The neurobiology of anxiety-related disorders is associated with impaired neuroplasticity. The glutamate/NO/cGMP pathway has been proposed to play a key role in neuroplasticity and neurodevelopment. It was demonstrated in recent reports that chronic co-administration of the phosphodiesterase type 5 (PDE5) inhibitor sildenafil and the antimuscarinic agent atropine exerts antidepressive-like activity in rats, and that this effect is related to PDE5 inhibition, with consequent elevation of cGMP levels and enhanced protein kinase G stimulation. The current study investigated possible anxiolytic effects of the chronic co-administration of sildenafil and atropine in stress-sensitive Flinders Sensitive Line (FSL) rats. FSL rats received vehicle control, fluoxetine (15 mg/kg), atropine (1 mg/kg), sildenafil (10 mg/kg) or sildenafil plus atropine via intraperitoneal administration, either acutely 30 minutes prior to testing (acutely) or daily for 14 days (chronically). FRL control rats received only vehicle. Thereafter anxiety-like behaviour was evaluated in the social interaction test (SIT - acute) and elevated plus maze (EPM - acute and chronic). The current study also compared to different ways to score the EPM, namely the percentage time spend in the open arms of the EPM and both the number of full and half body open arm entries, and also implemented defecation on the EPM as a measure of anxiety. Vehicle-treated FSL rats exhibited more anxiety-like behaviour than FRL rats in both the SIT and EPM following acute treatment, and in the EPM following chronic treatment. Acute treatment with fluoxetine exerted anxiogenic activity in the SIT and EPM, but anxiolytic activity following chronic administration, as observed in the EPM. In acute treatments neither sildenafil nor sildenafil plus atropine yielded any significant effects on anxiety-like behaviour. However, following chronic treatment, sildenafil exerted anxiolytic activity in the EPM by increasing the time spend in the open arms (45.72% ± 9.94% vs. 20.80% ± 9.94%, P<0.001). Atropine exerted a small anxiolytic response (30.71% ± 8.40% vs. 20.80 ± 9.94%), whereas atropine co-administration was additive to sildenafil alone and yielded an enhanced anxiolytic effect in the elevated plus maze (59.56% ± 4.95% vs. 20.80% ± 9.94%, P<0.001), relative to vehicle control. The percentage time spend in the open arms was scored in the EPM, the results suggested that the chronic treatment with sildenafil plus atropine exert an anxiolytic-like effect in FSL rats and the number of fecal droppings did not increase which is also an indication of an anxiolytic-like effects of the treatment. The current study demonstrated that the chronic treatment with sildenafil, alone or in combination with atropine, exhibit an anxiolytic-like action in stress-sensitive rats. In addition, the data support the clinical potential of using PDE5 inhibitors as antidepressant and anxiolytic strategy and warrant further investigation. Furthermore the study supports the previously proposed key role of the glutamate/NO/cGMP pathway in the neurobiology of anxiety-like disorders, and as an important target for drug development. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2011

Exposure to microorganisms is considered an environmental factor which can contribute to diabetes mellitus via cytotoxicity or autoimmune responses against pancreatic cells. Firstly, the effects on rat insulinoma pancreatic β-cell line of secondary metabolites pyrrolnitrin (Burkholderia spp), phenazine compounds (Pseudomonas aeruginosa and Burkholderia spp) were investigated. Both compounds separately showed significant cytotoxicity after 24 h and at concentrations of 10 & 100 ng/ml potentiated insulin gene transcription, Ca2+ content and glucose-stimulated insulin secretion (GSIS). Furthermore, the outward membrane current was inhibited by phenazine (100 ng/ml) or pyrrolnitrin (10 or 100 ng/ml). Secondly, the capacity of 45 microbial species to bind insulin was screened in order to assess how common insulin binding was amongst microorganisms Burkholderia multivorans, B. cenocepacia and Aeromonas salmonicida bound insulin. A genomic library of B. multivorans was constructed in λ Zap Express and screened successfully for insulin binding recombinants. Recombinant phagemids p1 & p2 were excised, p1, encoded an insulin binding protein (IBP1 30 kDa) with homology to the iron complex siderophore receptor. For p2, two IBPs were detected at 20 & 30 kDa (IBP2 & IBP3), representing an intracellular and outer membrane peptide transporter. Comparison of IBP1 and human insulin receptor (HIR) produced 6 linear epitopes, and for IBP2 & IBP3 produced 3 epitopes. Thirdly, glutamic acid decarboxylase GAD65 is a major pancreatic autoantigen contributing to autoimmune diabetes. To assess the likelihood that microorganisms possess epitopes that mimic regions on GAD, 45 microbial species were tested for homology. This was facilitated by purifying recombinant GAD protein which was used to produce GAD antiserum. Four E. coli cross-reacting proteins were identified using mass spectrometry, outer-membrane protein A, formate dehydrogenase, superoxide dismutase and DNA starvation protein. Epitopes occurred at the C-terminal region of GAD65 (residues 419–565), a region previously reported to be targeted by autoantibodies. This study suggests that pyrrolnitrin and phenazine are cytotoxic to pancreatic β-cells and B. multivorans IBPs linear epitopes may be diabetogenic, particularly in patients with cystic fibrosis related diabetes (CFRD) who suffer a long term infections with Pseudomonas and Burkholderia species. Furthermore, microbial GAD epitopes could potentially induce an autoimmune response leading to diabetes.

Medulloblastomas are tumours of cerebellar origin and are thought to arise from the malignant transformation of progenitor cells in the developing cerebellum. A number of developmental signalling pathways are required for the precise cell specification, proliferation, migration and differentiation involved in forming the mature cerebellum and it is the dysregulation of these processes that can lead to the eventual formation of a tumour. Genes encoding components of the canonical Wnt/β-catenin signaling pathway are mutated in around 15% of medulloblastomas and germline mutations that activate this pathway are known to predispose to medulloblastoma. Despite this, the contribution of Wnt/β-catenin signaling to normal cerebellum development is not yet well understood and the developmental origins of medulloblastoma arising from activation of this pathway are only beginning to be revealed. Therefore, the aims of this thesis were to characterise the spatio-temporal nature of Wnt/β-catenin signalling during cerebellum development and to investigate its function, with the broad goal of informing our understanding of how medulloblastoma arises from oncogenic activation of Wnt/β-catenin signalling. To address the first aim I utilised a LacZ expressing Wnt/β-catenin signalling reporter mouse to characterize the spatio-temporal pattern of Wnt/β-catenin pathway activation during cerebellum development. Analysis of LacZ reporter expression revealed a pattern of transient Wnt/β-catenin activity in discrete cell populations throughout cerebellum development. I found that Wnt/β-catenin activity is present during the early specification of granule cells at the cerebellar rhombic lip but not during the expansion of this cell population at later stages. During perinatal development Wnt/β-catenin activity shifts to the cerebellar ventricular zone, a known germinal centre for GABAergic interneurons and glia, and was observed in cells radiating out from this region. By early postnatal development the expression of the Wnt/β-catenin reporter became progressively restricted to the developing Bergmann glia population. To investigate the function of Wnt/β-catenin in these cell lineages and how its dysregulation could contribute to medulloblastoma, I used a combination of ex vivo organotypic culture, in utero electroporation and tissue-specific gene targeting to manipulate components of the pathway. Culturing slices of E18.5 cerebellum in the presence of small molecule activators of the Wnt/β-catenin pathway revealed a reduction in the expression of glial markers Sox9 and GFAP. In addition, interneuron lineage marker Pax2 was also reduced, supporting the conclusion that dysregulation of Wnt/β-catenin signalling affects the generation of cell lineages from the ventricular zone. To investigate this hypothesis further, I constitutively activated the Wnt/β-catenin signalling pathway in the developing cerebellum using Cre-Lox gene targeting to knock out Apc, a negative regulator of the pathway, in ventricular zone derived lineages. Cre-induced recombination of Apc resulted in nuclear accumulation of β-catenin, a sign that the pathway had become ectopically activated. Furthermore, a reduction in the expression of Sox9 and Pax2 was also observed in these mutant cells. From these data, I conclude a potential role for Wnt/β-catenin signaling in the regulation of glial/interneuron progenitors. Combined, these data support a model where Wnt/β-catenin signalling could perform multiple functions in specification of the granule lineage, regulation of glial/interneuron progenitors and in glial differentiation/maturation. Importantly, dysregulation of progenitor self-renewal and differentiation is widely acknowledged to promote tumourigenesis. Thus, the data in this thesis support a potential mechanism for the development of medulloblastoma from the dysregulation of ventricular zone progenitors.

Methanococcus jannaschii is a thermophilic methane producing archaebacterium. In this organism genes encoding the aspartate transcarbamoylase (ATCase) catalytic (PyrB) and regulatory (PyrI) polypeptides were found. Unlike Escherichia coli where the above genes are expressed from a biscistronic operon the two genes in M. jannaschii are separated by 200-kb stretch of genome. Previous researchers have not been able to show regulation of the M. jannaschii enzyme by the nucleotide effectors ATP, CTP and UTP. In this research project we have genetically manipulated the M. jannaschii pyrI gene and have been able to assemble a 310 kDa E. coli like enzyme. By using the second methionine in the sequence we have shown that the enzyme from this organism can assemble into a 310 kDa enzyme and that this enzyme is activated by ATP, CTP and inhibited by UTP. Thus strongly suggesting that the second methionine is the real start of the gene. The regulation of the biosynthetic pathway in Pseudomoans aeruginosa has previously been impossible to study due to the lack of CTP synthase (pyrG) mutants. By incorporating a functional uridine (cytidine) kinase gene from E. coli it has been possible to isolate a pyrG mutant. In this novel mutant we have been able to independently manipulate the nucleotide pools and study its effects on the enzymes in the biosynthetic pathway. The enzyme asapartate transcarbamoylase was repressed 5-fold when exogenous uridine was high and cytidine was low. The enzyme dihydroorotate was repressed 9-fold when uridine was high. These results suggest that a uridine compound may be the primary repressing metabolite for the enzymes encoded by pyrB and pyrC. This is the first study to be done with the proper necessary mutants in the biosynthetic pathway of P aeruginosa. In the past it has been impossible to vary the internal UTP and CTP pools in this organism.

ER positive (ER+) and HER2 negative (HER2-) breast cancers are routinely treated based on estrogen dependence. CDK4/6 inhibitors in combination with endocrine therapy have significantly improved the progression-free survival of patients with ER+/HER2- metastatic breast cancer. Gene expression profiling in ER+/HER2- models was used to define the basis for the efficacy of CDK4/6 inhibitors and develop a gene expression signature of CDK4/6 inhibition. CDK4/6 inhibition robustly suppressed cell cycle progression of ER+/HER2- models and complements the activity of limiting estrogen. Chronic treatment with CDK4/6 inhibitors results in the consistent suppression of genes involved in cell cycle, while eliciting the induction of a comparable number of genes involved in multiple processes. The CDK4/6 inhibitor treatment shifted ER+/HER2- models from a high risk (luminal B) to a low risk (luminal A) molecular-phenotype using established gene expression panels. Consonantly, genes repressed by CDK4/6 inhibition are strongly associated with clinical prognosis in ER+/HER2- cases. This gene repression program was conserved in an aggressive triple negative breast cancer xenograft, indicating that this is a common feature of CDK4/6 inhibition. Interestingly, the genes upregulated as a consequence of CDK4/6 inhibition were more variable, but associated with improved outcome in ER+/HER2- clinical cases, indicating dual and heretofore unknown consequence of CDK4/6 inhibition. Interestingly, CDK4/6 inhibition was also associated with the induction of a collection of genes associated with cell growth; but unlike suppression of cell cycle genes this signaling was antagonized by endocrine therapy. Consistent with the stimulation of a mitogenic pathway, cell size and metabolism were induced with CDK4/6 inhibition but ameliorated with endocrine therapy. Together, the data herein support the basis for profound interaction between CDK4/6 inhibitors and endocrine therapy by cooperating for the suppression of cell cycle progression and limiting compensatory pro-growth processes that could contribute to therapeutic failure.

No description available.

B lymphocytes are an important type of immune cell that contributes to pathogen clearance. When dysregulated, these cells contribute significantly to diseases such as autoimmunity, allergy and B cell cancers. Here we examine an important regulatory circuit that involves the lipid phosphatase SHIP, a key regulator of the PI3K signaling pathway. SHIP was first described as the major effector of inhibitory IgG receptor FcγRIIB, which downregulates B cell antigen receptor (BCR) signaling pathways when co-engaged. However, it is also known to inhibit signaling downstream of several other receptors, both activating and inhibitory. Here we examine the regulation and function of SHIP in B cells, focusing on the inter-related influences of binding partners, tyrosine phosphorylation and subcellular localization dynamics. First, we assess interaction of SHIP to selected candidate binding partners using an in vitro screening approach. The two most robust interactions were further characterized with respect to dissociation constant. These were: a novel interaction between SHIP phospho-Tyr944 and the SH2 domain of Nck, and a known interaction between the SH2 domain of SHIP and FcγRIIB phospho-Y292. Next, we perform the first examination of SHIP Tyr944. We provide evidence that it contributes to interaction with Nck after BCR engagement and is required for inhibition of actin turnover by SHIP. Finally, we perform the first detailed examination of the mechanisms controlling SHIP localization in human B cells stimulated through the BCR with and without co-engagement of FcγRIIB. We discover that SHIP is recruited to the plasma membrane equally in both stimulation contexts, however FcγRIIB co-ligation results in reduced mobility of SHIP molecules at the cell periphery. We identify a novel and essential role for Syk kinase in promoting SHIP membrane localization, tyrosine phosphorylation, and interaction with known binding partner Shc1. Together, these results provide significant and exploitable insight into the molecular control of a clinically important regulator of B cell responses. / February 2017

No description available.

Master of Science in Biomedical Sciences / Department of Anatomy and Physiology / Yongming Sang / Animal metabolic and immune systems integrate and inter-regulate to exert effective immune responses to distinct pathogens. The signaling pathway mediated by mechanistic target of rapamycin (mTOR) is critical in cellular metabolism and implicated in host antiviral responses. Recent studies highlight the significance of the mTOR signaling pathway in the interferon (IFN) response. Type I IFNs mediate host defense, particularly, against viral infections, and have myriad roles in antiviral innate and adaptive immunity. In addition to their well-known antiviral properties, type I IFNs also affect host metabolism. However, little is known about how animal type I IFN signaling coordinates immunometabolic reactions during antiviral defense. Therefore, understanding the interaction of mTOR signaling and the type I IFN system becomes increasingly important in potentiating antiviral immunity. Tissue macrophages (MФs) are a primary IFN producer during viral infection, and their polarization to different activation statuses is critical for regulation of immune and metabolic homeostasis. Using porcine reproductive and respiratory syndrome virus (PRRSV) as a model, we found that genes in the mTOR signaling pathway were regulated differently in PRRSV-infected porcine alveolar MФs at different activation statuses. Therefore we hypothesize that: 1) the mTOR signaling pathway involves host anti-PRRSV regulation; 2) mTOR signaling interacts with IFN signaling to modulate the antiviral response; and 3) different type I IFN subtypes (such as IFN-α1 and IFN-β) regulate mTOR signaling differently. We show that modulation of mTOR signaling regulated PRRSV infection in MARC-145 cells and porcine primary cells, in part, through regulating production and signaling of type I IFNs. In addition, expression and phosphorylation of two key components in the mTOR signaling pathway, AKT and p70 S6 kinase, were regulated by type I IFNs and PRRSV infection. Taken together, we determined that the mTOR signaling pathway, a key pathway in regulation of cell metabolism, also mediates the type I IFN response, a key immune response in PRRSV infection. Our findings reveal that the mTOR signaling pathway potentially has a bi-directional loop with the type I IFN system and implies that some components in the mTOR signaling pathway can serve as targets for augmentation of antiviral immunity and therapeutic designs.

Like its enteric counterpart, aspartate transcarbamoylase (ATCase) from Pseudomonas putida is a dodecamer of two different polypeptides. Unlike the enterics, the Pseudomonas ATCase lacks regulatory polypeptides but employs instead inactive dihydroorotases for an active dodecamer. Previous work showed that PyrB contains not only the active site but also the effector binding sites for ATP, UTP and CTP at its N-terminus. In this work, 11 amino acids were deleted from the N-terminus of PyrB and the ATCase with the truncated protein was expressed in E. coli pyrB- and purified. The wild type enzyme was similarly treated. Velocity-substrate plots without effectors gave Michaelis-Menten kinetics in all cases. Deleting 11 amino acids did not affect dodecameric assembly but altered effector responses. When carbamoylphosphate was varied, the mutant enzyme was inhibited by UTP while the wild type enzyme was activated 2-fold. When the aspartate was varied, CTP had no effect on the mutant enzyme but strongly inhibited the wild type enzyme.