P53 is a tetrameric transcription factor which exhibits DNA binding activity through its core domain which encompasses the conserved domains (known as Box II, III, IV and V). The N-terminal domain of p53 provides a scaffold for binding of components of the transcriptional machinery. Phosphorylation at residues within the N-terminal transactivation domain of p53 such as Serine 20 is a crucial event in the activation of p53. It stabilises the binding of the co-activator p300, reduces the binding of the inhibitory partner Mdm2 and enhances activation of p53 target genes. The identification of enzymes that phosphorylate p53 transactivation domain is an important development in the ongoing mapping of signaling pathways that control p53-dependent transcription and resultant tumour suppression. Environmental and physiological stresses activate p53 which has led to the creation of several hypothetical models in which tumour suppressor kinases mediate p53 activation by phosphorylation at Serine 20. Although much researched the identity of the main Serine 20 kinase in cells remains undefined. In this study we have identified Calcium Calmodulin kinase superfamily (CAMK) members as potent Serine 20 kinases in cells and show that the co-transfection of p53 peptides derived from the conserved domains can modify this response. Moreover, we show that the multi-protein docking site, p53 Box V domain, is required for Serine 20 phosphorylation and ubiquitination of p53. To further define the domains required for the interaction of p53 with CAMK superfamily members, mutagenesis of p53 was performed. Using transcriptional and binding based assays we were able to establish that p53 does indeed form an interaction with Chk1 and DAPK1. Development of cell models and gene expression studies demonstrated that depletion of Chk1 and DAPK1 results in activation of the p53 signalling pathway. There may therefore be a role for kinases as negative regulators of p53 and a potential for the development of kinases as drug targets for reactivation of the p53 pathway.

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Human tissue kallikrein-related peptidases (KLK) are fifteen genes located on chromosome 19q13.4, encoding hormonally regulated, secreted serine proteases with trypsin/chymotrypsin-like activity. I identified expression of many KLKs in tissues throughout the female reproductive system and in cervico-vaginal fluid (CVF). The female reproductive system is hormonally regulated during the menstrual cycle, suggesting KLKs may also be regulated by these hormones. Measurement of KLKs levels in CVF and saliva samples throughout the menstrual cycle revealed a peak in expression following ovulation in both fluids. Progesterone levels rise during this period suggesting KLK regulation by progesterone during the menstrual cycle. Using proteomic techniques, I resolved the CVF proteome to identify potential KLK substrates. Among 685 proteins identified, several cell-cell adhesion molecules, cervical mucins and defense-related proteins were found. KLKs play a role in the desquamation of skin corneocytes through cleavage of cell-cell adhesion proteins. The vaginal epithelium undergoes cyclical changes during the menstrual cycle involving desquamation of cells upon rising progesterone levels. The post-ovulatory peak in KLK expression suggests that KLKs may contribute to cell desquamation during the menstrual cycle. Cervical mucus acts to block the uterus from vaginal microorganisms. Around ovulation, cervical mucus loses viscosity to facilitate sperm passage through the cervix. Proteolytic enzymes are thought to aid in this mucus remodelling. Our immunohistochemical studies localized KLK expression to the mucus secreting cervical epithelium and I investigated KLK processing of cervical mucin proteins in vitro. KLKs 5 and 12 were found to cleave mucins, suggesting their potential involvement in cervical mucus remodelling. CVF plays a role in host defense. KLKs are known to process the antimicrobial cathelicidin protein in skin and I investigated whether KLKs may also process antimicrobial proteins found in CVF. KLK5 was found to cleave defensin-1 alpha, in vitro, suggesting KLKs may aid in defense of the female reproductive system. Here I provide evidence of potential physiological roles for KLKs in the female reproductive system: in desquamation of vaginal epithelial cells, remodelling of cervical mucus and processing of antimicrobial proteins. These findings suggest KLKs may function in female fertility, in pathological conditions such as vaginitis and in host defense.

Schizophrenia is characterized by positive and negative symptoms along with cognitive symptoms that include impairment in working memory (WM). WM is the storage of relevant information for short intervals of time to guide thoughts and actions. The neural correlate of WM is thought to be the persistent activity exhibited during the retention interval of WM tasks. Persistent activity is hypothesized to be mediated by the activation of NMDA receptors (NMDAR) within recurrent neuronal circuits. Consistent with this hypothesis, studies with healthy humans and monkeys have shown that the administration of the NMDAR antagonist ketamine induces memory-load dependent deficits in WM, along with increasing response time. In parallel to this, the pathophysiology of schizophrenia has been hypothesized to rest on the hypofunction of NMDAR. Previous studies in humans indicate that blockade of NMDAR induces schizophrenia-like symptoms. In addition, symptoms of schizophrenia patients are alleviated with sub-chronic treatments focusing on the activation of the NMDAR co-agonist site. Based on these observations, I tested the hypothesis that increasing the activation of NMDAR with co-agonist stimulation has beneficial effects on WM. D-serine (100mg/kg/day-6 weeks) was orally administered to two female macaque monkeys performing a visual sequential comparison task (VCST), which allows the manipulation of memory load. In this task, the monkeys had to identify the location of a colour change within an array of 2 to 5 coloured stimuli following a retention interval of 1 second. I hypothesized that sub-chronic treatment with D-serine produces a gradual improvement in the monkeys’ performance on the VSCT. Specifically, I predicted that the improvement would scale with memory load due to increased demands on WM resources at higher loads. Contrary to my hypothesis, D-serine produced minute changes in response accuracy, which were not memory load-dependent. Also, the response latency of the monkeys was found to increase, which is commonly observed following NMDAR antagonist treatments. These findings suggest that D-serine has a limited role in increasing the activation of NMDARs to improve WM per se. The beneficial effects reported by NMDAR co-agonists in schizophrenic patients could be a general reduction in cognitive symptoms, not specifically related to WM. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2013-10-01 17:45:20.643