Thesis advisor: Laura Anne Lowery / Wolf Hirschhorn Syndrome (WHS) is a neurodevelopmental disorder characterized by craniofacial malformations, heart and skeletal defects, intellectual disability as well as seizure disorders. While this disorder is thought to arise from a deletion of a region on the short arm of chromosome 4, which includes the four genes WHSC1, WHSC2, LETM1 and TACC3, the mechanism by which loss of these genes results in WHS is not understood. Given that these genes have been linked to cell migration and that affected tissues include those derived from the neural crest, we propose that WHS results from a defect in neural crest cell migration. Here, we show that WHSC1, WHSC2, TACC3 and LETM1 are all expressed along the neural tube and developing neural folds during Xenopus embryonic development. These genes are additionally enriched in the pharyngeal arches, which are migrating neural crest cells. The knockdown of these WHS-related genes leads to variable defects in craniofacial and cartilage morphology. Moreover, the loss of WHS gene expression causes defects in forebrain and midbrain development. This implicates these four genes in the WHS phenotype. Further analysis of both WHSC1 and TACC3 function show that their individual knockdown causes defective neural crest cell migration both in vivo and in vitro. This supports the notion that the WHS phenotype is a result of erroneous neural crest cell motility. Our analysis shows that the WHS related genes; WHSC1, WHSC2, LETM1 and TACC3, play a role in the WHS phenotype of craniofacial malformation, skeletal abnormality, and microcephaly. Further analysis of these genes will determine the combinatorial effects of their knockdown on neural crest cell migration during embryonic development to further elucidate the mechanism through which WHS develops. / Thesis (BS) — Boston College, 2018. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Biology.

Organophosphate (OP) pesticides, as a group, are the most widely used insecticides in Australia. Approximately 5 000 tonnes of active ingredient are used annually (Radcliffe, 2002). The OP pesticide group consists of around 30 identifiably distinct chemicals that are synthesised and added to approximately 700 products (Radcliffe, 2002). OP pesticides are used on fruit, vegetable, grain, pasture seed, ornamental, cotton, and viticultural crops, on livestock and domestic animals, as well as for building pest control. OP pesticides all act by inhibiting the nervous system enzyme acetylcholinesterase (AChE) and as such are termed anticholinesterase insecticides. The phosphorylation of AChE and the resultant accumulation of acetylcholine are responsible for the typical symptoms of acute poisoning with OP compounds. In addition to acute health effects, OP compound exposure can result in chronic, long-term neurological effects. The traditional method of health surveillance for OP pesticide exposure is blood cholinesterase analysis, which is actually biological effect monitoring. However, there are several drawbacks associated with the use of the blood cholinesterase test, including its invasive nature, the need for baseline levels and a substantial exposure to OP pesticide before a drop in cholinesterase activity can be detected. OP pesticides are metabolised fairly rapidly by the liver to form alkyl phosphates (DAPs). Approximately 70% of OP pesticides in use in Australia will metabolise into one or more of six common DAPs. During the last 30 years, scientists have developed a urine test that detects these six degradation products. However, unlike the blood cholinesterase test, there is currently no Biological Exposure Index (BEI) for the urine DAP metabolite test. Workers in the agricultural industry - particularly those involved with mixing, loading and application tasks - are at risk of exposure to OP pesticides. It is therefore important that these workers are able to assess their risk of health effects from exposure to OP pesticides. However, currently in Queensland, workplace health and safety legislation exempts the agricultural industry from hazardous substance legislation that incorporates the requirement to perform risk assessments and health surveillance (blood cholinesterase testing) for OP pesticide exposure. The specific aim of this research was to characterise OP pesticide exposure and to assess the feasibility of using urine DAP metabolite testing as a risk assessment tool for agricultural and related industry workers exposed to OP pesticides. An additional aim among farmers was to conduct an in-depth evaluation of their knowledge, attitudes and behaviours related to handling OP pesticides and how they assess the risks associated with their use of OPs. A cross-sectional study design was used to assess exposure to OP pesticides and related issues among four groups: fruit and vegetable farmers, pilots and mixer/loaders, formulator plant staff and a control group. The study involved 51 farmers in the interviewer-administered questionnaire and 32 in urine sample provision. Eighteen pilots and mixer/loaders provided urine samples and 9 exposed formulation plant staff provided urine and blood samples. Community controls from Toowoomba Rotary clubs provided 44 urine samples and 11 non-exposed formulation plant staff provided blood and urine samples; all groups also provided responses to a self-administered questionnaire. Participant farmers were drawn from the main cropping areas in south-east Queensland - Laidley/Lowood, Gatton, and Stanthorpe. The farmer group was characterised by small owner-operators who often had primary responsibility for OP pesticide mixing and application. Farmers had good knowledge of pesticide-related safety practices; however, despite this knowledge, use of personal protective equipment (PPE) was low. More than half of the farmers did not often wear a mask/respirator (56%), gloves (54%) or overalls (65%). Material Safety Data Sheets were never or rarely read and 88.2% of farmers never or rarely read OP pesticide labels before application. There were also problems with chemical suppliers providing farmers with MSDSs. The majority of farmers (90.2%) reported that they had never had any health surveillance performed and three-quarters had never read about or been shown how to perform a formal risk assessment. The main inhibitors to the use of PPE in the farmers' group included the uncomfortable and cumbersome nature of PPE, especially in hot weather conditions, and the fear of PPE use triggering neighbours' complaints to Government authorities. Factors associated with better PPE use included having positive attitudes and beliefs toward PPE use, higher knowledge scores and low risk perception. Farmers' use of OP pesticides was infrequent, of short duration and involved application via a boom on a tractor, a lower risk application method. Consequently, urine DAP metabolite levels in this group were generally low, with 36 out of 96 samples (37.5%) containing detectable levels. Detectable results ranged from 9.00-116.00 mol/mol creatinine. Formulators exposed to OP pesticides were found to have the highest urine DAP metabolite levels (detectable levels 13.20-550.00 mol/mol creatinine), followed by pilots and mixer/loaders (detectable levels 8.40-304.00 mol/mol creatinine) and then farmers. Despite this, pilots and mixer/loaders (particularly mixer/loaders) had the greatest number of samples containing detectable levels (94.4% of samples). The DAP metabolite most frequently detected across all groups was DMTP, which was the only metabolite found in control samples. Levels found in this study are similar to those reported in international research (Takamiya, 1994, Stephens et al., 1996, Simcox et al., 1999, Mills, 2001, Cocker et al., 2002). The observed DAP levels were not associated with a drop in cholinesterase activity among the formulation plant workers, as expected from the literature. Such exposure also is unlikely to be associated with acute health effects. In contrast, there is insufficient scientific knowledge to know whether levels recorded in this study and elsewhere may be associated with long-term, chronic health effects. Notably, DMTP levels also were observed among the presumably 'unexposed' comparison groups. Environmental background level exposures to OPs producing the DAP metabolite DMTP are therefore of potential significance and may be related, at least in part, to consumption of contaminated fruit and vegetables. There is also emerging evidence to suggest that exposure to DAP metabolites themselves through diet and other sources may contribute to the concentration of DAPs, including DMTP in urine, potentially complicating assessment of occupational exposures. Nevertheless, the urine DAP metabolite test was a useful, sensitive indicator of occupational OP pesticide exposure among agricultural workers and may be of use to the industry as part of the risk assessment process. Future research should aim to establish a BEI for the urine DAP test.