Autism is a severe childhood neuropsychiatric condition characterized by impairments in socialization and communication, and by restricted and repetitive behaviours. Autism spectrum disorder (ASD) is a complex and largely unknown disease with a strong genetic basis, multiple genes involved and environmental factors determining its phenotype. Interestingly, the DLX1/DLX2 and DLX5/DLX6 bigene clusters are located in autism susceptibility loci and Dlx genes are involved in GABAergic interneurons differentiation and migration to the cortex during forebrain development. Dlx gene expression is controlled by different cis-regulatory elements. Of these, 4 are active in the forebrain, URE2, I12b, I56ii and I56i. In order to determine the role of the DLX genes in ASD, variants were found in gene exons and in cis-regulatory elements in autistic individuals. A single nucleotide polymorphism (SNP), a change of an adenine for a guanine, was identified in I56i enhancer. Finding a SNP in I56i was very surprising considering that it is located in a Dlx binding motif highly conserved among >40 species. We showed, using in vitro approaches, that the presence of this SNP affects the affinity of Dlx for their binding site and reduces the transcriptional activation of the enhancer. The SNP also affects activity of the I56i enhancer in transgenic mice. In order to determine the real impact of the SNP in vivo, mutant mice harboring the SNP in their I56i enhancer were produced. That involved the insertion of the I56i enhancer with the SNP, using homologous recombination in mouse embryonic stem cells to replace the wild type version of the enhancer. With these mutant mice, we demonstrated that, in vivo, this SNP reduces Dlx5 and Dlx6 expression in the forebrain. Furthermore, this decrease in Dlx5/Dlx6 expression could affect the differentiation and/or migration of specific populations of inhibitory interneurons in the forebrain. No distinct iv behavioural phenotypes were observed between wild type mice and those carrying the SNP, during social interaction and anxiety tests. Therefore, these results suggest that even a subtle change in a regulatory element can have an impact in the development of the forebrain and may even contribute to disorders such as autism.

Vitamin A and its derivatives, collectively termed retinoids, are essential for proper growth and development as well as maintenance of homeostasis in the adult. Retinoic acid (RA), the major biologically active vitamin A metabolite, is well characterized for its crucial roles in gene activation during embryogenesis. Our lab had previously performed a microarray analysis to identify genes induced by exogenous RA in the tailbud of early mouse embryos. Three genes were chosen from the microarray results for further investigation; Germ Cell Nuclear Factor (GCNF/NR6A1), Prickle1 (Pk1) and Ror2, the latter of which are known members of the planar cell polarity (PCP) pathway. These genes were further examined for RA regulation by embryo culture and RT-PCR, which strongly supported a direct regulatory mechanism of NR6A1 by RA. Further analysis aiming to identify a functional response element in the promoter of the targets was attempted, including chromatin immunoprecipitation (ChIP), made possible by the generation and characterization of a highly specific antibody against RARγ. This antibody was used in a ChIP promoter walk, which identified regions on target gene promoters that are occupied by RARγ in vivo, and therefore likely harbor RA response elements.

IRF2BP2 is a corepressor of IRF2, a transcription factor involved in the immune response. IRF2BP2 is also a coactivator of the VGLL4/TEAD4 complex in muscle. Given its functional duality, we asked how IRF2BP2 deletion would affect mouse development and adult muscle regeneration. Most Irf2bp2-/- mice die prior to birth, those that survive develop lymphoma in adulthood. Microarray profiling of Irf2bp2 knockout liver, heart, and skeletal muscle revealed a shared program of upregulated genes involved in inflammation and immunity. The function of IRF2BP2 in adult skeletal muscle recovery from cardiotoxin-induced injury was evaluated. Compared to WT mice, mice with macrophage-specific ablation of IRF2BP2 (Irf2bp2flox/LysMCre) or muscle-specific ablation of Irf2bp2 (Irf2bp2flox/MckCre) mice showed increased inflammation and impaired muscle regeneration. Global deletion of Irf2bp2 in mice results predominantly in embryonic death or lymphoma in adults. Irf2bp2 suppresses genes that mediate inflammation in mouse liver, heart, and in skeletal muscle, where IRF2BP2 promotes regeneration.

Human congenital heart disease (CHD) is the most common cause of non-infectious neonatal death affecting 1-2% of live births (Hoffman and Kaplan, 2002). Treatment of CHD requires major surgery and quality of life is often significantly reduced despite treatment. With the discovery of single gene mutations that cause CHD in model animals (Lyons et al., 1995), the role of genetics in CHD has become appreciated. The genetic basis of CHD is poorly understood, with different members of the same family presenting with different types of CHD (Schott et al., 1998), suggesting the causes of CHD are multifactorial. Cardiogenesis is intimately associated with the establishment of the left-right (L-R) body axis, with the two processes sharing several important transcription factors. Heart looping, in which the heart turns dextrally, is the earliest physical manifestation of L-R asymmetry. L-R patterning disorders are associated with an increased risk of CHD; heterotaxy (in which L-R asymmetry is neither normal nor mirror image) accounts for about 3% of all CHD (Zhu et al., 2006).Investigating cardiogenesis and the causes of CHD necessitates the use of animal models, typically mice, chicks, zebrafish and Xenopus. Recently a strain of mouse with a mutation in a gene essential for cardiac development was isolated from an ENU mutagenesis screen (Kile et al., 2003) using mice carrying a balancer chromosome. It has been subsequently found that the most likely candidate gene codes for the protein Prpf8, an integral component of the spliceosome. The mutation is homozygous lethal, with homozygous mice having a grossly deformed heart, developmental delay and a high incidence of heart looping reversal, indicative of a L-R patterning disorder. In depth characterisation of homozygous mutant embryos revealed defects in the morphology of the embryonic node, nodal cilia and the expression pattern of L-R axis genes. We also investigated the expression of Prpf8 during embryogenesis, and the effect that the point mutation we found in our homozygous embryos has on splicing kinetics.

Fetal growth restriction (FGR), when a fetus fails to reach its genetic growth potential, affects up to 10 % of pregnancies and is a major risk factor for both neonatal and adulthood morbidity and mortality. There are currently no treatments for FGR except for delivery of the fetus; resulting in premature delivery which, in itself, is linked to poor outcome. Therefore, the focus of current research is to examine whether therapies successfully used to treat diseases with similar aetiologies to FGR can also be used to treat FGR. Sildenafil citrate (SC), a selective phosphodiesterase-5 inhibitor, is one such candidate. With the recent announcement of the STRIDER international clinical trial for the treatment of severe FGR with SC, it is imperative to determine the efficacy and safety of SC treatment on both fetus in utero and long-term adult health. Mouse models that mimic characteristics of human FGR represent an attractive model to perform pre-clinical studies. Recent studies in mice have demonstrated that SC increased fetal and placental weight and normalised umbilical artery blood flow velocity in FGR but no studies have assessed effects of antenatal SC on offspring health. The aims of this study were to assess the effect of antenatal SC treatment on a) fetal weight b) fetal vascular reactivity b) pup viability and d) long-term effects on postnatal development/physiology in a mouse model of FGR.All experiments were performed in the placental-specific insulin-like growth factor 2 knockout mouse (Igf2 P0+/- mice) which have mixed litters of wild-type (WT) and growth restricted (P0) mice. It has been reported that SC administered in the drinking water was able to increase P0 fetal weight and thus this mouse model was chosen to assess the effects of SC on the fetus and offspring. SC was administered to pregnant dams in two regimens; orally (120 – 160 mg.kg-1) and subcutaneously (10 mg.kg-1) between E12.5 and E18.5. WT and P0 fetal abdominal aortas were isolated at E18.5 and ex vivo vascular function was assessed using wire myography. Fetal abdominal aortas demonstrated reliable and reproducible vasocontraction and vasorelaxation; there were some sex- and genotype-specific differences. SC demonstrated dose-dependent effects on fetal aortic function. Offspring from dams treated with a subcutaneous injection of SC or saline were assessed for postnatal growth (week 5 – week 12), systolic blood pressure (week 8 and week 13), glucose tolerance (week 12) and mesenteric / aortic vascular function (week 14 – week 16). These experiments demonstrated that;• A supratherapeutic concentration of antenatal SC (120 – 160 mg.kg-1) did not increase fetal weight but significantly blunted relaxation responses of fetal abdominal aortas at E18.5. • A subcutaneous injection of antenatal SC (10 mg.kg-1) did not increase fetal weight or alter fetal abdominal aortic function in mice but led to increased systolic blood pressure in both WT and P0 offspring. Additionally, glucose sensitivity was significantly reduced in female offspring from SC treated dams. In conclusion, the studies outlined in this thesis have demonstrated that antenatal SC treatment can cause alterations in fetal blood vessel function and also lead to changes in metabolic and cardiovascular function in mouse offspring. Using ex vivo wire myography, mouse fetal abdominal aortas were able to be assessed at E18.5. This methodological advance will be beneficial as it can be applied to assessing putative treatments in mice that show characteristics of human FGR. In addition, this technique will allow for investigation of the underlying mechanisms of in utero programming of adulthood cardiovascular diseases such as hypertension. Future work must focus on the mechanisms leading to increased systolic blood pressure in offspring from SC treated dams and whether such effects are noted in other animal models of FGR using a variety of SC dosing regimens. These studies will provide information with which to increase efficacy, and ensure the safety, of SC treatment in pregnancy complications.

Philosophiae Doctor - PhD / Introduction: Biocompatibility of all kinds of dental materials is of paramount importance In order to prevent/limit irritation or degeneration of the surrounding tissues where it is applied. Some researchers suggested that dentine bonding agents may be used for pulpal protection, while pulpal inflammation and inhibition of pulpal repair following the use of dentine bonding agents were also reported. Objectives: The first part of this study compared the cytotoxicity of human pulp cell lines to a mouse 3T3 cell line to cytotoxic challenges from dentine bonding agents. The second part of the study compared the cytotoxicity of recent dentine bonding agents namely, Scotchbond 1, Prime & Bond NTand Xeno III through artificial membranes as well as thin dentine discs (after its reaction with apatite) and Clearfil Protect Bond (CPB)as such, as well as the primer part of CPBand the bond part of CPB separately. Methods and Materials: Near confluent human pulp cells and 3T3 cells were exposed to culture medium (DMEM)extractions from the various polymerized agents mentioned above and the cell viability (survival rate) was measured using the standard MTTassay and related to the non-exposed controls. Results: Two human pulp cells lines were more sensitive to 3T3 cell lines while the other human cell line was less sensitive to the 3T3 cell line. All bonding agents as such were found to be cytotoxic towards the 3T3 cells with Xeno III (25%survival rate) and CPB (35%)the most cytotoxic. Of the two parts from CPB the bond part was the least toxic (91% survival rate), but the primer part (containing the anti-bacterial pyridinium molecule) was very toxic (30% survival rate). ScotchBond 1 (59% survival rate) and Prime & Bond NT (62% survival rate) were not statistically different (Kruskal-Wallis Test, p>0.05). However,the survival rate of Xeno III (25% through membrane as well as dentine discs) and Clearfil Protect Bond (35%) were significantly lower than that of the other two bonding agents, with Xeno III significantly the most toxic (p<0.05 ) Conclusion: In general, all 4 dentine bonding agents were cytotoxic of which Xeno III was the most toxic even after its reaction with apatite (through dentine discs). The most toxic part of CPB was found to be the primer part containing the pyridinium linked molecule. If human pulp fibroblasts are used for cytotoxicity testing of dentine bonding agents many cell lines must be used.

African rock rats of the genera Aethomys Thomas, 1915 and Micaelamys Ellerman, 1941, are endemic to East, Central and southern Africa but extend marginally into West Africa. In the past 16 subspecies have been described in the Namaqua rock mouse M. namaquensis Smith, 1834. Recent morphometric and morphological patterns of intraspecific variation suggested the recognition of only four subspecies: M. n. namaquensis, M. n. alborarius, M. n. monticularis and M. n. lehocla, of which the distributions appeared to coincide with the major phytogeographical zones of southern Africa. In the present study earlier analyses of mitochondrial DNA (mtDNA) cytochrome b (cyt b) gene variation were extended. Taken together these results show that M. namaquensis represents a polytypic species complex but with much more diversity than detected using morphology. Phylogenetic and phylogeographic analyses revealed 14 genetically distinct lineages of which several show strong geographic association with particular vegetation biomes or bioregions. The distributional ranges of eight of these lineages showed some correspondence with the type localities of previously described subspecies of M. namaquensis. Some clades displayed considerable within-lineage variation indicating possible fine-scale population structuring, while others showed very little differentiation. Divergence times between lineages varied between 7.26 MYA and 2.70 MYA, corresponding to a Late Miocene to Pliocene radiation. Cytochrome b sequences alone do not fully resolve the evolutionary relationships among the lineages and the phylogenetic analysis was thus supplemented with nuclear Recombination Activating Gene 1 (RAG1) sequences. This gene was successfully sequenced for 11 of the identified lineages. Independent analyses of the two genes were not congruent possibly as a result of incomplete lineage sorting of the nuclear gene. The combined dataset yielded good support for six of the lineages. Finally, a more detailed phylogeographic analysis was conducted among ten localities of the Eastern Kalahari Bushveld lineage based on mitochondrial cyt b sequences to elucidate processes underlying diversification in this species complex. A genetic pattern of phylogenetic continuity with a lack of spatial separation was observed. The mismatch distribution analysis suggests that the lineage has expanded its population size and the geographical expansion may have followed environmental changes in the recent past. Estimates of female gene flow indicate connectivity among localities but not to the extent expected for a panmictic population. Instead a combination of a stepping-stone model and metapopulation dynamics may be applicable to this lineage. Examination of type material of described subspecies is needed to resolve the identity of the unique lineages which will allow us to better understand the phylogeography and mode of speciation in M. namaquensis. In addition, localities of sympatry (lineages in sympatry) should also be studied in more detail in order to help resolve the current taxonomic uncertainties within M. namaquensis. Future research should therefore include a multidisciplinary approach, such as cytogenetics, morphology and more gene regions. Copyright / Thesis (PhD)--University of Pretoria, 2010. / Genetics / unrestricted

Necroptosis is a pathway of inflammatory cell death that is associated with several pathologies and is induced by ligation of surface TLR or cytokine receptors in macrophages. Many signaling pathways depend on endocytosis, a process mediated by GTPases such as dynamin. We evaluated the role of dynamin-dependent endocytosis in the necroptosis of macrophages using various dynamin inhibitors. Using flow cytometry, we confirmed that during necrosome signaling, various dynamin inhibitors (e.g. Dyngo 4a and Dynasore) blocked the internalization of TLR4, which also resulted in the inhibition of cytokine production. Despite the similar impact of Dynasore and Dyngo 4a on TLR4 endocytosis and cytokine production, only Dyngo 4a prevented TLR4-induced necroptosis of macrophages. Further studies indicated that Dyngo 4a was a potent stimulator of the p38 MAPK pathway, and activation of this pathway by Dyngo 4a was responsible for the inhibition of necroptosis of macrophages following TLR4 signaling. Thus, these studies reveal the previously unknown role of the p38 MAPK pathway in regulating the activation of necrosome signaling.

<p>  </p> <p>Recent advancements in genetics have revealed that <em>SCN2A</em> is one of the leading genes associated with neurodevelopmental disorders including autism spectrum disorder and epilepsy. In particular, loss-of-function and truncation variants account for a majority of cases. As there are no current treatments specific for <em>SCN2A</em>, the neuropharmacogenomics field has strived to further elucidate the role of <em>SCN2A</em> in neurodevelopment to identify intervention targets. Rodent models offer <em>in vivo</em>, pre-clinical insight into the effects of genetic variation on behavior, biochemistry, and electrophysiology as well as the mechanisms on molecular, cellular, and circuitry levels. Due to <em>SCN2A</em>’s critical involvement in the initiation and propagation of action potential neuronal firing early in neurological development, full null homozygous knockout of <em>Scn2a</em> in mice is perinatal lethal. Furthermore, canonical heterozygous knockout of <em>Scn2a </em>in mice does not render phenotypes that recapitulate <em>SCN2A</em> deficiency in humans. Therefore my dissertation aims at developing a mouse model that better parallels the human condition, then using that pre-clinical platform to explore precision medicine.</p> <p>  </p> <p>Using the unconventional strategy of gene trapping, we generated mice with a severe reduction in <em>Scn2a</em> expression, resulting in significant behavioral and electrophysiological differences from neurotypical wild-type mice with full <em>Scn2a</em> expression, but enough residual expression that the <em>Scn2a</em>-deficient mice survived into adulthood. The severely decreased sociability accompanied by increased high and low order repetitive behaviors observed with the <em>Scn2a</em>-deficient mice suggest autism-like phenotypes. In addition, <em>Scn2a</em>-deficient mice also displayed other co-morbidities of neurodevelopmental disorders including atypical innate behavior, increased anxiety, increased sensitivity to stimuli, motor discoordination, and impaired learning and memory. On the electrophysiological level, these mice displayed enhanced intrinsic excitabilities of principal neurons in the prefrontal cortex and striatum, brain regions known to be involved in seizures and social behavior. This increased excitability was autonomous and reversible by the genetic restoration of <em>Scn2a</em> expression in adult mice. Further, RNA-sequencing revealed a downregulation of multiple potassium channels as well as differential expression of glutamate excitatory and GABA inhibitory signaling, which led to the pursuit of targeting these pathways. Indeed, the use of potassium channel openers alleviated the hyperexcitability of <em>Scn2a</em>-deficient neurons, thus supporting the pursuit of these targets.  </p> <p>Since characterization of the <em>Scn2a</em>-deficient mouse model revealed disruption in excitatory and inhibitory pathways, excitatory/inhibitory balance was examined further as a precision medicine target. Increasing <em>Scn2a</em> expression throughout the whole brain by excising the gene trap, as well as specific targeting of the striatum and the neurons that project to it using a retrograde viral vector, rescued social deficits. However the striatum-specific injection did not lead to a social rescue. This shifted the focus to the neurons that project to the striatum such as the medial prefrontal cortex. Using chemogenetics to reduce excitatory signaling in the prelimbic region of the medial prefrontal cortex, we were able to increase the social behavior in <em>Scn2a</em>-deficient mice. Synthesizing the results from the retrograde striatum and prelimbic-specific rescue, the next hypothesis tested was a circuity-level manipulation of the medial prefrontal cortex projections to the striatum. Retrograde control (striatum) of chemogenetics (medial prefrontal cortex) decreased the excitatory signaling in the medial prefrontal cortex neurons that project to the striatum, which also led to improved sociability. On the other side of the excitatory/inhibitory balance, increasing inhibitory signaling through acute exposure to small-molecule GABA receptor positive allosteric modulators, clonazepam and AZD7325, rescued sociability.</p> <p>This dissertation opens up new avenues of research by supporting the use of a pre-clinical mouse model of <em>Scn2a</em> deficiency to advance the study of underlying mechanisms behind <em>SCN2A</em>-related neurodevelopmental disorders. Although the results of this dissertation need additional validation such as cellular support, the data and results in this dissertation can serve as a guide to further explore excitatory/inhibitory balance as a neuropharmacogenomics precision medicine target to treat <em>SCN2A</em>-related neurodevelopmental disorders. </p> <p><br></p> <p><br></p>

A simple method for IV self-administration of drugs by mice is described. When morphine (0.5 mg/kg) was made contingent on a nosepoke response, naive mice increased their rate of nosepoking when compared either with animals receiving contingent saline vehicle injectionsor yoked control animals receiving noncontingent morphine.