendix / Parent Information Sheet --- p.222 / 家長資訊 --- p.223 / Consent Form --- p.224 / Licence to Conduct Animal Experiments --- p.225 / Bibliography --- p.227

The ability to raise an immune response is essential for all life. Despite this, the evolution of immune systems is poorly understood, as immune genes confound many bioinformatic analyses. This is driven by the rapid rates at which immune genes evolve due to the incessant arms race between host and pathogen often causing standard phylogenetic approaches to fail to accurately model the evolutionary history of immune gene families. Many phylogenetic lessons have been learned since the dawn of the phylogenomics era however, and genome sequences of non-model organisms have now been assembled, permitting improved immune gene detection and hence taxon sampling. In this thesis, I have paired sophisticated phylogenomic tools, including outgroup-free rooting methods, and substitution models that account for structural and functional constraints on protein evolution, with new genome and transcriptome sequence data from taxa that allow inference of the ancestral immune state in vertebrates and animals. Using this approach, I have managed to identify the origins of several key immune genes and families. My results support ancestral complexity in the genes that regulate the functioning of vertebrate adaptive immune systems. My findings also support the presence of a complement system, a front-line innate immune defence, in the ancestor of all animals. I show that this system later underwent a period of major remodelling early in vertebrate evolution, generating novel complement systems in at least three major vertebrate taxa. It is clear from my findings that combining sophisticated phylogenetic models with enriched taxon sampling represents a powerful approach with which to gain understanding of the evolutionary history of the immune system, even in the face of gene loss and the inherent complexity of immune gene evolution.

The poultry industry has successfully applied breeding and production programmes to meet growing consumer demands for chicken meat and eggs. Over the last four decades, poultry breeders have selected birds not only for productivity, but also for improved health, welfare, fitness and environmental robustness. Intensive production settings contribute to faster spread of diseases and greater losses in production due to increased morbidity and mortality of the flock. Traditional methods of disease treatment and prevention have played a critical role in control of disease. However, growing resistance of pathogens to therapeutic measures and consumer concerns led to the withdrawal of antibiotics as growth promoting additives in chicken feed. In addition, some vaccines have been overcome by increasing variation and virulence of pathogens and are no longer successful in disease prevention. The emergence of virulent and drug resistant pathogens have emphasised the need to focus on other solutions to disease, particularly natural genetic resistance. Genetic loci or gene expression patterns associated with the differential resistance of lines to specific pathogens have been identified, providing valuable markers for selective breeding. However, to date relatively few of these have been successfully incorporated into commercial lines. An ability to suppress or resist multiple pathogens, by selection for improved innate immune robustness has also been studied but it has not been introduced in commercial production, partly as the phenotype is ill-defined. Previous studies that focused on pro-inflammatory cytokines and their mRNA levels expressed by innate immune effector cells (heterophils and macrophages) identified differences between resistant and susceptible chicken lines, with the former producing stronger responses, supporting efforts to select poultry with an efficient early innate response. Here, small-scale qPCR screening and cellular techniques were evaluated with the conclusion that a more rapid, cheaper and reproducible method needs to be applied. The main objective of this project was therefore to design and validate a diagnostic tool that could be used to phenotype the immune responses of chickens at the level of innate immunity. For this purpose, a panel of 89 genes was selected based on previously published infection studies and on RNA-seq results obtained from stimulation of heterophils, macrophages and dendritic cells with lipopolysaccharide (LPS). Target genes were cloned and sequenced to optimise the design of qPCR reactions and primers. A multiplex qPCR platform, the Fluidigm 96.96 Dynamic Array, was selected as the tool of choice with the capacity to measure transcription of 96 genes of interest in 96 samples simultaneously. The preamplification reaction was optimised and the platform validated using a commercial line of chickens housed in clean or pathogen-challenged environments. Lymphoid tissues, including bursa of Fabricius, spleen, ileum with Peyer’s patches, caecal tonsils, and blood leukocytes were isolated and transcript levels for immune-related genes defined between organs, birds and farms. For qPCR analysis, a panel of reference genes was normalised and TBP, ACTB and GAPDH genes were selected and validated as the most stable. The high-throughput qPCR analysis identified peripheral blood leukocytes as a potentially reliable indicator of immune responses among all the tissues tested with the highest number of genes significantly differentially expressed between birds housed in varying hygienic environments. The research described here could potentially aid the selection of poultry for improved immune robustness. The technical optimisation and validation of a new tool to simultaneously quantify expression of tens of relevant immune-related genes will prime research in many areas of avian biology, especially to define baseline immune gene expression for selection, the basis of differential resistance, and host responses to infection, vaccination or immuno-modulatory substances.

This thesis examines the costs and benefits of maternal allocation to both mother and offspring in gamebirds, specifically ring necked pheasants (Phasianus colchicus) and the Chinese painted quail (Coturnix chinensis). Maternal allocation of compounds from the mother to the young during early development can potentially have both positive and negative effects. For example the transfer of nutrients to the offspring can help to increase post-partum survival and subsequent life time reproductive success. In contrast, the transfer of stress hormones can potentially have long term negative effects on the offspring’s development. The first part of this thesis investigates the effects of the transfer of immune components from mother to offspring, which are known to have positive short term effects (protection against pathogens in early development). There is therefore considerable interest in whether this response can be induced by maternal vaccination to confer protection to young birds in commercial situations. However, it is unclear how this affects life-history trade offs in the mother and the development of the offspring post-hatching. These effects are especially important in the gamebird industry where birds are commercially reared on a large scale before being released into the wild. Chapter 2 therefore examines the costs and benefits to the mother of producing a maternally transmitted immune response to the CoxAbic vaccine, such as the impact of vaccination on egg laying characteristics and body condition. Chapter 3 looks at these costs and benefits to the chicks by examining growth rates and survival after a challenge of live coccidia in offspring from vaccinated and unvaccinated mothers. Chapter 4 examines the immune response to vaccination in adult pheasants. Finally, chapter 5 considers other factors that have been shown to affect the allocation decisions of females, specifically the effect of male characteristics on female reproductive effort.

The tumour necrosis factor (TNF) superfamily is a group of cytokines that orchestrate a variety of functions, both in the development of the architecture of immune organs and of the immune response. The mammalian TNF superfamily consists of 19 ligands and 29 receptors, whereas in the chicken only 10 ligands and 15 receptors are present. Chickens do not develop lymph nodes, possibly due to the absence of the lymphotoxin genes (TNF superfamily members) in their genome. New members of the chicken TNF superfamily have recently been identified in the genome, namely chicken receptor activator of NF-κB ligand (chRANKL), its signalling receptor, chRANK, and its decoy receptor, osteoprotegerin (chOPG). In mammals, RANKL and RANK are transmembrane proteins expressed on the surface of Th1 cells and mature dendritic cells (DC), respectively. OPG is expressed as a soluble protein from osteoblasts and DC, regulating the interaction between RANKL and RANK. To investigate the bioactivity of this triad of molecules, the extracellular soluble domains of chRANKL and chRANK and full-length chOPG were identified and cDNAs cloned. ChRANKL, chRANK and chOPG mRNA are ubiquitously expressed across non-lymphoid and lymphoid tissues and immune cells in the chicken. Similar to mammals, chRANK and chOPG mRNA expression levels are upregulated in mature bone marrow-derived DC (BMDC). ChRANKL transcription is regulated by Ca2+-mobilisation and is further enhanced by the activation of the protein kinase C pathway, as seen in mammals. The biological activities of chRANKL, chRANK and chOPG were investigated by the production of recombinant soluble fusion proteins. The extracellular, TNF-homology, domain of chRANKL (schRANKL) was sub-cloned into a modified pCI-neo vector expressing an in-frame isoleucine zipper to encourage trimer formation. FLAG-tagged schRANKL produced in COS-7 cells predominantly forms homotrimers and chOPG is expressed as homodimers, both signatures of their mammalian TNF superfamily orthologues. SchRANKL enhances the mRNA expression levels of pro-inflammatory cytokines in mature BMDC and BM-derived macrophages (BMDM). Pre-incubation with soluble chRANK-Fc or chOPG-Fc blocked the schRANKL-mediated increase in pro-inflammatory cytokine mRNA expression levels in BMDC. Expression of surface markers on BMDC and BMDM were not affected by schRANKL treatment. SchRANKL enhances the survival rates of BMDC and BMDM and can drive osteoclast differentiation from monocyte/macrophage progenitor cells. The chRANKL signalling receptor, chRANK, does not contain an intracellular catalytic domain but requires the binding of intracellular TNF receptor-associated factors (TRAF) to initiate signalling. TRAFs are a family of seven proteins (TRAF1-7) grouped due to their highly conserved RING domains, zinc finger domains, TRAF-N and TRAF-C domains. ChRANK possesses four of the five TRAF peptide-binding motifs found in mammalian RANK. The "missing" chRANK TRAF peptide-binding motif is TRAF6-specific, a vital protein for RANKL-mediated osteoclastogenesis. All seven members of the mammalian TRAF family are present in the chicken genome. To investigate the conservation of RANK-specific TRAF signalling proteins, chicken TRAF2 (chTRAF2), chTRAF5, chTRAF6 and a newly found member, chTRAF7, were identified and their cDNAs cloned. ChTRAF5, chTRAF6 and chTRAF7 had mRNA expression patterns, in non-lymphoid and lymphoid tissues and in a number of immune cells, similar to their orthologues in mammals. Interestingly, chTRAF2 has two variants, the full-length chTRAF2 and a novel isoform (chTRAF2S) lacking exon 4. ChTRAF2S lacks a portion of zinger finger one, all of zinc finger two and a portion of zinc finger three, producing a protein with a hybrid of zinc fingers 1 and 3 and intact zinc fingers 4 and 5. RT-PCR analyses indicated differential expression of both of the chTRAF2 isoforms in a number of non-lymphoid and lymphoid tissues, splenocyte subsets and in a kinetic study of ConA-stimulated splenocytes. ChTRAF2S is biologically active compared to chTRAF2, inducing higher levels of NF-κB activation. Co-transfections indicate that chTRAF2 may regulate chTRAF2S bioactivity as no synergistic effect was identified when cells were transfected with both isoforms. Knowledge gained from this study will help work to further dissect the interactions between chRANKL-expressing T cells and chRANK-expressing DC to drive Th1 immune responses and to understand how the chicken mounts an effective immune response while expressing a minimal essential repertoire of the TNF superfamily.

Cells defend their cytosol against pathogen invasion using cell-autonomous immunity. When pathogens enter the cytosol they can damage host endomembranes, causing the mislocalisation of host molecules not normally found in the cytosol that are sensed as Danger Associated Molecular Patterns (DAMPs). Glycans exposed on damaged endomembranes are detected by danger receptors such as Galectin8. Galectin8 is recognised by the autophagy cargo receptor NDP52, specifically targeting the bacteria to autophagy. I hypothesised that other proteins would also be recruited to damaged endomembranes, which may initiate downstream mechanisms involved in cell-autonomous immunity or endomembrane repair. Identifying novel damage recruited proteins (DRPs) is difficult due to the short-lived and dynamic nature of damaged endomembranes. Therefore, I developed an unbiased approach for the identification of novel DRPs by proximity-dependent biotinylation using the ascorbate peroxidise enzyme APEX. This approach preferentially labels proteins located at damaged endomembranes for subsequent identification by TMT mass spectrometry. Four enriched proteins CCDC50, FBXO21, STAMBP and PDCD6 were identified as novel damage recruited proteins, recognising damaged SCVs. An alternative form of cell-autonomous immunity is the induction of cell death, for example by pyroptosis. Cell death destroys the bacteria's replicative niche and exposes them to the extracellular space where they may be phagocytosed. I hypothesised that host cells might tag cytoplasmic bacteria with intracellular opsonins to assist in their phagocytosis following their release from host cells. However, my work revealed that intracellular Salmonella Typhimurium acquire phagocytosis protection, thus becoming internalised by phagocytes less efficiently than control bacteria. Phagocytosis protection was acquired rapidly after S.Typhimurium infection and was not observed with dead bacteria. Phagocytosis protection is only partially reversed by opsonisation in human serum. My results indicate that intracellular S.Typhimurium-induces an evasion mechanism to prevent its subsequent recognition by extracellular phagocytes.

Il est connu depuis de nombreuses années que le métabolisme des cellules cancéreuses diffère de celui des cellules saines. La Restriction Calorique (RC) est connue pour prolonger la durée de vie et pour limiter l’oncogenèse. Ainsi, il a été montré que la RC et ses mimétiques comme le 2-deoxyglucose (2DG) augmentent l’efficacité de la chimiothérapie et peuvent aussi induire une immunité anti-tumorale. J’ai pu montrer qu’en régulant le métabolisme via la restriction calorique (ou des mimétiques) nous pouvions moduler l’expression de la protéine anti-apoptotique Mcl-1. Ainsi nous avons établi in vivo et in vitro que la RC restaure la sensibilité des cellules de lymphome à l’apoptose induite par un inhibiteur de Bcl-2/XL, l’ABT-737. Nous avons aussi établi que ces effets sont indépendants de la protéine p53 et/ou des « protéines BH3-only ». La deuxième partie de mon travail a été d’élucider les mécanismes moléculaires mis en place lors de la Chimiothérapie Hyperthermique Intra péritonéale (CHIP) pouvant expliquer les effets bénéfiques observés chez les patients atteints d’une carcinose péritonéale (CP). Une partie de ces bénéfices sont dus à la mise en place d’une immunité anti-tumorale. En utilisant des modèles in vivo et in vitro j’ai mis en évidence l’implication de la protéine du choc thermique 90 (Hsp90) dans l’effet observé. Ainsi, l’inhibition spécifique de la Hsp90 réverse les effets protecteurs de la CHIP, soulignant l’importance de cette protéine dans notre modèle d’immunité anti-tumorale. / The link between cell metabolism and cancer at the cellular level has long been known. Caloric restriction (CR) is known to prolong lifespan and to protect from cancer incidence. The molecular mechanisms involved in these benefic effects have been evaluated and may offer new opportunities for therapeutic intervention. Moreover, CR and CR-mimetics such as 2-deoxyglucose (2DG) has been shown to enhance chemotherapy efficiency and to induce an anti-cancer immune response. During the period of my PhD I demonstrated how the modulation of metabolism through caloric restriction or through its mimetics could significantly reduce the expression of the anti-apoptotic protein Mcl-1 and sensitize lymphoma-bearing mice to apoptosis induced by a Bcl-2/XL inhibitor, ABT-737. We have demonstrated that CR can control Mcl-1 translation and sensitize cells to ABT-737-induced death regardless of the presence or absence of p53 and/or of the main “BH3-only proteins”. Then, I focused on deciphering the molecular mechanisms allowing the Hyper-thermic Intra-Peritoneal Chemotherapy (HIPEC) to be beneficial to patients suffering from peritoneal carcinomatosis. Part of the protective effect was mediated through the induction of an efficient anti-cancer immune response. Next, I showed the involvement of heat shock proteins 90 (Hsp90) in the observed effect. Indeed, when Hsp90 was blocked we lost the protection induced by the HIPEC-treated cells, therefore underling the role of Hsp90 in this HIPEC-dependent induction of anti-cancer immune response.

Orientador: Fabiana Pilarski / Banca: Diogo Teruo Hashimoto / Banca: Geovana Dotta Tamashiro / Banca: Luiz Carlos Kreutz / Banca: Antonio Augusto Mendes Maia / Resumo: As enfermidades têm se destacado como um dos principais entraves para o desenvolvimento da aquicultura, e apesar do tambaqui Colossoma macropomum ser a espécie nativa mais produzida na América do Sul, pouco conhecemos sobre as enfermidades que o afetam e como o seu mecanismo imune reage frente às infecções. A septicemia hemorrágica é causada pela bactéria Aeromonas hydrophila, e se destaca como uma das principais enfermidades na aquicultura, principalmente em espécies tropicais. Por isso, confirmamos a patogenicidade de A. hydrophila pelo Postulado de Koch e estabelecemos as doses letais até 80%, estimando a DL50 em 5, 57 x 107 a 1, 41 x 108 UFC/ml. Não recomendamos o verde malaquita como recurso profilático e indicamos ceftriaxona, florfenicol, oxitetracyclina, sulfazotrim e tianfenicol como potenciais antimicrobianos para o controle desta bactéria, mas consideramos os óleos essenciais de cravo Eugenia caryophyllata e canela Cinnamomum zeylanicum como melhores opções para potencial tratamento da bacteriose, com forte atividade inibitória. Descrevemos um perfil leucocitário incomum de severa leucopenia em período agudo de infecção, devido à liberação de armadilhas extracelulares pelos leucócitos (ETosis). ETosis é um mecanismo de suicídio leucocitário, ainda não descrito para a maioria dos peixes, e que pode ser considerado como um dos últimos recursos imunes adotado pelo hospedeiro para tentar conter a infecção. Este mecanismo pode ser visualizado através de uma metodologia ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Diseases have emerged as one of the main obstacles to aquaculture development, and although tambaqui Colossoma macropomum is the most produced native species in South America, little is known about the diseases that affect this fish and how its immune mechanism reacts to infection. Hemorrhagic septicemia is caused by the bacterium Aeromonas hydrophila, and stands out as one of the major diseases in aquaculture, especially in tropical species. Therefore, we confirmed the pathogenicity of A. hydrophila by the Koch's Postulate and established the lethal doses up to 80%, estimating the LD50 at 5.57 x 107 to 1.41 x 108 CFU/ml. We do not recommend malachite green as a prophylactic resource and we recommend ceftriaxone, florfenicol, oxytetracycline, sulphazotrin and thiamphenicol as potential antimicrobials for this bacterium control, but we consider the essential oils of clove Eugenia caryophyllata and cinnamon Cinnamomum zeylanicum as the best options for potential treatment of bacteriosis, with strong inhibitory activity. We describe an unusual leukocyte profile with severe leukopenia during acute infection due to the release of leukocyte extracellular traps (ETosis). ETosis is a mechanism of leukocyte suicide, not described yet for most of fish species, and it can be considered as one of the last immune mechanisms adopted by the host to try to contain the infection. This mechanism can be visualized through a low-cost and of easy-execution methodology, developed in this thesis. A... (Complete abstract click electronic access below) / Doutor