Puerperal Psychosis is the most severe form of postnatal psychiatric illness, and is a psychiatric emergency. Human-based research to find a means of genetically predicting a woman's risk of puerperal psychosis has so far failed to reliably or reproducibly identify candidate genes or pathways, due to problems common within the field of psychiatric genetics, leading to the proposal of using an animal model in the form of Porcine Maternal Infanticide. In this project, the author has aimed to understand the pathophysiology of PMI using NGS technologies in order to 1) extend the validity of PMI as a model for PP; 2) determine future steps for development of the PMI model; and 3) generate insights into the management of PMI (and by extension PP) via prediction and detection of a puerperal trigger. These aims have been pursued via two experiments. In the first, the author has created RNA-Seq libraries from archival RNA, and then performed differential gene expression analysis. The results indicated that RNA-Seq technologies can be used with archival RNA samples, but using such samples introduces the risk of degradation-based bias. The substantial influence of outliers, confounding factors and sample size on the results prevented reliable identification of candidate genes" but provide concrete guidelines development of the Porcine Maternal Infanticide model. In the second, the author has created MBD-Seq libraries from archival tissue, and then performed differential methylation analysis. The results indicated that it is possible to use MBD-Seq technologies with Sus scrofa brain tissue. Once again, the effect of confounding factors and sample size on the results prevented reliable identification of candidate genes, but provide guidance for development of the Porcine Maternal Infanticide model.

Concern over kidney disease has increased dramatically in recent years within the medical community. It is estimated that approximately one in fifteen Americans, nearly 20 million people, experience chronic kidney disease with most of those diagnosed progressing to kidney failure. The ultimate treatment available for end stage renal failure is whole kidney transplantation. However, there are very few kidneys available for patients to receive and those patients who are fortunate enough to receive an organ must remain on immunosuppressive medication for the remainder of their lives. The United States Department of Health & Human Services have reported that 18 people die every day while on the waiting list for organ donations. The treatment is fairly successful as 69% of patients who receive a kidney transplant are still alive 5 years after the transplant. Tissue engineered organs could be a promising alternative for whole organ transplantation. The overall objective is to repopulate appropriate decellularized scaffolds from pigs, which are not immunogenic, with a patient's own cells to achieve a functional organ. Therefore, there would be an inexhaustible source of organs ready for transplantation without the risk of immune rejection. The naturally obtained scaffolds devoid of immunogens are a potential matrix to create artificial kidneys. Repopulation of decellularized rat kidneys with renal progenitor cells has been reported in previous studies. This dissertation reports the scale-up of the previous technology and building of partially functional human-sized kidneys. In the first step, we investigated various cell lysing agents and developed an automated decellularization procedure for whole porcine kidney decellularization. We also developed a preservation method for native and decellularized kidneys to avoid spoilage before and after decellularization. We also developed a decontamination procedure for whole porcine kidneys. Finally, we recellularized whole porcine kidney scaffolds with renal epithelial cells and achieved partial repopulation of the renal structure.

The recurrent laryngeal nerve (RLN) innervates all the intrinsic muscles of the larynx that are responsible for human vocalization and language. The RLN runs along the tracheoesophageal groove bilaterally and is often accidentally damaged or transected during head and neck surgical procedures. RLN palsy and vocal cord paralysis are the most common and serious post op complications of thyroid surgeries. Patients who suffer from RLN injury can develop unilateral or bilateral vocal fold paralysis (BVFP). Theoretically, selective reinnervation of the posterior cricoarytenoid muscle would be the best treatment for BVFP. The phrenic nerve has been shown in several studies to be the best candidate to anastomose to the distal end of a severed RLN to restore glottal abduction. Successful PCA reinnervation has been sporadically achieved in both human patients and in animal models. Another notable ramification of recurrent laryngeal nerve injury is vocal instability caused by the alteration of mechanical properties within the larynx. In phonosurgery, alterations to the position and framework of the laryngeal apparatus are made to improve voice quality. Accurate and realistic synthetic models are greatly needed to predict the outcome of various adjustments to vocal cord tension and position that could be made surgically. Despite the sporadically successful attempts at PCA reinnervation, thus far, there are still several deficits in our anatomical familiarity and technological capability, which hinder the regularity of successful PCA reinnervation surgeries and our capacity to generate synthetic models of the human larynx that are both realistic and functional. We will address three of these deficits in this project using the porcine larynx as a model. Firstly, we will identify the anatomical variations of the porcine recurrent laryngeal nerve branches. A microscribe digitizer will be used to create three-dimensional mapping of the recurrent laryngeal nerve branches that are relevant to the posterior cricoarytenoid muscle and the abduction of the vocal folds. Secondly, we will develop a magnetic resonance imaging technique to correlate recurrent laryngeal nerve branching patterns with high-resolution MR images that can be used to determine the branching patterns present in a given specimen without surgery. Lastly, we will determine the distribution and composition of different tissue types found within human vocal folds. High resolution MRI, and Mallory's trichrome and H&E histological staining will be used to distinguish and identify the tissue composition of the vocal folds and surrounding laryngeal structures. Detailed information regarding vocal fold tissue composition and histological geometry will enable laryngeal modelers to select more sophisticated and life-like materials with which to construct synthetic vocal fold models.

In mammals, the trophoblast lineage of the embryo is specified before implantation. It is restricted to become the fetal portion of the placenta. We have isolated and cultured trophoblast-derived cells from day 10 and day 13 porcine embryos. These cells demonstrate morphological and biological characteristics that make them unique. We have demonstrated that these cells can grow in vitro in a defined, serum-replacement medium for over a year without showing any signs of senescence. Trophoblast-derived cells placed into serum-containing medium, however, rapidly senesce and fail to proliferate. Gene expression analysis by RT-PCR and Fluidigm analysis of cells in culture from 0-30 days confirmed expression of genes involved in trophoblast function (CDX2, TEAD4, CYP17A1, HSD17B1, FGFR2, PLET, HAND1) as well as some genes known to mediate pluripotency (POU5F1, KLF4, CMYC). These experiments revealed changes in gene expression over time and in response to serum-containing medium. We have demonstrated that these trophoblast-derived cells are easily stably transfected with an exogenous transgene (eGFP) by a variety of methods, and show the ability to survive and to be passaged repeatedly after transfection. Also, immunofluorescence analysis results demonstrated that these cells do not only demonstrate epithelial characteristics by the expression of KRT18, but also they show expression of VIMENTIN which is a protein found in mesenchymal cells. These findings contradict studies done by Ramsoondar in 1993 and Flechon in 1995 which reported the negative expression of VIMENTIN in similar cells. In summary, early embryonic porcine trophoblast-derived cells have demonstrated unique characteristics which have taken us to the conclusion that they could be used as valuable tools for laboratory work. Anticipated applications include the study of trophoblast physiology as well as possible solutions for improving efficiency of transgenesis by somatic cell nuclear transfer and for pluripotency reprogramming of cells.

The initial focus of this PhD project was on comparative gene mapping. Comparative gene mapping is facilitated by consensus PCR primers which amplify homologous gene fragments in many species. As a part of an international co-ordinated programme of comparative mapping in pigs, 47 CATS (Comparative Anchor Tagged Sequence) consensus primer pairs for loci located on human chromosomes 9, 10, 20, and 22, were used for amplifying homologous loci in pigs. After optimization of PCR conditions, 23 CATS products have confirmed by comparison with homologous sequences in GenBank. A French somatic cell hybrid panel was used to physically map the 6 porcine CATS products distinguishable from rodent background product, namely ADRA1A, ADRA2A, ARSA, GNAS1, OXT and TOP1. Of these, the map location of ADRA1A and OXT showed inconsistency with the previously recognised conserved relationship between human and pig. The other four loci mapped to positions consistent with known syntenic relationships. Despite low levels of polymorphism, frequently indistinguishable rodent and porcine products in somatic hybrids and some confusion of identity of gene family members, these CATS primers have made a useful contribution to the porcine-human comparative map. The focus of the project then changed to genetic and molecular characterisation of endogenous retroviruses in pigs and their relatives. Pigs are regarded as a potentially good source of organs and tissues for transplantation into humans. However, porcine endogenous retroviruses have emerged as a possible problem as they can infect cultured human cells. Two main types of pig retrovirus, determined by envelope protein, PERV-A and PERV-B, are widely distributed in different pig breeds and a third less common type, PERV-C, has also been recognised. Endogenous retroviruses were analyzed from the Westran (Westmead transplantation) inbred line of pig, specially bred for biomedical research. Thirty-one 1.8 kb env PCR product clones were sequenced after preliminary screening with the restriction enzymes KpnI and MboI. Five recombinant clones between A and B were identified. 55% of clones (17/31) sequenced had stop codons within the envelope protein-encoding region, which would prevent the retrovirus from making full-length envelope protein recognizable by cell-surface receptors of the virus. The endogenous viruses were physically mapped in Westran pigs by FISH (Fluorescence In Situ Hybridisation) using PERV-A and PERV-B envelope clones as probes. Preliminary FISH data suggest that there are at least 22 PERVs (13 PERV-A and 9 PERV-B) and the chromosomal locations of these in the Westran strain are quite different from European Large White pigs. The sequences and mapping results of inbred Westran pig suggest that there are relatively few PERV integration sites compared with commercial pigs and further that a large proportion of clones are defective due to premature stop codons in the envelope gene. To investigate the relationship of endogenous retroviruses in peccaries and pigs, a set of degenerate primers was used to amplify peccary retroviral sequences. The sequences of two putative retroviral clones showed close homology, albeit with a 534 bp deletion, to mouse and pig retroviral sequences. Also, four non-target sequences were amplified from peccary with the degenerate retroviral primers. They are a part of the peccary cofilin gene, a SINE, and a sequence containing a microsatellite. The peccary endogenous retroviral sequences are significant in that they are the first such sequences reported in peccary species and repudiate old claims in the literature that peccaries do not have C-type retroviral sequences.

Immunological stressors, in the form of clinical and sub-clinical disease are currently controlled using both prophylactic antibiotics in-feed, and therapeutic antibiotic treatment. Respiratory disease, primarily Actinobacillus pleuropneumoniae (App) infection, is recognised as a major factor causing reduced productivity in pigs. This thesis reports investigations into the use of novel immunomodulators in particular Interleukin 4 (IL-4) and Interleukin 1 receptor antagonist (IL-1ra) as alternatives to antibiotics to treat App infection. Immunological and molecular biological assays were used to investigate and accumulate data. An in vitro study undertaken to find potential anti-inflammatory substances, revealed that Interleukin 8 (IL-8) mRNA production stimulated by PMA or LPS in whole pigs' blood was suppressed by IL-4. IL-1ra also suppressed stimulated IL-8 mRNA production by heat killed App bacteria (KB) in vitro. An acute LPS challenge in pigs in vivo however, showed no variation in illness or weight loss between pigs treated prophylactically with anti-inflammatory substance (IL-4 and IL-1ra) and saline treated pigs. The use of plasmids as a delivery system for anti-inflammatory substance did not show promise since it did not enhance growth or prolong the expression of the substances in the pigs. However, in the chronic App challenge model IL-4 and IL-1ra administered prophylactically in vivo showed an ability to improve growth. The therapeutic administration of IL-4 and IL-1ra to App challenged pigs showed no difference in pigs' growth, regardless of the treatment or control administered. To conclude, IL-4 and IL-1ra showed promise when administered prophylactically and improved growth and abrogated disease under conditions of App challenge. However when IL-4 and IL-1ra where administered therapeutically they did not perform as well. Moreover these compounds have potential as a commercial application to reduce the growth reduction caused by disease such as App.

The porcine proliferative enteropathies (PPE) are a group of diseases ranging from intestinal adenomatosis (PIA), a chronic condition causing reduced growth rates in post weaning pigs, to the often fatal proliferative haemorrhagic enteropathy (PHE), resulting in intestinal haemorrhage. PHE predominantly occurs in older and heavier pigs than the chronic disease PIA. This thesis examined whether the age when susceptible pigs are infected affects the clinical response to L.intracellularis infection. The characteristic pathologic lesion of PPE is the abnormal proliferation of crypt epithelial cells in the ileum and colon. Closely associated with this proliferation is the presence of an obligately intracellular bacterium, Lawsonia intracellularis. Characterisation of L.intracellularis was performed in in-vitro co-cultures of L.intracellularis extracted from PHE-affected mucosa. The efficacy of antimicrobials to inhibit the growth of L.intracellularis in-vitro was evaluated and compared with isolates cultured in the United Kingdom. The results were analysed with respect to medication strategies currently used to control PPE in piggeries. PPE occurs in virtually all piggery management systems, including newly developed systems that are aimed at improving the herd health, such as segregated early weaning and multiple site production. PPE is currently controlled in Australia with the routine addition of antimicrobials in pig feed, in particular olaquindox. Recommendations to reduce the use of feed-based antibiotics in Australia require the development of alternate strategies to control diseases such as PPE. Sequential outbreaks of PHE reported in minimal disease herds suggested that pigs could develop immunity to disease. An experimental model of L.intracellularis infection was developed in this thesis to demonstrate that immunity to re-infection with L.intracellularis could be developed. Infection was monitored by detection of faecal shedding of L.intracellularis and serum IgG antibodies against L.intracellularis. Two in-feed antimicrobial strategies were analysed in this thesis for their ability to induce the development of immunity to L.intracellularis, while avoiding clinical signs of disease. The first strategy evaluated the use of low levels of in-feed antimicrobials to allow subclinical infection and the development of immunity. The second strategy evaluated the use of high levels of in-feed antimicrobials to terminate infection two weeks after exposure to L.intracellularis. Gaining a greater understanding of how L.intracellularis infection is spread both within and between piggeries will enable the development of management strategies to control the spread of infection. This thesis examined the possibility that other species in contact with pigs and piggeries such as rats, mice and birds may transmit infection to pigs. The transmission of infection between pigs via the faecal/oral route was also examined, as was the survival and infectivity of L.intracellularis over time. Ultimately this thesis aimed to understand the pattern of L.intracellularis infection and the survival and transmission of L.intracellularis in order to develop effective control measures for PPE, especially in minimal disease herds.

Together with the advancements in animal cloning, the ability to efficiently target specific genes in somatic cells would greatly enhance several areas of research. While it has been possible for quite some time to target specific genes in the germ cells of mice, the advancements in somatic cell gene targeting has been slowed for two main reasons. First, the finite lifespan of somatic cells, due mainly to the inability of the somatic cells to regenerate or maintain their telomeres, poses a major problem given the lengthy selection process needed to identify a targeting event. The second problem is the overall inefficiency of homologous recombination. A double strand break or introduction of foreign DNA into a cell can be processed either through the homologous recombination or non-homologous end joining pathways. Of these two, non-homologous end joining is dominant in somatic cells. A two plasmid recombination system was used to study the effects of the manipulation of several non-homologous end joining and homologous recombination pathway molecules on the rates of homologous recombination in porcine fetal fibroblasts. In addition, the effect of telomerase expression, cell synchrony, and DNA nuclear delivery was examined. Results indicate a strong positive relationship between inactivation of p53, cell synchronization, and efficient DNA nuclear delivery in enhancing the rate of homologous recombination. These findings were then applied to an actual locus in the pig, the α1,3 galactosyltransferase gene. Results from these transfections are compared to published accounts of successful targeting at this locus and possibilities for the differences found are discussed.

In mammals, the trophoblast lineage of the embryo is specified before implantation. It is restricted to become the fetal portion of the placenta. We have isolated and cultured trophoblast-derived cells from day 10 and day 13 porcine embryos. These cells demonstrate morphological and biological characteristics that make them unique. We have demonstrated that these cells can grow in vitro in a defined, serum-replacement medium for over a year without showing any signs of senescence. Trophoblast-derived cells placed into serum-containing medium, however, rapidly senesce and fail to proliferate. Gene expression analysis by RT-PCR and Fluidigm analysis of cells in culture from 0-30 days confirmed expression of genes involved in trophoblast function (CDX2, TEAD4, CYP17A1, HSD17B1, FGFR2, PLET, HAND1) as well as some genes known to mediate pluripotency (POU5F1, KLF4, CMYC). These experiments revealed changes in gene expression over time and in response to serum-containing medium. We have demonstrated that these trophoblast-derived cells are easily stably transfected with an exogenous transgene (eGFP) by a variety of methods, and show the ability to survive and to be passaged repeatedly after transfection. Also, immunofluorescence analysis results demonstrated that these cells do not only demonstrate epithelial characteristics by the expression of KRT18, but also they show expression of VIMENTIN which is a protein found in mesenchymal cells. These findings contradict studies done by Ramsoondar in 1993 and Flechon in 1995 which reported the negative expression of VIMENTIN in similar cells. In summary, early embryonic porcine trophoblast-derived cells have demonstrated unique characteristics which have taken us to the conclusion that they could be used as valuable tools for laboratory work. Anticipated applications include the study of trophoblast physiology as well as possible solutions for improving efficiency of transgenesis by somatic cell nuclear transfer and for pluripotency reprogramming of cells.

Porcine parvovirus (PPV) is considered to be one of the most important causes of reproductive failure in swine. Fetal death, mummification, stillbirths and delayed return to estrus are some of the clinical signs commonly associated with PPV infection in a herd. The virus genome is considered to be conservative, with substitution rates near to that of their host. However, it has been shown that some parvoviruses exhibit a substitution rate close to that commonly determined for RNA viruses. In this scenario, new PPV phenotypes may reduce the effectiveness of the currently used vaccines, recommending the continuous monitoring of the currently prevalent PPV strains. In addition, a number of novel porcine parvoviruses have been described during the last decade, but the importance and characteristics of these viruses remain unknown. In the present dissertation, three studies were performed to address the PPV genetic variability, to monitor the emergence of new PPV strains and the prevalence of novel parvoviruses. In the first study, recent PPV field isolates from Austria, Brazil, Germany and Switzerland were sequenced and analyzed. These samples, together with sequences retrieved from GenBank, were included in three datasets (viral protein complete gene, viral protein partial gene and non-structural protein complete gene). For each dataset, the nucleotide substitution rate was determined and a molecular clock estimated. The analysis revealed that for the new strains, the amino acids substitutions were located mainly in the viral capsid loops. Only the capsid protein datasets present the higher suitability for phylogenetic analysis. In them, a higher divergence was found, with three well defined clusters. By inferring the evolutionary dynamics of the PPV sequences, a nucleotide substitution rate of approximately 10 -4 substitutions per site per year was found for these datasets. An association of the phylogenetic tree with the molecular clock revealed that the main divergence of the PPV strains for the viral protein ocurred in the last 30 years. In the second study, the population dynamic of PPV isolates from swine herds was analyzed using PPV complete protein gene and partial sequences deposited in GenBank. The population dynamic of the virus was calculated using a Bayesian approach with a Bayesian skyline coalescent model. Additionally, an in vitro model was performed by twenty-one consecutives passages of the Challenge strain (a virulent field strain) and NADL2 strain (a vaccine strain) in PK15 cell-line supplemented with polyclonal antibodies raised against the vaccine strain (negative control was not supplemented). The Bayesian analysis indicated a decrease in the population diversity over the years and the predominance of some PPV strains. In agreement, the in vitro study revealed that a lower number of mutations appeared for both viruses in the presence of anti-PPV antibodies in comparison with the control passages without antibodies. In the third study, tonsils and hearts from 100 pigs were collected in a German slaughterhouse in 2010 and tested for PPV, porcine parvovirus 2 (PPV2), porcine parvovirus 3 (PPV3) and porcine parvovirus 4 (PPV4). Positive samples of PPV, PPV2 and PPV3 were sequenced. PPV was observed in 60/100 hearts and 61/100 tonsils and PPV2 in 55/100 hearts and 78/100 tonsils. PPV3 and PPV4 could not be detected in the heart samples but 20/100 and 7/100, respectively, of the tonsils were tested positive. The phylogenetic analysis of the PPV, PPV2 and PPV3 sequences revealed that the German samples could be divided in at least two clusters or clades for each virus. Altogether, it can be concluded that PPV is continuously evolving. Apparently, PPV vaccines largely used in the last 30 years probably have reduced the genetic diversity of the virus and induced the predominance of strains with distinct capsid profile from the original vaccine-based strain. Moreover, the high prevalence of the PPV, PPV2 and PPV3 and their genetic diversity highlight the importance of the continuous monitoring of these viruses.