Infection with Campylobacter jejuni is one of the leading causes of bacterial gastroenteritis, causing an estimated 2.1 million cases annually. Although infections with C. jejuni resolve naturally, over 13,000 hospitalizations and 100 deaths are attributed to this organism. Despite these alarming numbers, relatively little is known about C. jejuni pathogenesis, when compared to other enteric pathogens. This dissertation outlines the identification and characterization of a novel virulence factor in C. jejuni, the protein expressed by the Cj1534c gene. Using microarray and RT real time PCR, Cj1534c was found to be greater than 10 fold over expressed in both swine and poultry. Employing immunoelectron microscopy, we determined that at least a subset of the protein is surface localized. Based on the surface localization and up regulation in poultry, colonization studies were performed. Results demonstrate a significant reduction in colonization by a Cj1534c deficient mutant as compared to wild type. In vitro binding assays using both biotic and abiotic surfaces indicate this protein is involved with attachment to surfaces, as well as the invasion of cultured epithelial cells. In vitro findings were confirmed in vivo using swine, with the Cj1534c mutant being highly attenuated as compared to wild type strains. Additionally, the Cj1534c protein was tested for its potential as a vaccine in poultry. Studies demonstrated that Cj1534c recombinantly expressed in a Salmonella expression vector partially protected chickens, reducing cecal colonization three logs as compared to wild type. Taken together, this data demonstrates a major role of Cj1534c in both chicken cecal colonization and infection of swine.

Impulsivity and stress are two of the most important determinants of drug addiction in that both factors predict the initiation and maintenance of drug use, as well as relapse to drug taking following abstinence. Despite this combined influence, the interaction between stress and impulsivity has never been examined systematically in animal models of addiction. The objective of the current study is to examine the role of acute stress on two different measures of impulsivity in rats: the Go/No-go test measures motor impulsivity, and the Delayed Reinforcement Paradigm measures cognitive impulsivity. To determine whether a 1 hr restraint stress is physiologically stressful, blood samples from rats in Experiment 1 were taken at 5 different sampling points: baseline (0 min), reactivity (15 and 60 min) and recovery (100 and 180 min). In Experiments 2 and 3, rats were tested in either the Go/No-go test or the Delayed Reinforcement test immediately following 1 hr of restraint stress. Results from Experiment 1 show that 1 hr of restraint stress increased plasma corticosterone concentrations at 15 min and 60 min; corticosterone concentrations returned to baseline levels by 100 min. Following stress, the percentage of Go interval responding was not altered during Go/No-go testing (Experiment 2), nor were there changes in the indifference point values during Delayed Reinforcement testing (Experiment 3). These results suggest that 1 hr of acute stress does not increase either motor or cognitive impulsivity, and stress may influence addiction via mechanisms that are independent of impulsivity. / Thesis (Master, Psychology) -- Queen's University, 2009-08-24 15:17:00.597

The effects of the rate of distraction and of local infusion of IGF-1 upon bone deposition during mandibular distraction osteogenesis was studied in a rabbit model. Five groups of rabbits were studied. All rabbits, except sham operated controls, underwent distraction to 15 mm. The variables studied were the rate of distraction (0.5 mm twice a day versus 1.5 mm twice a day) and the effects of local IGF-1 infusion via osmotic infusion pumps. Analysis by DEXA scanning and three point bending 28 days after the end of distraction demonstrated no difference in density or strength of bone between the experimental groups. Histological examination demonstrated non-union across the distraction gap to be more common in rapidly distracted rabbits. Histomorphometric analysis demonstrated higher mineral apposition rates and less un-mineralised osteoid with slow as opposed to rapid distraction (p = 0.0001). Infusion of exogenous IGF-1 also resulted in a small increase in mineral apposition rate which was significant at slow but not a rapid distraction. Bone densitometry and three point bending results did not reveal any effect of distraction rate or IGF-1 infusion other than greater stiffness associated with IGF-1 infusion during rapid distraction (p = 0.01). It seems probable that the overwhelming stimulus to new bone formation produced by distraction renders the anabolic effects of IGF-1 less significant making it detectable by only the more sensitive analysis. This may be due to maximal stimulation of IGF-1 production by slow distraction thus rendering the administration of exogenous IGF-1 relatively superfluous. Rapid distraction may produce less stimulus to growth factor synthesis or the level of production maybe insufficient to cope with a higher requirement. The complete union of rapidly distracted rabbits who received exogenous IGF-1 may be as a result of levels being restored to optimal.

No description available.

The recent history of autoimmune neurology is marked by the discovery of many central nervous system (CNS) antibody-mediated diseases. These disorders are caused by antibodies that target important proteins expressed in the neuronal surface, which are believed to be directly pathogenic. These antibodies are immunoglobulin G (IgG) isotype and, as such, have the potential to cross the placenta during gestation. Foetal exposure to CNS-targeting antibodies could alter developing neuronal circuits, leading to disease. However, the consequences of exposure to these antibodies during neurodevelopment has hardly been considered. To study the relationship between maternal antibodies towards neuronal surface proteins and neurodevelopmental disorders in the foetus a dual approach was undertaken. First, pregnancy serum samples from mothers of children later diagnosed with a neurodevelopmental disorder and from mothers of children with typical development were screened for the presence of neuronal surface antibodies. Next, the effects of pathogenic neuronal surface antibodies in the offspring were assessed in a maternal-to-foetal transfer mouse model. Antibodies to neuronal surface proteins in the gestational serum, particularly CASPR2 antibodies, were found to associate with an increased risk of mental retardation and disorders of psychological development in the progeny. The animal model showed that mice exposed in utero to CASPR2 antibodies have long term behavioural sequelae and histological findings suggestive of abnormalities in brain development. These findings support a model in which maternal antibodies towards foetal neuronal proteins cause long-term behavioural deficits and permanent abnormalities at the cellular and synaptic level in a subset of children with neurodevelopmental disorders.

Polycystic ovary syndrome (PCOS) is a common and complex endocrine condition with reproductive and metabolic complications, affecting up to 10% of reproductive-age women. Hyperandrogenemia, ovulatory dysfunction, and luteinising hormone hypersecretion are characteristic traits of PCOS however, it seems that the most concerning long-term key issues are metabolic problems associated with the syndrome, such as hyperinsulinemia, insulin resistance, obesity, dyslipidaemia and non-alcoholic liver disease. Despite the numerous studies on PCOS, its origin and pathophysiology are still not fully understood. However, there is increasing evidence that the adult PCOS phenotype is programmed in fetal life by androgen excess. Exposure to increased levels of testosterone in utero in rodents, sheep and monkeys result in adult reproductive and metabolic pathologies that parallel those seen in PCOS women. Since hyperandrogenemia is a hallmark of PCOS and daughters of PCOS mothers have elevated levels of androgens at birth, it is likely that prenatal androgenisation during early life predispose to the future development of PCOS. Animal models of PCOS provide an opportunity to examine the developmental aetiology and molecular mechanisms underlying the pathogenesis of this condition. Over last 10 years our lab has successfully utilised a well-established ovine model of PCOS, where pregnant ewes were treated with testosterone propionate (TP) through mid-gestation. From this model, we had a large sample bank of fixed and frozen tissues from the fetal, lamb and adolescent prenatally androgenised animals that allowed to carry a broad range of experiments. In addition, a new cohort of prenatally androgenised adult sheep enabled additional in vivo analysis. Past research documented that prenatal androgenisation result in hyperinsulinemia with altered pancreas structure and function, and early fatty liver without difference in body weight in adolescent sheep. This thesis examines the effects and consequences of increased in utero androgen exposure on metabolic dysregulation in adolescent and adult female sheep. During puberty, but not fetal or early life, there was decreased adipogenesis in subcutaneous adipose tissue (SAT), but not visceral adipose tissue (VAT), accompanied by decreased circulating concentrations of fibroblast growth factor 21 (FGF21), leptin and adiponectin, and increased concentrations of fasting free fatty acids (FFA) in prenatally androgenised sheep. This was countered by upregulated expression of FFA transporters in liver. As adults, TP-exposed animals had increased body weight, elevated fasting insulin and FFA concentrations but normal FGF21, leptin and adiponectin levels. Histological analysis revealed that adult TP-exposed animals had SAT hypertrophy, which was associated with increased expression of inflammatory markers and correlated with increased fasting FFA. Therefore, it is likely that impaired preadipocyte differentiation in SAT during adolescence resulted in hypertrophy and inflammation of adult SAT. This consequently lowered capacity of SAT to safely store fat and potentially explains metabolic perturbations observed in PCOS-like female sheep. To further investigate potential causes of obesity in adult PCOS-like sheep postprandial thermogenesis (PPT), an important constituent of energy expenditure, was measured through implantation of datalogger thermometers into interscapular adipose tissue. Adult prenatally androgenised sheep had decreased amplitude of PPT, without difference in basal body temperature, despite receiving the same caloric intake, and independent of obesity. These findings indicate that adult PCOS-like sheep have reduced capacity for energy expenditure, which is mirrored in women with PCOS. This reduced capacity for postprandial thermogenesis was correlated with hyperinsulinemia decreased noradrenaline levels and reduced thermogenic potential of brown and/or beige adipose tissue. This suggests that women with PCOS might be prenatally programmed to become obese. In summary, findings documented in this thesis provide better understanding into the pathophysiology of PCOS from puberty to adulthood and give opportunities for early clinical intervention to ameliorate the metabolic phenotype of PCOS.

Improved understanding of how the liver regenerates would be of great value, particularly given the dearth of therapies for end-stage liver disease. Currently, the only effective treatment for total liver failure is transplantation. Such an invasive, costly and specialised intervention is unable to address the enormous global impact from diseases of the liver. Ironically, the liver has the greatest regenerative potential of any organ in the mammalian body. However, this capacity for repair is overwhelmed in the face of massive or repeated injury. Understanding the key factors driving or inhibiting successful liver regeneration offers the potential for novel, targeted therapies to promote regeneration of a patient’s own liver. Animal models are widely used when studying complex, dynamic, multicellular processes such as liver injury and regeneration. Continued progress in transgenic modification of mice, combined with ongoing advances in microscopy techniques, means that the opportunity now exists to observe labelled cells, and subcellular structures, in real time and in vivo, with previously unobtainable resolution and fidelity. Not only does this afford the opportunity for novel insights into both normal physiology and the response to injury or disease, it can vastly expand the amount of biologically relevant information that can be obtained from each experimental animal. Hence, it is possible to advance scientific knowledge and reduce experimental animal use simultaneously. This thesis examines the role of αv integrins in liver regeneration. Integrins are expressed on the surface of cells and can perform a range of functions, including signalling and extracellular matrix adhesion. The most well-characterised role for αv integrins is activation of transforming growth factor beta, a molecule which has been shown to inhibit hepatocyte proliferation and liver regeneration. Partial hepatectomy was used as an experimental model of liver injury and regeneration. It was performed in mice, in which one or more αv integrins had been genetically depleted from specific cell types in the liver, namely hepatocytes, hepatic stellate cells or liver sinusoidal endothelial cells. These investigations revealed that depletion of integrin αvβ8 from hepatocytes led to increased hepatocyte proliferation and accelerated liver regeneration. The possible mechanisms through which hepatocyte integrin αvβ8 may exert its braking effect on liver regeneration following injury were also explored. In parallel, a novel experimental system to permit intravital multiphoton microscopy of the regenerating liver following partial hepatectomy in mice was developed and validated. Intravital imaging of mouse liver was performed with a range of cellular labels, combined with a fluorescent cell cycle reporter and label-free imaging modalities. This demonstrated the enormous potential of the system to study the dynamics of hepatocytes and non-parenchymal cells in the regenerative niche, reconstruct the sinusoidal vascular network in three dimensions during angiogenesis, and measure sinusoidal blood flow and parenchymal lipid deposition. Advances in experimental animal models such as this drive forward our understanding of the cellular and molecular mechanisms of liver regeneration whilst refining and reducing experimental animal use. Novel insights into the process of liver regeneration will permit the development of innovative therapeutic strategies to allow this remarkable organ to heal itself even in the face of massive or sustained insult.

Onchocerciasis, caused by Onchocerca volvulus remains a major public health and socio-economic problem across the tropics, despite years of mass drug administration (MDA) with Ivermectin to reduce disease burden. Through modelling, it has been shown that elimination cannot be achieved with MDA alone and additional tools are needed, such as vaccination, which remains the most cost-effective tool for long-term disease control. The feasibility behind vaccination against O. volvulus can be demonstrated in the Litomosoides sigmodontis mouse model, which shows that vaccine induced protection can be achieved with immunisation using irradiated L3, the infective stage of L. sigmodontis and with microfilariae (Mf), the transmission stage of the parasite. There is further evidence of protective immunity in humans, with individuals living in endemic areas that show no signs of infection despite being exposed to the parasite (endemic normal). The protective efficacy of promising vaccine candidates were evaluated using an immunisation time course in the L. sigmodontis model, using either DNA plasmid or peptide vaccines. In immunisation experiments in L. sigmodontis, Mf numbers are used as a measure of protection and marks the end of an immunisation time course. However, when changes in gene expression were measured at the end of an immunisation time course, in attempts to identify gene signatures that could be used as markers of protection (correlates of protection) in the blood, no gene signatures were found to be associated with protection. This suggest that at the end of an immunisation time course, when protection is measured (change in Mf numbers), it is too late in infection to measure changes in immune pathways being triggered. Changes in gene expression were therefore measured in blood samples collected throughout an immunisation time course in the L. sigmodontis model, in order to identify the time point in an immunisation experiment which are the most indicative of protection. Two independent immunisation time courses were used, either using irradiated L3 or Mf as vaccine against L. sigmodontis, as these elicit the greatest protection. This generated a large high dimensional dataset, that was too large and complex for a differential fold-change analysis. Therefore, an analysis pipeline was created using machine learning algorithms, to detect changes in gene expression throughout the time courses to detect markers of protection. The 6 hour time point following immunisation showed the greatest change in gene expression, with the analysis pipeline identifying known pathways associated with vaccine-induced immunity. The pipeline was applied to gene expression data from human samples obtained from individuals living in endemic areas who were either infected with O. volvulus or endemic normal (naturally protected), this was to identify pathways associated with protective immunity in humans. When comparing vaccine induced immunity seen in mice and natural protective immunity in humans there was some overlap in pathways being triggered, suggesting that similar pathways are needed for protection and that if a vaccine can trigger the right pathways in mice, it is likely to be effective in humans. Overall the machine learning analysis of the gene expression data, not only shows that it is feasible to measure change in gene expression in blood during filarial infections, but that during an immunisation time course it is the early time points following immunisation that are the most predictive of vaccine efficacy (protection outcome). One of the vaccine candidates, cysteine protease inhibitor-2 (CPI), is a known immuno-modulator that inhibits MHC-II antigen presentation on antigen presenting cells such as dendritic cells (DC). This candidate has consistently been shown to induce protection if its immuno-modulatory active site was modified. In in vitro studies, it was shown that modification of the active site of CPI rescues antigen presentation in DC. This shows the importance of DC activation before the onset of infection, demonstrating the importance of triggering protective responses early in infection, and provides insight on how one of the vaccine candidates achieves protection.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A esquizofrenia à um transtorno psiquiÃtrico grave e, talvez, por isso, um dos mais pesquisados. Apesar dos importantes avanÃos realizados nos estudos sobre a fisiopatologia da doenÃa, durante o sÃculo passado, poucos benefÃcios foram significativos no tratamento pacientes. A elaboraÃÃo de novos modelos animais de esquizofrenia à uma ferramenta importante para a compreensÃo da fisiopatologia da doenÃa e para o desenvolvimento de novas terapias, uma vez que atà mesmo os antipsicÃticos atÃpicos nÃo mostraram os resultados esperados em relaÃÃo à melhora dos sintomas negativos e cognitivos. O objetivo desta pesquisa foi investigar os efeitos da cetamina no sistema nervoso central como um possÃvel agente em um modelo neurodesenvolvimental da esquizofrenia. Procurou-se determinar as alteraÃÃes de comportamento e estresse oxidativo pela administraÃÃo de cetamina neonatal, bem como a reversÃo e prevenÃÃo destes efeitos pelo tratamento com clozapina, um antipsicÃtico atÃpico. TambÃm realizou-se a avaliaÃÃo do sexo no desenvolvimento da esquizofrenia, dividindo as fÃmeas pelo ciclo estral, cujas fases apresentam concentraÃÃes altas e baixas de estrogÃnio, proestro e diestro, respectivamente, visto que estrogÃnio apresenta um efeito neuroprotetor. O delineamento experimental seguiu os critÃrios para determinar um modelo animal: a validade de face (onde se procura imitar o comportamento caracterÃstico da doenÃa no animal), validade do constructo (a fisiopatologia da doenÃa) e validade preditiva (se os medicamentos estabelecidos para a doenÃa sÃo capazes de inverter e prevenir os efeitos que a droga pesquisada induz). Os animais utilizados no protocolo experimental foram ratos Wistar, divididos em seis grupos e tratados com soluÃÃo salina ou cetamina intraperitoneal (i.p.) durante cinco dias (7Â- 11 dias apÃs o nascimento). Grupo 1 (machos tratados com soluÃÃo salina); Grupo 2 (fÃmeas tratadas com soluÃÃo salina) ; Grupo 3 (machos tratados com cetamina 2,5 mg/kg), Grupo 4 (fÃmeas tratadas com cetamina 2,5 mg/kg), grupo 5 (machos tratados com cetamina 5 mg/kg); Grupo 6 (fÃmeas tratadas com cetamina 5 mg/kg). Os testes de inibiÃÃo prÃ-pulso e y-maze (testes comportamentais) foram realizados na adolescÃncia (35 dias apÃs o nascimento), e repetidos na fase adulta (65 dias apÃs o nascimento). Na fase adulta, as fÃmeas foram divididas de acordo com o ciclo menstrual (diestro e de proestro) para observar a interferÃncia do estrogÃnio. No 66 dia apÃs o nascimento, os animais tratados com cetamina 5 mg/kg foram submetidos ao tratamento de reversÃo pela administraÃÃo de clozapina 10 mg/kg (i.p.), uma vez ao dia, durante 10 dias. ApÃs este perÃodo, os testes comportamentais e neuroquÃmicos (dosagem de BDNF e GSH) foram realizados. Os animais tratados com cetamina 2,5 mg/kg nÃo apresentaram qualquer diferenÃa significativa nos testes comportamentais realizados na fase adulta, quando comparados com os animais tratados com soluÃÃo salina, e, portanto, a dose escolhida para medir os outros parÃmetros foi de 5 mg/kg. O tratamento com cetamina 5 mg/kg reduziu significativamente os parÃmetros observados nos testes comportamentais, em machos e fÃmeas no diestro, na fase adulta. Os machos e as fÃmeas no diestro tambÃm apresentaram uma reduÃÃo significativa nos nÃveis de GSH. Os machos adultos tratados com cetamina 5 mg/kg apresentaram um aumento significativo de BDNF, enquanto que as fÃmeas no diestro os apresentaram uma reduÃÃo neste parÃmetro. As fÃmeas no proestro alcanÃaram um melhor desempenho em todos os testes. No protocolo de prevenÃÃo, o tratamento com clozapina 10mg/kg reverteu os efeitos da cetamina para os ensaios de inibiÃÃo prÃ-pulso e nos testes neuroquÃmicos, em machos e fÃmeas no diestro, tratados com cetamina 5 mg/kg. Os resultados observados nos testes comportamentais e neuroquÃmicas demonstraram que a cetamina induziu os parÃmetros da esquizofrenia, apresentando-se como uma nova ferramenta para a pesquisa de modelos animais esquizofrenia neonatal. O modelo desenvolvido conseguiu, pela primeira vez, mimetizar as diferenÃas entre as fases do ciclo estral dos animais, simulando o que acontece em humanos. / Schizophrenia is a severe psychiatric disorder, and maybe, because of this, one of the most researched. Despite the important advances achieved in the studies about the physiopathology of the disease during the last century, few benefits were significant in the treatment of these patients. The construction of new animal models of schizophrenia is an important tool for the comprehension of the physiopathology of the disease and the development of new therapies, since even the atypical antipsychotics did not show the expected results regarding the improvement of negative and cognitive symptoms. The objective of this research was to investigate the effects of ketamine in the central nervous system as a possible agent in a neurodevelopmental model of schizophrenia. We sought to determinate the behavioral changes and oxidative stress by the neonatal administration of ketamine, as well as the reversion and prevention of these effects by the treatment with clozapine, an atypical antipsychotic. We also conducted an analysis of the role of sex at the development of schizophrenia, dividing the females by the estrous cycle, that includes phases presenting high and low concentrations of estrogen, proestrus and diestrus, respectively, since estrogen presents a neuroprotective effect. The experimental design followed the criteria to determine an animal model: the face validity (where one seeks to mimic the characteristic behavior of the disease in the animal), construct validity (the physiopathology of the disease) and predictive validity (if the established medicines for the disease are able to reverse and prevent the effects that the researched drug induces). The animals used at the experimental protocol were Wistar rats, divided in six groups and treated with saline or ketamine intraperitoneal (i.p.) for five days (7th-11th day after birth). Group 1: (males treated with saline) Group 2 (females treated with saline); Group 3 (males treated with ketamine 2,5mg/kg); Group 4 (females treated with ketamine 2,5mg/kg); Group 5 (males treated with ketamine 5 mg/kg); Group 6 (females treated with ketamine 5 mg/kg).The tests of prepulse inhibition and y-maze (behavioral tests) were conducted in adolescence (35 days after birth), and repeated at the adult phase (65 days after birth). At the adult phase, the females were divided according with the estrous cycle (diestrus and proestrus) to observe the influence of estrogen. At the 66th day after birth, the animals trated with ketamine 5 mg/kg were submitted to a reversion treatment by clozapine 10 mg/kg i.p. once a day during 10 days. After this period, the behavioral and neurochemical (dosage of BDNF and GSH) tests were performed. The animals treated with ketamine 2,5 mg/kg did not show any significant difference in the behavioral tests conducted in the adult phase when compared with the animals treated with saline and, therefore, the dose selected to measure the other parameters was 5,0 mg/kg. The treatment with ketamine 5 mg/kg significantly reduced the parameters observed at the behavioral tests, both in adult males and females at the diestrus. The males and females at the diestrus also presented a significant reduction in the GSH levels. The adult males treated with ketamine 5 mg/kg presented a significant increase in BDNF, whereas the females at the diestrus presented a reduction in this parameter. The females at the proestrus achieved a better performance in all tests. In the prevention protocol, the treatment with clozapine 10mg/kg reverted the effects of ketamine in the tests of prepulse inhibition and in the neurochemical testes in males and females in the diestrus that were treated with ketamine 5 mg/kg.The results observed in the behavioral and neurochemical tests showed that ketamine induced the parameters of schizophrenia, presenting itself as a new tool available to the research of animal models of neonatal schizophrenia. The developed model succeeded for the first time in mimicking the differences between the phases of the estrous cycle of the animals, in a close approximation of what happens in humans

Schizophrenia is a debilitating psychiatric disorder that affects approximately 1% of the global population. Schizophrenia is highly comorbid with other psychiatric disorders such as Alcohol Use Disorder (AUD) with a prevalence rate of 27% - 65%, which is significantly higher than AUD exhibited by the general population (6%). Research indicates that a higher rate of AUD in individuals suffering from schizophrenia may be related to the common neuronal pathways that underlie the expression of both disorders. The present study will determine whether the neonatal quinpirole (NQ) rodent model of schizophrenia will approximate the human condition and exhibit increased EtOH consumption. Rats will be treated neonatally with quinpirole or saline. Following the treatment period, rats will be tested for EtOH consumption using a 24-hour two-bottle free-access paradigm. The proposed research will test the hypothesis that rats neonatally treated with quinpirole will consume significantly greater amounts of EtOH than their saline counterparts.