Thesis (M.S.)--Brandeis University, 2009. / Title from PDF title page (viewed on May 29, 2009). Includes bibliographical references.

Thesis (M.S.)--Brandeis University, 2009. / Title from PDF title page (viewed on August 9, 2009). Includes bibliographical references.

Thesis (M.A.)--Indiana University, 2007. / Department of Philosophy, Indiana University-Purdue University Indianapolis (IUPUI). Advisor(s): Jason T. Eberl, Peter H. Schwartz, William H. Schneider. Includes vitae. Includes bibliographical references (leaves 71-81).

More than a decade after the completion of the human genome project, the function of a large number of genes remains to be elucidated. Forward and reverse genetic approaches have proven to be powerful tools to study gene function and have provided insights into fundamental biological processes. Furthermore, functional genetic screening can lead to a better understanding of the action of endogenous and exogenous stimuli such as hormones or drugs on biological systems. Thus far, systematic and unbiased studies have largely been limited to model organisms. However, complex disease-relevant genotypes and phenotypes cannot be studied in entirety in lower organisms creating a need for systematic approaches in human cells. This thesis describes a series of studies using forward and reverse genetic approaches combined with state-of-the-art technology in haploid human cells. The first chapter describes the development of a quantitative phenotypic read-out using a novel application of RNA-sequencing that allows the functional annotation of genes in signalling pathways. The presented data demonstrate that the employed shallow RNA-sequencing method is scalable and suitable as a read-out for reverse genetic screening. The second chapter focuses on the implementation of this method in a large reverse genetic study in human cells to functionally annotate tyrosine kinases in signalling pathways upon stimulation with a set of ten polypeptides and small molecules. The screens revealed known and unexpected interactions between different signalling molecules and pathways, validating the technical approach in a biological context. The third chapter presents a pilot study describing the set-up of a forward genetic technique for compound mode-of-action screening using a pooled human mutant cell line collection. The chemical genetic approach displayed sufficient sensitivity and allowed to monitor thousands of gene-drug interactions simultaneously. Together, this thesis combines elements to advance technological and biological aspects of functional genomics and chemical genetics.

Soybeans are among seeds the common plant foods that contains high protein contents and high oil. The protein provides about 35 to 38 percent of the seeds calories compared to around 20 to 30 percent in other legumes and many animal products. The quality of soy protein is notable and approaches the quality of meat and milk. Unlike many other good sources of protein, soybeans are low in saturated fat and are cholesterol-free. Its proteins provide all the important amino acids, most the amounts needed by humans (NSRL, 2010). As the most consumed vegetable oil in the world, soybean oil has been used substantially in the food industry (Soystats, 2010). Its utilization is determined by its fatty acid composition, with commodity soybean oil typically 13% palmatic acid (16:0), 4% stearic acid (18:0), 20% oleic acid (18:1), 55% linoleic acid (18:2), and 8% linolenic acid (18:3). The change of fatty acid profiles to improve soybean oil quality has been a long time goal of many researchers throughout the world. Biodiesel is an up and coming trend in energy production. Breeding effort can be undertaken in order to produce a higher energy profile soybean oil. Using ethyl-methanesulfonate (EMS) mutagenesis effects on DNA, significant changes to the genes and gene network underlying the protein and oil profile can be achieved. These changes are hard to accomplish using standard breeding techniques. In addition, high amount of linolenic and stearic acid are very important for fuel and biodiesel production, but are not good for food production due to the fact that such oil is oxidized easily and the food goes rancid quickly. However, soybean oil with elevated amount of oleic acid is desirable for food, because this monounsaturated fatty acid improves the nutrition and oxidative stability of soybean oil compared to other oils. In order to improve the quality of soybean oil and processed foods, chemically mutagenized soybeans have been developed in this project. Seeds harvested from individual M3 and M4 plants (from 2 successive years 2012 and 2013) were analyzed for protein content, oil composition, and content. Moreover, seven phenotypic traits including oil analysis (stearic, palmitic, oleic, linolenic and linoleic), seed protein content, weight of the seeds (High yield), seeds color, stem length, germination rates, and branch architecture were collected and analyzed in this project of soybean `Forrest' mutagenized population. The result of this research showed that there were 25 significantly different lines (p< 0.05) compare to the wild type, which is useful for developing mutants with altered oil and fatty acid compositions in soybean.

Introduction: In recent years, the average age of first reproduction has risen significantly, the mean now standing at around 30 years in many countries. The adverse effects of maternal age on fertility and reproduction have been well documented. However, the influence of paternal age on fertility, reproduction and postnatal health is relatively poorly understood, and 50% of all male infertility cases are classed as idiopathic or unexplained infertility. Methods: The aim of this study was to investigate factors that contribute to male infertility, split into two main parts. The first part focused on analysing data collected from patients who had undergone fertility treatment to assess the influence of different factors on infertility, especially at the genome level. The second part attempted to deal with some of the technical challenges of screening and diagnostic methods to study the genome, with the aim of providing tools that would assist future studies in pinpointing genetic factors responsible for infertility, especially in cases of idiopathic infertility. Results: Based on data from the first part of the study, it was determined that advanced paternal age can affect sperm progressive motility, sperm DNA integrity and the fertilisation rate of in vitro fertilisation (IVF) cycles, as well as the development of embryos. Direct analysis of sperm DNA fragmentation (SDF) and degradation levels revealed an association between elevated SDF and impaired embryo development. Furthermore, a correlation was shown between chromosome aneuploidy and variance in SDF and sperm DNA degradation. Moreover, aneuploidy can influence abnormal sperm morphology and consequently also progressive motility. Also, embryo development rate of IVF cycles on day three, demonstrated a significant decline in cycles where the sperm used for fertilisation had a high aneuploidy rate, which can highlight the reduced developmental capacity of aneuploid embryos. From the lifestyle factors assessed, only alcohol consumption significantly correlated with the sperm DNA damage. Therefore, poor semen quality may highlight damage that has been incurred by the sperm DNA. When the semen quality is suboptimal, the intracytoplasmic sperm injection (ICSI) technique is suggested as a standard strategy to improve the prognosis of ART. However, when the progressive motility is poor, the ICSI approach is not as effective. Based on our findings and in line with other studies, the only sperm parameter that can be affected by paternal age is sperm motility, which could be an indicator of SDF. Therefore, the decline in ICSI fertilisation rate in patients with impaired sperm progressive motility could be due to sperm DNA damage, and even ICSI cannot improve the fertilisation rate considerably. Discussion: The aim of the second part of this project was to establish a robust workflow for whole- genome amplification (WGA) and whole-genome sequencing of single cells to improve the coverage rate and fidelity, with the aim of providing means of detecting any mutation in the genome that might be responsible for reduced embryonic developmental competence. Towards this end, the efficiencies of two different WGA protocols (REPLI-g and TruePrime) were compared. Multiple technical factors required optimisation in order to create a suitable protocol. Our results demonstrated the overall superiority of REPLI-g compared to TruePrime in almost all the assessed parameters. The amplification rate of REPLI-g was much faster than that of TruePrime, and prolonged incubation led to overamplification and an increased duplication rate. However, the TruePrime method has a slower amplification rate and therefore, by increasing the incubation time, it was possible to improve the quality of the data. The modified protocol with reduced volume also had the most promising outcome in terms of the data produced, and could fulfil our expectations by being fast, cost-effective and efficient. Conclusion: In conclusion, the results from the first part of this study confirmed the negative impact of male age on assisted reproductive treatments, which can result in decreased success rates of fertilisation. Other factors such as sperm DNA damage may also contribute to this age effect, suggesting that assessing this parameter prior to fertility treatment, and attempting to mitigate elevated levels of sperm DNA damage, may be of value to older patients. Additionally, overcoming the technical challenges in studying genetic contributory factors in infertility is a promising step toward better understanding of the mutations and variations that are involved in this phenomenon.

A doença de Tay-Sachs (DTS) é uma doença neurodegenerativa, de herança autossômica recessiva, que se manifesta a partir do sexto mês de vida. Crianças afetadas desenvolvem degeneração física e mental intensa, levando à morte até os 5 anos de idade. Não há atualmente cura ou tratamento disponível. Na população judaica, 1 em cada 31 indivíduos é portador da DTS, e a incidência da doença (aproximadamente 1 em cada 4.000 nascimentos) é cerca de 100 vezes maior nesta do que em outras populações. O advento do diagnóstico pré-natal para a DTS e o concomitante desenvolvimento de programas de detecção e orientação de heterozigotos da DTS em populações de judeus Ashkenazitas, iniciados em massa desde 1970, levaram a uma diminuição de 90% da incidência da doença nesta população. Estes programas são realizados em Israel, EUA e no Canadá, na população francocanadense. Três mutações no gene HEXA, codificador da sub-unidade ? da enzima hexosaminidase A, são responsáveis por 98% dos casos da DTS na população judaica Ashkenazita. Esse fato possibilita a utilização de um teste de DNA para a identificação de portadores da DTS nesta população. Segundo o censo do IBGE de 1991, a população judaica no Brasil é de 86.416 indivíduos. Este trabalho visou analisar a necessidade e a aceitação de um programa de detecção e orientação de portadores da DTS nesta população. Em particular, procurou-se (1) estabelecer a freqüência das 3 principais mutações causadoras da DTS na população judaica brasileira; (2) avaliar a reação de indivíduos desta população à proposta de um programa de triagem de portadores da DTS, com o objetivo de orientação para casais em risco de terem crianças afetadas. Este estudo foi realizado em escolas judaicas de ensino médio, em São Paulo e no Rio de Janeiro, seguindo o modelo canadense. Dentre 581 alunos (>=16 anos) que assistiram palestras informativas, 404 participaram da pesquisa, indicando uma taxa de participação de 70%. De acordo com os dados dos formulários de consentimento, aproximadamente 65% dos cromossomos analisados eram de origem judaica Ashkenazita. A análise das 3 mutações comuns no gene HEXA nestes 404 indivíduos detectou 8 portadores da DTS (7 da mutação InsTATC1278 e 1 de IVS12+1). Assim, a freqüência de portadores na amostra foi de 1 em cada 51 indivíduos. Considerando somente os cromossomos de origem judaica Ashkenazita, nossos dados indicam que a freqüência de portadores da DTS na amostra é de 1 em cada 33 indivíduos. As freqüências encontradas são equivalentes àquelas descritas em outras populações judaicas (P>0,05). Baseando-se nestes dados, concluí-se que justifica-se a implementação de um programa de identificação e orientação de portadores da DTS na população judaica brasileira. A contemplação dos diversos aspectos éticos envolvidos são parte essencial deste programa. / Tay-Sachs disease (TSD) is an autossomal recessive disease of lysossome storage characterized by progressive nervous degeneration. Children affected by TSD manifest first symptoms around 6 months and die before 5 years of age. TSD is caused by mutations in the HEXA gene, coding for the ? subunit of the hexosaminidase A enzyme. In the absence of the enzyme, its substrate, GM2 ganglioside, accumulates in the neurons of the central nervous cortex. Late onset TSD (chronic form) is a rare variant phenotype with appearance of first symptoms during the second or third decade of life. In the Ashkenazi jewish population, 1 in every 31 individuals is a TSD carrier. The disease incidence (1 in every 4.000 newborns) is 100 times higher in this population. The advent of pre-natal diagnosis for TSD and the development of preventive screening programs massively adopted by Ashkenazi jewish populations led to a 90% decrease in the disease incidence in this group. Three mutations in the HEXA gene are responsible for 98% of the disease incidence in Ashkenazi jews. This lead to the establishment of a DNA test for detection of TSD carriers in this population. The brazilian Ashkenazi jewish populations is around 100.000 individuals. This work aimed to establish the need and the acceptance of a screening program for this population. Specifically, it established the frequency of the 3 most common TSD mutations in a sample of the brazilian jewish population and evaluated the reaction of the community to the offer of a preventive program. This work was undertaken in jewish senior high-school students, in São Paulo and Rio de Janeiro, following the canadian preventive model. From 581 students (>=16 years old) that attended educational sessions, 404 volunteered to the test, indicating a 70% participation rate. According to the data of the consent form, approximately 65% of the chromosomes were associated to Ashkenazi jewish ancestry. The molecular analysis of the 3 most common mutations in the HEXA gene from the 404 participants detected 8 carriers (7 of InsTATC1278 and 1 of IVS12+1), thus indicating a carrier frequency of 1/51. In the Ashkenazi fraction, the estimated carrier frequency is 1 in 33 individuals. Both frequencies are similar to those described for other jewish communities (P>0,05). Based in this findings, it was concluded that the implementation of a screening program for TSD in the brazilian jewish population is feasible. The ethical aspects involved are an essential part of such a program.

Nephrotoxicity is a dose-limiting side effect of gentamicin that accounts for a significant portion of clinical acute kidney injury (AKI). The mechanism of gentamicin-induced nephrotoxicity is uncertain and effective therapy for gentamicin-induced renal cell injury is limited by incomplete mechanistic insight. To address this challenge, we hypothesized that RNAi signal pathway screening can identify both a unifying mechanism of gentamicin-induced cell injury and a therapeutic that ameliorates its toxicity. Dual shRNA screens of 5,000 individually barcoded signal pathway genes were performed in gentamicin-exposed human proximal tubule cell lines and differentially expressed shRNAs were analyzed by Ingenuity Pathways Analysis (IPA) software. Computational analysis of RNAi signal screens identified the Cross-Organelle Stress Response (CORE), the Unfolded Protein Response (UPR), and cell chaperones as key injury targets of gentamicin-induced proteotoxicity. To validate these injury mechanisms, we assessed the effect of gentamicin on the CORE, UPR and cell chaperone function, and tested the therapeutic efficacy of enhancing cell chaperone content. Early gentamicin exposure disrupted the CORE, evidenced by a rise in the ATP:ADP ratio, mitochondrial-specific H2O2 accumulation, Drp-1 associated mitochondrial fragmentation, and endoplasmic reticulum-mitochondrial dissociation. CORE disruption preceded measurable increases in whole cell oxidative stress, misfolded protein content, transcriptional UPR activation and its untoward downstream effects: CHOP expression, PARP cleavage and cell death. Geranylgeranylacetone, a therapeutic that increases cell chaperone content, prevented mitochondrial H2O2 accumulation, preserved the CORE, reduced the burden of misfolded proteins and CHOP expression, and significantly improved survival in gentamicin-exposed cells. We identify CORE disruption as an early and remediable cause of gentamicin proteotoxicity that precedes downstream cytosolic UPR activation and cell death. Preserving the CORE is associated with improved renal cell survival likely by reducing organelle-specific proteotoxicity during gentamicin exposure.

N-Acylethanolamines (NAEs) are fatty acid derivatives in plants that negatively influence seedling growth. N-Lauroylethanolamine (NAE 12:0), one type of NAE, inhibits root length, increases radial swelling of root tips and reduces root hair numbers in a dose dependent manner in Arabidopis thaliana L. (ecotype Columbia). A forward genetics approach was employed by screening a population of T-DNA “activation-tagged” developed by the Salk Institute lines for NAE resistance to identify potential genes involved in NAE signaling events in Arabidopsis thaliana L. (ecotype Columbia). Seeds of the activation tagged lines were grown at 0, 25, 30, 50, 75 and 100 µM N-lauroylethanolamime (NAE 12:0). Ten plants which displayed NAE tolerance (NRA) seedling phenotypes, compared with wildtype (Columbia, Col-0) seedlings were identified. I focused on one mutant line, identified as NRA 25, where the tolerance to NAE 12:0 appears to be mediated by a single dominant, nuclear gene. Thermal asymmetric interlaced (TAIL) PCR identified the location of the T-DNA insert as 3.86 kbp upstream of the locus At1g68510. Quantitative PCR indicated that the transcript level corresponding to At1g68510 is upregulated approximately 20 fold in the mutant relative to wildtype. To determine whether the NAE tolerance in NRA 25 is associated with overexpression of At1g68510 I created overexpressing lines of At1g68510 with and without GFP fusions behind the 2X35S CaMV promoter. As predicted, results with overexpressing lines of At1g68510 also exhibited enhanced resistance to NAE when compared with the wildtype. Confocal images of the fusion proteins suggest that GFP-At1g68510 is concentrated in the nucleus and this was confirmed by counterstaining with 4', 6-Diamidino-2-phenylindol (DAPI). Futhermore, At1g68510 overexpressing lines and NRA 25 line also exhibited tolerance to abscisic acid (ABA) during seedling germination. The findings suggests that At1g68510 overexpression mediates seedling tolerance to both ABA and NAE, a mechanism independent of fatty acid amide hydrolase in which its overexpression leads to NAE resistance but hypersensitivity to ABA. The next steps are to identify the association of At1g68510 with specific genomic regions or interacting proteins that may be additional components of NAE signaling in plants.

Background: There are few studies on the uptake of non-invasive prenatal screening, but those available suggest substantial variation in uptake in the initial years in which it was offered. There is a need to update the earlier evidence and determine whether there has been any change in usage trends as the number of users have increased. This will help inform policy makers about NIPT uptake under currently existing policies and guidelines which can help inform whether to maintain or refine policies on NIPT. Objectives: The primary objective of this thesis was to investigate recent trends in NIPT utilization, and the secondary objective was to identify differences between pregnant individuals aged 40 years and above and/or with a history of previous aneuploidy who opted for first-tier (first-line screening) or second-tier (contingent screening) NIPT and pregnant individuals aged less than 40 years with no history of previous aneuploidy. Methods: This retrospective cohort study used a province wide birth registry from Ontario and the population studied comprised pregnant individuals with an expected date of delivery from August 1st, 2016 to March 31st, 2020. Results: Of 536,748 pregnant individuals resident in Ontario during the study period, 27,733 were classified as high-risk of giving birth to a baby with a chromosomal aneuploidy and 509,015 were classified as low-risk of giving birth to a baby with a chromosomal aneuploidy. Uptake of NIPT has increased every year since 2016. We found substantial variation in NIPT between regions within the province. Highest uptake was found in urban areas, highest neighbourhood of income and education quintiles, high-risk population, among those with a prenatal care visit in the first trimester, multiple pregnancy, multigravidity, body mass index within the normal range (18.5-24.9 kg/m2), and OHIP funding. Conclusion: Our results suggest a need to provide more education/training about NIPT and funding eligibility to health professionals and pregnant individuals, including low-risk pregnant individuals in the first-tier (first-line screening) NIPT funding policy, to ensure equitable assess.