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published_or_final_version / abstract / Biochemistry / Doctoral / Doctor of Philosophy

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The gonad of the snail Helix aspersa is innervated by a branch of the intestinal nerve. Here it is demonstrated that nerve stimulation causes peristaltic contractions and the acceleration of cilia beating in the proximal part of the hermaphroditic duct. Acetylcholine and serotonin induced peristaltic contractions when applied without nerve stimulation. As well, serotonin induced the acceleration of cilia beating. The neuropeptide FMRFamide caused dilation of the hermaphroditic duct. Pharmacological blocking of acetylcholine and serotonin receptors with concurrent nerve stimulation induced a dilation similar to that caused by FMRFamide application. It is suggested that all three transmitters are released from intestinal nerve terminals to facilitate oocyte transport during ovulation. Nerve stimulation induced an increase in gamete transport rates. Because several candidate chemical messengers failed to induce ovulation when injected into the circulatory system, Helix aspersa appears to initiate ovulation differently from related species. Whereas Aplysia and Lymnaea use hormones, Helix apparently signals ovulation via the intestinal nerve.

This Thesis follows the portfolio format and a brief overview is given here. Chapter one is a systematic review of the literature on the relationship between emotion regulation, anxiety and depression in older adults. Chapter two is a research journal which explores the relationship between fear of falling and emotion regulation in community dwelling older adults. The systematic review is written up for publication in the Journal of Affective Disorders. The research article is written up for publication in Aging and Mental Health. Their respective style guidelines were followed. Purpose The aim of the thesis was to explore the relationship between emotion regulation and psychological distress in older adults. The aim of the systematic review was to explore the relationship between self-reported emotion regulation, anxiety and depression in older adults. The empirical study aimed to look at the relationship between fear of falling, a common type of psychological distress in older adults, and emotion regulation. It also aimed to look at the relationship between fear of falling related avoidance behaviour and emotion regulation. Methods The literature was systematically searched for research which has explored the relationship between emotion regulation, anxiety and depression in older adults. The papers which met the inclusion criteria were rated according to predetermined quality criteria. An overview of the results and implications were discussed. The empirical research used a cross-sectional design to examine the research hypothesis. Older adults completed self-report measures of emotion regulation, fear of falling, fear-related avoidance behaviour, anxiety and depression. Correlational analysis explored the relationship between the study variables. A linear regression model examined the unique contribution of emotion regulation to fear of falling after controlling for age, falls history, anxiety and depression. Results 12 studies met the inclusion criteria for the systematic review. The most prevalent relationship explored was that between rumination and depression with consistent evidence that higher levels of rumination were related to higher levels of depressive symptoms. Common methodological limitations were the lack of valid and reliable emotion regulation measures for older adults, non-random sampling, and failure to control for important confounding factors. Within the empirical research, a significant relationship between emotion regulation and fear of falling was found. There was also a significant relationship between emotion regulation and fear of falling avoidance behaviour. After controlling for age, number of falls, depression and anxiety, emotion regulation was no longer significantly associated with fear of falling. Depression was the only modifiable variable that retained a significant association to fear of falling.

Indiana University-Purdue University Indianapolis (IUPUI) / Stem cell factor (SCF) mediated c-Kit signaling, and downstream activation of Phosphatidylinositol-3 Kinase (PI-3K) is critical for multiple biological effects mediated by mast cells. Mast cells express multiple regulatory subunits of PI-3Kinase, including p85α, p85β, p50α and p55α. In the present study, we have examined the relationship between p85α and p85β subunit in mast cell development and show that loss of p85α in mast cell progenitors impairs their growth, maturation and survival whereas loss of p85β enhances this process. To further delineate the mechanism (s) by which p85α provides specificity to mast cell biology, we compared the amino acid sequences between p85α and p85β subunits. The two isoforms share significant structural homology in the two SH2 domains, but show significant differences in the N-terminal SH3 domain as well as the BCR homology domain. To determine whether the c-Kit induced reduction in growth of mast cells is contributed via the N-terminal SH3 or the BCR homology domain, we cloned and expressed the shorter splice variant p50α, and various truncated mutant versions of p85α in p85α deficient mast cells. We demonstrate both invitro and invivo that while the SH3 and the BH domains of p85 are dispensable for mast cell maturation; they are essential for normal growth and survival. In contrary to existing dogma on redundant functional role of PI-3K regulatory subunits, this study proves that p85α and p85β regulatory subunits of PI-3K have unique roles in mast cell development. We prove that p85α deficiency impairs the expression of multiple growth, survival and maturation related genes whereas p85β deficiency inhibits c-Kit receptor internalization and degradation. This novel finding on negative role of p85β in mast cell development has significant clinical implication, as this knowledge could be used to develop treatments for mast-cell-associated leukemia and mastocytosis.

Better understanding difficulties in emotion regulation may help integrate a conceptualization for the etiology of a number of emotional disorders, such as depression or anxiety, and personality disorders. However, one deficit in extant literature has been in identifying a widely accepted measure in assessing problems with emotion regulation. A number of emotion regulation measures are currently used in the literature. Because each measure addresses regulation from a different perspective, the definition of emotion regulation remains unclear. Gratz and Roemer (2004) provide an integrated conceptualization for emotion regulation that accounts for the many ways in which emotions may affect the expression of psychological disorders. Gratz and Roemer constructed the Difficulties in Emotion Regulation Scale (DERS) to assess various areas in which one might experiences problems with emotion regulation. The DERS is a 36-item scale that has six factors related to emotion regulation: Awareness, Non-acceptance, Clarity, Impulsivity, Strategies, and Goals. The purpose of this study was to perform a confirmatory factor analysis on the DERS, and to examine its predictive validity for disorders that are associated with difficulties in emotion regulation. The data for this study was collected from part of a larger study on emotion regulation. Participants consisted of undergraduates at a rural mid-western university who completed a survey packet including the DERS, the Trait Meta-Mood Scale (TMMS) the Center for Epidemiological Studies Depression Scale (CESDS), and the Posttraumatic Stress Disorder Checklist (PCL). It hypothesized that a six factor solution would be found, validating the findings of Gratz and Roemer (2004). It was also expected that total DERS scores will be associated with CESDS and PCL scores, suggesting that participants who scored higher on these measures of depression and PTSD would also report difficulties with emotion regulation. The DERS factors were expected to be related to corresponding factors on the TMMS. Results indicated that although a 6-factor solution was supported, several modifications were needed to the original model in order to achieve acceptable goodness of fit index values. Additionally, a number of other suggestions for changes to the measure, including re-wording several of the items or the possible removal of the Awareness factor, are discussed.

Proneural proteins are transcription factors of the bHLH family and have a conserved role in directing neurogenesis from invertebrate to mammals. In Drosophila, proneural proteins are required for early developmental specification of precursor cells of sense organs (SOPs). Despite considerable progress having been made in this field, it remains unknown how proneural proteins organise the well-orchestrated process that facilitates each type of SOP to acquire both generic neuronal properties and individual neuronal subtype identity during the progression from specification to differentiation. To approach this question, we investigate the gene regulatory network by proneural protein Ato by means of the microarray analysis. Ato directs the formation of the Drosophila chordotonal organs (Ch), important proprioceptive sense organs (Jarman et al., 1993b). The microarray study generated a list of candidate Ato target genes (Cachero et al., 2011). My PhD project entails the characterisation of two potential Ato target genes arising from this screen: Rfx and dila. To determine their positions in the gene regulatory network, I analysed the regulation and function of these genes. First, I demonstrated that both Rfx and dila are activated during Ch neurogenesis as direct targets of Ato. This was established by characterising their expression patterns, cis-regulation analyses and identifying the potential Ato binding sites by site-directed mutagenesis. RFX is a well-known ciliogenic regulator (Dubruille et al., 2002; El Zein et al., 2009; Swoboda et al., 2000), and its activation by Ato is consistent with Ch neurons having ciliated dendrites. However, the role of dila was completely unknown, but its sequence suggested that it may be involved in neuronal differentiation rather than gene regulation. I generated several dila mutant alleles and demonstrated that dila mutants exhibit severe uncoordination, due to a series of defects in ciliated neurons. These defects were linked to a disruption in the ciliogenesis machinery, particularly in the process known as intraflagellar transport (IFT). dila mutants also display reduced male fertility because of aberrant basal body function, which leads to a disorder in sperm individualisation. Thus DILA is required for the differentiation of all ciliated cells in Drosophila. Visualisation of tagged protein localised DILA to the basal body and transition zone of the sensory cilia. Further analysis revealed the genetic interaction between DILA and UNC (another basal body protein) during ciliogenesis. Taken together I propose that DILA regulates IFT at the base of the cilia in collaboration with UNC. Given that dila is an evolutionarily conserved gene, dila homologues could be candidate genes for human ciliopathies. Rfx is essential for ciliogenesis in both Ch and the external sense (ES) organs, which have distinctive cilia. Despite of this common role of RFX, I discovered that Rfx is expressed differently in Ch and ES lineages, which led me to hypothesise that the difference in Rfx expression modulates ciliogenesis in these two lineages. I obtained preliminary data that support this hypothesis. Overall, my study demonstrates important links between Ato and the regulation of ciliogenesis, which is an important process in Ch neuron differentiation. The data support a model in which Ato controls ciliogenesis both directly (e.g. via activating a ciliary genes like dila) and indirectly (e.g. via regulating the transcriptional factors essential for ciliogenesis, like RFX).

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