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Type 2 diabetes mellitus is a metabolic disease that affects one in ten people in the United States. It is caused by a combination of genetics and lifestyle factors. Disease progression begins with insulin resistance in peripheral tissues followed by pancreatic beta-cell failure. The mechanisms behind disease progression are not completely understood. PH domain leucine rich repeat protein phosphatase 1 (PHLPP1) is a known regulator of Akt and other members of the AGC kinase family. Akt has been established to play a role in numerous metabolic signaling pathways, including insulin action. It is hypothesized that as a regulator of Akt, PHLPP1 would have an important function in glucose homeostasis. Glucose tolerance tests performed on 8-week old Phlpp1-/- mice revealed no significant difference in glucose tolerance compared to wild type, however these mice did exhibit increased fasting blood glucose levels. Glucose tolerance tests were repeated at 20 weeks on the same mice and, interestingly, they displayed impaired glucose tolerance compared to wild type. Insulin tolerance tests showed that 8-week old mice have increased insulin sensitivity, however, the 20-week old mice were insulin-resistant compared to control animals. The 20-week old knockout mice also had significantly higher fasting blood glucose levels compared to 8-week old mice. To determine if the increased fasting blood glucose levels are due to increased hepatic glucose output, pyruvate tolerance tests were performed on both the 8 & 20 week old mice. Old mice displayed significantly increased hepatic glucose production compared to wild type. EchoMRI done on 24-week old mice showed significantly increased fat mass and decreased lean mass in the Phlpp1-/- mice compared to wild type littermates. Western blot analysis of liver samples from 32 week old Phlpp1-/- mice indicates loss of Akt signaling accompanied by a decrease in IRS2 protein levels, a common indicator of insulin resistance. These data suggest that Phlpp1-/- mice mimic the development of type 2 diabetes in humans, and provide a unique animal model to study the progression of type 2 diabetes and diabetes-associated complications.

Um die Beeinflussbarkeit des osteoporotischen Lendenwirbelkörpers der Ratte durch vertikale und horizontale Ganzkörpervibrationen (WBV) unterschiedlicher Frequenzen zu untersuchen, wurde ein Kollektiv aus 90 drei Monate alten Ratten (Gattung Sprague Dawley) gebildet. Von diesen Tieren bildeten 15 Ratten die gesunde SHAM-Gruppe. 75 Tiere wurden ovariektomiert und zu je 15 Tieren auf die Gruppen OVX und die einer WBV unterzogenen Gruppen 35 Hz vertikal, 70 Hz vertikal, 35 Hz horizontal, 70 Hz horizontal aufgeteilt. 61 Tage nach erfolgter Ovariektomie wurde mit der WBV (zweimal täglich für 15 Minuten) über einen Zeitraum von 31 aufeinander folgenden Tagen begonnen. Nach Durchführung der WBV wurden die Versuchstiere durch Dekapitation getötet, die einzelnen Wirbelkörper präpariert und den jeweiligen Untersuchungen zugeführt. Die Knochenmineraldichte und der Anteil an Kalzium und Phosphat wurden am zweiten Lendenwirbelkörper durch Veraschung bzw. Flammatomabsorptionsspektroskopie/Photometrie ermittelt. Um die biomechanischen Parameter zu untersuchen, wurde der vierte Lendenwirbelkörper in toto einem axialen Kompressionstest unterzogen. Durch Analyse des dritten Lendenwirbelkörpers mittels Mikro-CT konnte Aufschluss über das trabekuläre Netzwerk sowie das Volumen und die Knochenmineraldichte erlangt werden. Anschließend wurden die Ergebnisse aus Veraschungsversuch und Mikro-CT mit den biomechanischen Parametern korreliert, um so die Determinanten eines stabileren Knochens zu identifizieren. In dieser Studie konnte ein signifikanter negativer Einfluss des Östrogenmangels auf die biomechanische Stabilität sowie die Knochenmineraldichte, das Knochenvolumen und Trabekeldicke des Knochens gezeigt und der Nutzen einer WBV als nicht-medikamentöse Therapieoption des osteoporotischen Knochens belegt werden. Die bei 35 Hz vibrierten Tiere erlangten bezüglich der biomechanischen Stabilität (Fmax, Streckgrenze, Steigung) und der Knochenmineraldichte (total BMD) das Niveau der gesunden SHAM-Tiere. Die horizontale WBV konnte keinen eindeutigen Effekt auf die erhobenen Parameter zeigen. Durch den berechneten Korrelationskoeffizienten konnte die Abhängigkeit der biomechanischen Stabilität des Wirbelkörpers von der mittels Mikro-CT ermittelten Trabekeldicke, dem Knochenvolumen und der Knochenmineraldichte dargestellt werden. Somit kann von den durch Bildgebung erhobenen Daten direkt auf die biomechanische Belastbarkeit und die Frakturneigung geschlossen werden. In dieser Studie konnten der Einfluss und Nutzen einer vertikalen 35 Hz WBV auf den osteoporotischen Lendenwirbelkörper der Ratte gezeigt werden. Die Bestätigung der Ergebnisse am Großtiermodell stellt ebenso wie die Durchführung klinischer Studien Ziele für die Zukunft dar, um den Nutzen einer WBV als schonende nicht-medikamentöse Therapieform der Osteoporose zu betätigen.

Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2008.

Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2008.

Canine Oral Papillomavirus (COPV) induces warts on the oral mucosa in domestic dogs and other canids. The canine oral papillomatosis (COP) is a well established animal model of mucosal papillomatosis. While the regression of a current infection is mediated by cellular immunity, humoral immunity does prevent reinfection. Question of the present study was, if unspecific stimulation of the immune system with the inducer of paramunity Baypamune® during the period of growth would have an influence on the course of canine oral papillomatosis. Clinical criteria for this purpose were period of growth, period of regression and the size and morphology of Papillomas at the end of growth. Additionally ALAT and ASAT were quantified in order to rule out deterioration of liver cells. PCV and leucocytes where monitored and a differentiation was performed. In a second part of the study, L1-antibodies and IFNg, TNF-a as well as IL-18 were determined by the Institute of Virology of the Faculty of Veterinary Medicine of the University of Leipzig. At the end of the study, biopsies of mucosa were taken for detection of viral genome. In a pre-study, three Labrador Retrievers at 14 weeks of age were challenged with 15 μl of virus suspension (40 μg of COPV-L1 Protein/ml) per site by scarification of oral mucosa. Papillomas developed at all challenged sites. These were removed during the period of growth and handed over to the Institute of Virology of the Faculty of Summary 70 Veterinary Medicine of the University of Leipzig for preparation of the suspension used in the main study. In the main study, 13 Labrador Retrievers at 14 weeks of age were challenged with 10 μl of virus suspension (40 μg of COPV-L1 Protein/ml) per site by scarification of three areas of the left oral mucosa. All animals developed at least at one site warts, totally 88 % of the challenged sites showed papillomas. 44 Days after infection, 12 dogs were divided into two groups which did not differ in time of incubation and size of the papillomas. Over a period of four weeks, one group received a total of five dosis of Baypamune®, the other group five dosis of placebo. There was no statistically significant difference between the groups regarding the period of growth, period of regression, size and morphology of the papillomas. The administration of Baypamune® during the period of growth had no effect on the clinical course of COP in this trial. However, time of application of the substances should be considered critically, since papillomas of five animals had been in regression before the first application of Baypamune® and placebo had taken place. No deterioration of liver cells could be ascertained. There was no difference between the groups regarding packed cell volume, white blood count and differentiation throughout the study. Parameters of unspecific immunity determined by the Institute of Virology, IFN-γ, TNF-a und IL-18, delivered no evaluable results. However, four animals of the placebogroup showed Papilloma-DNA in the Biopsies taken, but none of the animals, that received Baypamune®, being this a possible indicator for stimulation of cellular immunity. Anti-L1-Antibodies rose earlier in the Baypamune®group than in the placebogroup. In conclusion therapeutic efficacy of Baypamune® in dogs with present COP could not be shown in this trial, making future investigation necessary.

Epidemiological evidence from season of birth and migrant studies has led to the proposal that developmental vitamin D3 (DVD) deficiency may be a risk factor for schizophrenia. Concurrently, there has been recent intense interest in vitamin D3 as a modifying factor in the development of immune responses. However, the effects of DVD deficiency on immune function remains unknown. Thus, a model of DVD deficiency has been developed in Sprague-Dawley rats. Briefly, female rats were maintained on a vitamin D3 deficient diet prior to and during gestation. At birth dams were returned to a vitamin D3 replete diet. Post-weaning their offspring, the DVD-deficient rats, were maintained on a vitamin D3 replete diet. Therefore, this model represents a transient, prenatal vitamin D3 deficiency. A number of structural and cellular changes have been described in the DVD-deficient rat brain, including ventriculomegaly, decreased growth factor expression, and increased rates of mitosis. These changes are correlated with altered adult behaviour in the DVD-deficient rat, including a locomotor sensitivity to novelty. This thesis will focus on extending these findings and characterising immune function in the DVD-deficient rat. Vitamin D3 can affect cellular differentiation and is anti-proliferative and pro-apoptotic in many tissues including the brain. The effect of DVD deficiency on brain development has been restricted to neuronal cells in vivo and in vitro. However, the effect of this exposure on glial cell maturation and phenotype was unknown. The experiments reported in this thesis demonstrated that primary cortical glial cell cultures from DVD-deficient rat neonates displayed a similar phenotype and maturational status compared with cells from control rats. Learning and memory was examined in this model by exploring the phenomenon of latent inhibition. DVD-deficient rats displayed normal latent inhibition, however, they exhibited a subtle performance acquisition deficit during the early stages of the conditioned avoidance learning task. Extended handling and pre-exposure were able to ameliorate this deficit, though with this treatment normal latent inhibition was also abolished in both control and DVD-deficient rats. Ultrasonic vocalization and nociceptive threshold testing confirmed that alterations in peripheral sensation could not explain this performance acquisition deficit. The results suggested that anxiety or attentional mechanisms may have contributed to this rate of learning deficit. Finally, as vitamin D3 is a powerful immunoregulator, there is the potential for a transient DVD deficiency to induce a persistent alteration in the development and function of the immune system. This hypothesis was supported by findings that showed DVD deficiency resulted in a primed immune system, as indicated by an enlarged spleen, thymus and peripheral lymph tissue as well as increased pro-inflammatory cytokine production in response to an in vitro stimulation. However, these findings did not lead to an alteration in cell mediated immune response in vivo. The results from the research reported in this thesis indicate that a transient, prenatal vitamin D3 deficiency had a subtle yet significant effect on the immune system and behaviour of the adult rat. These findings add further weight to the body of evidence that link prenatal vitamin D3 status to various adverse health outcomes. The DVD-deficient rat model is an integral step in understanding prenatal vitamin D3 deficiency as a potential environment risk factor in the development of immune and psychiatric disorders.

Epidemiological studies indicate that maternal vitamin D deficiency may be a candidate developmental risk factor for schizophrenia. For example, people born in winter/spring, urban environments and dark-skinned individuals whose parents migrated to cooler climates are all at increased risk of developing schizophrenia later in life. The biological plausibility that a low prenatal level of vitamin D has an adverse impact on the developing brain has been studied using a developmental vitamin D (DVD) deficient rat model. These animals display molecular and anatomical abnormalities in brain development and alterations in behaviour as adults. Compared with control rats, neonatal DVD-deficient rat brains are different in shape; displaying a thinner cortex and larger lateral ventricles. Moreover, the brains appear to be less differentiated. At adulthood, DVD-deficient rats show an enhanced sensitivity to novelty-, antipsychotic- and psychomimetic- induced locomotion. These observations have lead to the hypothesis that dopamine and/or glutamate neurotransmission may be altered in DVD-deficient rats. Thus, the main aim of this thesis was to further characterise the dopamine and glutamate neurotransmitter systems in DVD-deficient rats. DVD-deficiency resulted in sex and age specific changes in dopamine signalling. At birth, DVD-deficient rats showed altered dopamine metabolism in the forebrain providing the first report of altered dopamine function after DVD-deficiency. Female DVD-deficient rats displayed a post-adolescent (at 3 months of age) enhanced response to amphetamine-induced locomotion. Accompanying this behavioural sensitivity were decreased levels of dopamine 1 and 2 receptor density in the nucleus accumbens. The altered behaviour in female DVD-deficient rats was not associated with increased dopamine release in the prefrontal cortex, caudate putamen or nucleus accumbens. Although a similar increase in the behavioural sensitivity to amphetamine was not observed in male DVD-deficient adult rats, increases in the density of the dopamine transporter were observed in the caudate putamen and nucleus accumbens. However, when examined at a mature adult age (6 months) neither the enhanced response to amphetamine, receptor or transporter changes persisted. These results suggest that after puberty a transient change in dopamine receptor signalling manifests as an altered response to amphetamine under certain environmental and experimental conditions. Glutamate signalling was probed with the N-methyl-D-aspartate receptor antagonist MK-801. Adult male DVD-deficient rats showed an enhanced locomotor response to MK-801 and this persisted when examined at a mature adult age. Female DVD-deficient rats showed an enhanced response but this was only observed at the mature adult age examined. No behavioral differences were observed prior to adolescence. This behavioural sensitivity did not appear to be due to altered dopamine release after MK-801 in the prefrontal cortex and caudate putamen. Taken together, male DVD-deficient rats develop a locomotor sensitivity to MK-801 at an earlier age than DVD-deficient females. This behavioural alteration is not associated with altered dopamine function. The combined results from the studies in this thesis present a complex phenotype that suggests altered dopamine and glutamate interactions in DVD-deficient rats that are dynamic; demonstrating both age and sex specific traits. I speculate that the development of these behavioural and neurochemical alterations in DVD-deficient rats follows a similar temporal profile to the symptomology observed in schizophrenia patients. Both behavioural sensitivities to amphetamine and MK-801 are observed in schizophrenia in addition to a delayed onset of symptoms in females. This provides further support for a role of vitamin D in the developing brain and suggests that a transient deficiency can result in long-term behavioural and neurochemical alterations. Together this suggests that the DVD-deficient rat model may be an informative model for exploring the developmental vitamin D deficiency hypothesis of schizophrenia.

Introdução: A técnica de criopreservação de tecido ovariano tem sido vista como tratamento promissor e se apresenta como a principal maneira de preservar a fertilidade em pacientes pré-púberes e em mulheres que necessitam de tratamento do câncer de imediato. Contudo, atualmente, ainda existem obstáculos em relação ao autotransplante de tecido ovariano criopreservado, devido a fatores como lesão isquêmica, assim como danos causados pelo processo durante o congelamento, bem como a escolha do melhor local para o enxerto. Objetivo: Verificar a possível restauração da função ovariana, analisando a histologia do ovário transplantado em ratas adultas estéreis, após transplante autólogo de tecido ovariano criopreservado em fase pré-púbere. Métodos: Foram utilizadas 45 ratas Wistar com 30 dias de idade, que foram divididas aleatoriamente em três grupos: Grupo Controle (n = 15), férteis normais; Sham (n = 15), submetidas à ooforectomia bilateral; Transplante (n = 15), submetidas à ooforectomia bilateral, seguida de transplante autólogo na região dorsal entre as escápulas. A partir do d35, foram realizadas observações quanto à maturidade sexual, através da análise da abertura vaginal e de esfregaços vaginais, para avaliação do ciclo estral. Após observação da fase do ciclo estral, os animais foram eutanasiados. E, amostras de tecidos foram coletadas e processadas para avaliação histológica dos implantes ovarianos; considerando: organização estrutural do tecido transplantado e adjacente, bem como o desenvolvimento folicular. Resultados: Quanto às avaliações de maturidade sexual, através das análises de abertura vaginal e da análise microscópica do material obtido dos esfregaços vaginais, foi possível observar que os animais do Grupo Controle, que eram férteis ciclaram normalmente. As ratas do Grupo Sham e Transplante não apresentaram ciclo regular, permanecendo em diestro. As avaliações histológicas das amostras de tecido de ovário pré-púbere, implantados em fêmeas adulto jovens, evidenciaram degeneração ovariana; uma vez que estes apresentaram fibrose e áreas de necrose, o que provavelmente impossibilitou o desenvolvimento folicular, nas ratas que receberam o transplante. Conclusão: A técnica de transplante de tecido ovariano em ratas é uma técnica relativamente simples de ser executada, e se mostrou eficaz na manutenção do massa corporal dos animais durante o período observado. Este achado sugere que houve produção hormonal, oriunda do ovário transplantado, fato este que encoraja as pesquisas neste sentido, a fim de se obter uma técnica que restaure a produção de folículos viáveis em pacientes estéreis. Apesar de ter apresentando indícios de falência do enxerto e isquemia no tecido transplantado, os resultados preliminares desta investigação precisam ser complementados com estudos adicionais, a fim de buscar as melhores condições para a obtenção de maior eficácia dos transplantes autólogos de tecido ovarianos criopreservados. / Introduction: Ovarian tissue cryopreservation is a promising treatment and it is presented as the main way to preserve fertility in prepubertal patients and women who need cancer treatment immediately. However still remain obstacles related to the ovarian tissue cryopreserved autograft due to ischemic injury, damage caused by the freezing process and selecting the best location for the graft. Objective: Investigate a possible restoration of the ovarian function by analyzing the histology of the ovary transplanted into sterile adult rats after autologous transplantation of ovarian tissue cryopreserved in prepubertal phase. Methods: 45 Wistar rats, 30 days old,which were randomly divided into three groups: control group (n = 15), normal fertile; Sham group (n = 15), underwent bilateral oophorectomy; Transplantation group (n = 15), underwent bilateral oophorectomy followed by autologous transplantation in the scapular area. From the d35, sexual maturity was observed by examining the vaginal opening and vaginal smears, for evaluation of the estrous cycle. After observing the phase of the estrous cycle, the animals were euthanized. The tissue samples were collected and processed for histological evaluation of ovarian implants; where structural organization of the transplanted tissue and adjacent as well as follicular development were analyzed. Results: Regarding sexual maturity evaluations, observed by vaginal opening analysis and microscopic analysis of material obtained from vaginal swabs, we could observe that the animals in the control group cycled normally. The rats of Sham and Transplant Group showed no regular cycle, staying in diestrus phase. The histological assessments of prepubertal ovarian tissue samples implanted in young adult females showed ovarian degeneration, since they had areas of necrosis and fibrosis, which probably impeded the follicular development in these rats. Conclusion: The ovarian tissue transplantation technique in rats is a relatively simple technique, and is effective in body mass maintenance of animals during the observed period. This finding suggests that there were hormone production originated from the transplanted ovaries, and this, encourages research in order to obtain a technique to restore the production of viable follicles in sterile patients. Despite presenting evidence of graft failure and ischemia in the transplanted tissue, the preliminary results of this investigation need to be supplemented with additional studies in order to get the best conditions for achieving greater effectiveness of autologous transplantation of cryopreserved ovarian tissue.

Intellectual disability (ID) and autism spectrum disorders (ASDs) can have a devastating impact on an individual’s functioning and quality of life. Insights from pre-clinical models of monogenic forms of ID and ASD are now revealing the biochemical pathways and aberrations in cellular and synaptic functioning involved. One monogenic cause of ID, ASD and epilepsy is SYNGAP1 ID which results from mutations in the SYNGAP1 gene on human chromosome 6. Although a variety of symptoms have been reported, many affected individuals have moderate to severe intellectual impairment and severe seizure phenotypes. Previous pre-clinical studies have mainly focussed on the effects of altered SynGAP expression in mice. This thesis is therefore the first to explore altered SynGAP expression in a rat model. It also adds to the body of research exploring the roles of SynGAP isoforms in glutamatergic synaptic function. The SynGAP_GAP deletion rat was engineered to have a deletion encompassing the enzymatically active GTPase activating protein (GAP) domain of the protein, via which SynGAP regulates multiple biochemical pathways by enhancing the slow intrinsic hydrolysis of GTP by GTP-binding proteins. SyngapGAP/GAP rats appeared small and failed to thrive. As with Syngap-/- mice, this complete loss of WT SynGAP proved lethal, whereas Syngap+/GAP rats appeared to develop normally. The electrophysiological data obtained from this new model reveals a reduction in the frequency of miniature excitatory post-synaptic currents (mEPSCs) in Syngap+/GAP cultured neurons. However the exaggerated hippocampal long-term depression identified in Syngap+/- mice was not seen in the rats. There was also no evidence of differences in intrinsic cell properties, excitatory and inhibitory currents or ratios of AMPAR / GABAAR and AMPAR / NMDAR between WT and heterozygous rats. In addition to the characterisation of the SynGAP_GAP deletion rat, the impact of the previously unstudied Eα1 isoform on forebrain neuronal synaptic function was examined through mEPSC recordings. A trend towards lower mEPSC frequency was found which supports previous research showing that α1 isoforms reduce synaptic strength. This body of work therefore adds to published evidence of isoform specific functions and provides the first evidence of the impact of SynGAP alterations in rats, the results of which show some intriguing differences from previous work in mice.