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Eukaryotes use proteins to carefully package and compact their genomes to fit into the nuclei of their individual cells. Nucleosomes are the primary level of compaction. Nucleosomes are formed when DNA wraps around an octamer of histone proteins and a nucleosome's position can limit access to genetic regulatory elements. Therefore, nucleosomes represent a basic level of gene regulation. DNA and its associated proteins, called chromatin, is usually classified as euchromatin or heterochromatin. Euchromatin is transcriptionally active with loosely packed nucleosomes while heterochromatin is condensed with tightly packed nucleosomes and is transcriptionally silent. In order to become active, heterochromatin must first be remodeled. We have studied the effects of nucleosome positioning on transgene expression in vivo using Caenorhabditis elegans as a model. We show that both location and polarity of the DNA sequence can influence transgene expression. We also discuss some considerations for working with CRISPR/Cas9. A major reason for doing in vitro nucleosome reconstitutions is to determine the effects of DNA sequence on nucleosome formation and position. It has previously been implied that nucleosome reconstitutions are stochastic and not very reproducible. We show that nucleosome reconstitutions are highly reproducible under our reaction conditions. Our results also indicate that a minimum depth of 35X sequencing coverage be maintained for maximal gains in Pearson's correlation coefficients. Communicating science with others is an important skill for any researcher. The rising generation of scientists need mentors who can teach them how to be independent thinkers who can carry out scientific experiments and communicate their finding to others. With this goal in mind, we have devised a scaffolding pedagogical method to help transform undergraduates into confident independent thinkers and researchers.

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Two rare Banksia species, B. chamaephyton A.S. George and B. elegans Meissner, were the subject of this study. B. chamaephyton is gazetted as rare under the Wildlife Conservation Act of Western Australia and B. elegans has been recorded by various authors as restricted in range and habitat.Data were collected on the distribution, habitat, reproduction and fire responses of each species. Both were found to occur predominantly in the Irwin Botanical District. B. chamaephyton is found in low heath on sand over laterite and B. elegans in scrub or thickets on deep, yellow sands. Data on species co-occurring with B. chamaephyton were gathered for the purpose of identifying habitats with actual or potential populations of the rare species. Several species appeared to be reliable indicators.Both species are represented in areas set aside for conservation although some populations are vulnerable and their loss would considerably reduce the range of each species. Of particular concern are the southernmost populations of B. chamaephyton and the northernmost populations of B. elegans. The latter appears to represent a size variant within the species. Although neither species is currently endangered, it is recommended that B. chamaephyton remain a gazetted rare species and that consideration be given to the gazettal of B. elegans.Both species are xenogamous and probably bird-pollinated although pollination by small mammals is a possibility. Both also have very low fruit and seed set. In B. chamaephyton, this is probably related to resource availability but B. elegans possesses a malformed stigma which may prevent the normal reproductive process from taking place. Most populations of B. elegans are sterile. Further research into the sexual reproduction and propagation of B. elegans is recommended.Fire is important to both species. In B. chamaephyton, fire, together with subsequent ++ / wet/dry cycles, is necessary for seed release from the follicles. Seedling recruitment is negligible in B. elegans. Mature individuals of both species survive fire and in B. elegans fire stimulates root suckering. Autumn burns appear to be most suitable for recruitment in both species, preferably at a minimum interval of ten years.

Polycyclic aromatic hydrocarbons (PAHs) are environmentally significant compounds due to the toxicity of some members. They are ubiquitous and are persistent bioaccumulative toxins(PBTs). The toxicity of PAHs represents a risk to human health, and there are varied risk assessment approaches to quantifying the risk posed by PAHs based on exposure routes and scenarios. PAHs are not carcinogenic until they are metabolically activated as the body attempts to break them down and forms reactive metabolites that bind to the DNA causing subsequent replication in the cells. Fundamental to assessing the risk posed by PAHs is understanding the metabolism of PAHs. Since exposure to PAHs is never to single PAHs, understanding what differences may occur in mixtures of PAHs gives accurate assessment of the dangers of PAHs. Understanding the dynamics of complex metabolism vis-a-vis single metabolism of PAHs and possible effects on the toxicity expression of PAHs is a necessary advancement to accurately impact and guide remediation strategies. Studies were carried out comparing the metabolism of the PAHs Phenanthrene (PHE), Flouranthene (FLA) and Benzo[a]pyrene (BAP) in single, binary and ternary mixtures by monitoring the disappearance of the parent compound. It was observed that PAH metabolism in the single PAH experiment differed from metabolism in both binary and ternary mixtures. Enzyme competition was evident in the metabolism of mixtures, changing significantly the metabolism patterns of individual PAHs. PAH structure was also seen to affect metabolism in mixtures and the possible creation of toxicity effects during mixture metabolism. PAH concentration changed over time with faster change during single PAH metabolism followed by ternary mixture metabolism and finally binary metabolism. These results affirm that substrate interactions must be considered in the risk assessment approaches to the dangers posed by exposure to PAHs.

Organisms must coordinate behavioral and physiological responses to changingenvironmental conditions. In the nematode C. elegans, the presence or absence of foodin the environment affects many metabolic and behavioral responses, including fathomeostasis, lifespan, and male mating. Specifically, male mating behavior normallyoccurs when a well-nourished male encounters a hermaphrodite, and is repressed if themale is under-nourished. To understand how environmental changes influence the driveto carry out specific behavioral tasks, I used C. elegans male mating as a model.Previously, mutants were isolated that display male mating behavior at inappropriatetimes, i.e. in the absence of mating cues. Loss of function mutations in the ERG K+channel, UNC-103, results in spontaneous seizures of the male sex muscles.Interestingly, I found that food deprivation can suppress unc-103(lf)-induced seizures,suggesting that pathways activated under this environmental condition can suppress theexcitability of the mating circuit.Using molecular, genetic, and behavioral assays, I identified sensory andmolecular mechanisms that reduce sex-muscle excitability under food-deprived conditions. I found that mutations that affect the muscular feeding organ, the pharynx,phenocopy the effects of food deprivation, and reduce sex-muscle excitability. Idemonstrated that mutations in the pharyngeal muscle protein, tropomyosin, cause thepharyngeal neurosecretory motor neurons (NSMs) to increase pharyngeal excitabilityand reduce sex-muscle excitability. Additionally, I found that olfactory neurons (AWCs)with sensory cilia exposed to the environment are up-regulated in the absence of foodstimuli, and also send inhibitory signals to the sex muscles. To determine howchemosensory and pharyngeal neurons in the head can signal to the genitalia, Ihypothesized that one mechanism could be via secretion of metabolic hormones. To testthis, I examined loss-of-function mutations in the insulin-like receptor, DAF-2, which isknown to regulate many behavioral and physiological responses to food. I demonstratedthat DAF-2 activity in the sex muscles is required for food-deprivation suppression ofunc-103(0)-induced seizures. I then identified components of a novel-insulin-like/DAF-2signaling pathway that reduces excitability. Specifically, I propose that ligand binding toDAF-2 activates PLC- and leads to increased cystolic Ca2+. This Ca2+ influx activatesCaMKII, which can phosphorylate/activate EAG-like K+ channels, thereby reducing cellexcitability.

Insulin signaling plays a prominent role in regulation of dauer formation and longevity in Caenorhabditis elegans. Here, I show that insulin signaling also is required in benzaldehyde-starvation associative plasticity, where worms pre-exposed to the odor attractant benzaldehyde in the absence of food subsequently demonstrate a conditioned aversion response towards the odorant. Animals with mutations in ins-1, daf-2, and age-1 which encode the homolog of human insulin, insulin/IGF-1 receptor, and PI-3 kinase, respectively, have significant deficits in benzaldehyde-starvation associative plasticity. Using a conditional allele I show that the behavioral roles of DAF-2 signaling in associative plasticity can be dissociated, with DAF-2 signaling playing a more significant role in the memory retrieval than in memory acquisition. I propose DAF-2 signaling acts as a learning specific starvation signal in the memory acquisition phase of benzaldehyde-starvation associative plasticity but functions to switch benzaldehyde-sensing AWC neurons into an avoidance signaling mode during memory retrieval.

Sperm play a pivotal role in determining the reproductive success of individuals whose sperm must compete directly with that of others. I used sperm precedence assays and experimental evolution to examine the role of sperm traits in the reproductive success of hermaphrodites and males in the androdioecious nematode Caenorhabditis elegans. First, sperm size and the rate of reproduction were analyzed, in the context of male-male sperm competition, for evidence of natural heritable variation. Sperm size proved to be a strong indicator of second-male sperm precedence in the genotypes examined. Second, I tested the theoretically predicted effect of larval development time on the number of self-sperm produced by hermaphrodites. I demonstrated that a short larval development period favored the evolution of fewer sperm, inline with theoretical predictions. These results provide important insights into C. elegans reproductive biology and more generally to our understanding of the evolution of reproductive systems.

E3 ubiquitin ligases are important regulators of several cellular processes, including apoptosis. To determine the extent to which E3 ligases regulate DNA damage-induced apoptotic signalling in C. elegans, a high-throughput RNAi screen was performed in our laboratory. We identified the E3 ubiquitin ligase EEL-1 as a positive regulator of DNA damage-induced germ cell apoptosis. ARF-BP1, the mammalian EEL-1 ortholog, negatively regulates both the tumour suppressor protein p53 and the anti-apoptotic protein Mcl-1. In C. elegans, we found that eel-1 regulates DNA damage-induced germ cell apoptosis by a mechanism downstream of cep-1/p53 and upstream of ced-9/mcl-1. My results show that unlike ARF-BP1, EEL-1 does not regulate CED-9/Mcl-1 protein levels, suggesting a novel mechanism of apoptosis regulation in C. elegans for this E3 ligase. Unexpectedly, eel-1 causes synthetic sterility in ced-9 loss-of-function mutants that is suppressed by ablation of the Apaf-1 orthologue ced-4, suggesting an additional role for these genes in oogenesis.

E3 ubiquitin ligases are important regulators of several cellular processes, including apoptosis. To determine the extent to which E3 ligases regulate DNA damage-induced apoptotic signalling in C. elegans, a high-throughput RNAi screen was performed in our laboratory. We identified the E3 ubiquitin ligase EEL-1 as a positive regulator of DNA damage-induced germ cell apoptosis. ARF-BP1, the mammalian EEL-1 ortholog, negatively regulates both the tumour suppressor protein p53 and the anti-apoptotic protein Mcl-1. In C. elegans, we found that eel-1 regulates DNA damage-induced germ cell apoptosis by a mechanism downstream of cep-1/p53 and upstream of ced-9/mcl-1. My results show that unlike ARF-BP1, EEL-1 does not regulate CED-9/Mcl-1 protein levels, suggesting a novel mechanism of apoptosis regulation in C. elegans for this E3 ligase. Unexpectedly, eel-1 causes synthetic sterility in ced-9 loss-of-function mutants that is suppressed by ablation of the Apaf-1 orthologue ced-4, suggesting an additional role for these genes in oogenesis.