The 21st century marks the defining point of human history in terms of technological advancement. In 2014, we were at the edge of acquiring a complete understanding of the fundamental construct to all life forms. The capability to manipulate and recreate lives as desired will soon be at our hands and will eventually lead to the redefinition of life and humanity. This brave new world, for better or worse, will be stitched together by scientific breakthroughs in many disciplines. Nanopore fluidic system – and microfluidic in general – might be one of the key puzzles towards the future. It is seen as a likely candidate for the next generation of rapid and low-cost genetic sequencing technology, which will allow us to gain thorough insight into the genetic code of every living organism on earth. It can also have the capability to individually detect and manipulate virtually any biological molecules, possibly allowing it to be a universal diagnostic tool or a bio-molecule synthesiser. The future of nanopore fluidic system is prosperous, but the difficulties are equally challenging. Currently, both biological and solid-state nanopores are non-trivial to create. For instance, a small solid-state nanopore can only be fabricated with expansive machinery in a low-yield, low-throughput manner. To overcome this challenge, a new set of methods involving high electric field to fabricate and enlarge a solid-state nanopore has been developed. It was found that a nanopore, when subjected to a high electric field, can be enlarged in angstrom increments and cleared of unidentified obstructions that cause low-frequency ionic current fluctuations. It was also found that an intact solid-state membrane, when subjected to a high electric field for a period of time, can leave a single nanopore imprinted onto it. The process of creation is best describe as a dielectric breakdown event and can be modeled by the percolation theory for dielectric breakdown. The resulting nanopores are cylindrical in shape and are shown to be equally capable of single molecule sensing compare to pores created by other methods. To accommodate future nanopore designs and applications and to examine the scope of applicability of the new fabrication approach, more advanced nanopore devices were created on some dual-layer solid-state membranes comprising of a metallic and a dielectric layer. Experiments indicated that the method could indeed create nanopore on such advanced membranes. It was further shown that the metallic layer receded further than the dielectric layer, forming a hollow conical shape at the opening of the dielectric nanopore. Such metalized bi-layer nanopore system was found to interact strongly with short single stranded DNA molecules, resulting in prolonged DNA translocation time. A simple picture of the mechanism was proposed to explain the observation. Lastly, to extend the limit of the new fabrication approach, I attempted to fabricate nanopore on complex multi-layer membranes involving a graphene film sandwiched in several dielectric materials. It was found that the quality of the graphene film and the transfer method were vital to the success of this project. Nevertheless, preliminary results indicated that the new method could create a nanopore through this complex multi-layer membrane. The new method to fabricate and tune both simple and complex nanopores is amongst the simplest, the least costly and the most efficient one that one can imagine. The research work has already sparked a dramatic increase in scientific throughput in our laboratory and other laboratories we had collaboration with. It fueled more than a dozen projects and involved close to a thousand nanopores in total. Such projects are far from possible if they were to rely on conventional fabrication methods. However, these are insignificant if we consider the new method is simple enough that, for the very first time, general public can easily access nanofabrication and single-molecule manipulation technology. The liberation of nanotechnology to the general public symbolically marks the beginning of a brave new world.

Lower motor neuron diseases and peripheral neuropathies are two groups of diseases that include multiple rare disorders where many causes are unknown and definitive treatments are unavailable. Understanding the molecular etiology of these genetic diseases provides an opportunity for rapid diagnosis, preconception genetic counseling and, in a subset, direction for the development of future treatment options. The recent introduction of whole exome sequencing (WES) marks a new era in Mendelian genetic disease research as the majority of the coding region of the genome can be sequenced in a timely and cost-effective manner. In this study, WES was used to investigate the molecular etiology of a cohort of 37 patients presenting with lower motor neuron disease or peripheral neuropathy. A molecular diagnosis was determined for seven patients informing the diagnostic utility of WES. Novel phenotypes were found for three genes originally associated with a different disorder. Finally, the foundation has been laid, through the use of functional studies and large scale data-sharing, to identify novel disease-causing genes for lower motor neuron disease and peripheral neuropathy.

Objective: The objective of this thesis was to estimate the impact on newborn screening (NBS) results of changing screening technology from tandem mass spectrometry (MS/MS) to an approach using targeted next-generation sequencing (T-NGS) and MS/MS in parallel. Methods: We integrated results of an analysis of MS/MS screening data for phenylketonuria (PKU) and medium-chain acyl-CoA dehydrogenase (MCAD) deficiency; and a query of genetic compendia for variants of genes associated with the two disorders. Results: The introduction of T-NGS into NBS may reduce nearly 80% of false positives that are generated using the current screening approach. Based on estimated NBS results, T-NGS may be applied using a second-tier approach, which may improve specificity while maintaining sensitivity at its current level. Discussion: T-NGS may enhance the performance of NBS for PKU by improving specificity when used as a second tier test, but may be limited by feasibility and cost under current circumstances. Future studies should consider the cost-effectiveness of T-NGS for all infants undergoing NBS.

A comprehensive molecular and phenotypic analysis of congenital and childhood cataractRachel L. Gillespie; The University of Manchester, Doctor of Philosophy, 2015Congenital and childhood cataract (CCC) is estimated to affect 3.5-6 per 10,000 children under 16 years in developed countries - a major cause of lifelong visual impairment. It is estimated that 25-50% of CCC cases are caused by genetic mutations. CCC demonstrates extreme heterogeneity with more than 100 associated genes, and may occur as an isolated anomaly of the eye (non-syndromic) or as a manifestation of a multisystem condition (syndromic). Limitations of conventional sequencing technologies have precluded precise genetic diagnosis and limited understanding of the epidemiological basis of the condition. Next generation sequencing (NGS) technologies have revolutionised the approach to the study of human disease. The aim of this research was to conduct a comprehensive molecular and phenotypic analysis of CCC using NGS.A disease-targeted NGS assay was designed to screen, in parallel, 115 genes associated with all forms of CCC. DNA from 36 patients, randomly selected from the study cohort, underwent cataract-targeted NGS. Putative cataract-causing variants were identified in 75% of individuals; 85% of non-syndromic patients and 63% of syndromic CCC patients. Cataract-targeted NGS was able to efficiently delineate disease sub-type and in some cases identified rare syndromic forms of the condition. These findings were envisaged to alter care and management of CCC patients demonstrating the potential clinical utility of the test. In a subset of cases, NGS identified CCC was a manifestation of an inborn error of metabolism. A number of these conditions were eligible for preventative treatment emphasizing the importance of early diagnosis. A strategic approach to the identification of novel recessive causes of CCC was also undertaken. Affected children from seven consanguineous families underwent pre-screening by cataract-targeted NGS to delineate those with mutations in known genes. Mutation negative patients underwent autozygosity-guided whole exome sequencing (WES) analysis. This strategic approach to disease gene discovery led to the identification three novel cataract-causing candidate genes, TRIM8, CCDC13 and GRWD1. It also led to the association of EIF2B2, known to cause adult-onset leukoencephalopathy with vanishing white matter (VWM) disease featuring pre-senile onset cataract, with childhood-onset cataract. This work demonstrated that cataract-targeted NGS offers an efficient and unbiased means of pre-screening, however, causation is difficult to assign to novel disease genes in the absence of experimental evidence. Correspondingly, in vitro analysis of a missense variant in HMX1 demonstrated the deleterious effect of the mutation on protein function. This work confirmed HMX1 as the cause of a rare oculoauricular phenotype and expanded the class of disease-causing mutations in this gene. In conclusion, this study has demonstrated that NGS is effective in the study of CCC and has provided a platform for future studies in to the genetic aetiology of the condition, as well as the molecular mechanisms underlying lens transparency and human development. The work adds to the increasing body of evidence that augurs an era of personalised genomic medicine in ophthalmology that will foresee improved patient outcomes attributable to the implementation of a stratified approach to medicine.

Knowledge of the diet of wildlife can aid wildlife biologists to better understand how a species functions within a given ecosystem. Numerous studies have identified various avenues to examine diet for species throughout the world. Wildlife biologists have used diet composition variables as a means to better understand habitat use and aid in the management and conservation of mule deer, Odocoileus hemionus. The complexity of deer diet is still unknown, and local wildlife management agencies could improve conservation strategies with more information regarding the breadth of plant selection in deer diet. Researchers have used non-invasive methods, such as microhistology via fecal analyses, to assess diet composition for mule deer. However, microhistology has several drawbacks that include accuracy in identification and differentiation of plant species, and even genus, as well as determination of accurate proportions of taxa ingested. Genetic techniques, such as next-generation sequencing (NGS), present new avenues for analyzing herbivore diets, especially through the amplification and analyses of specific regions of chloroplast DNA (cpDNA). Additionally, few studies have directly compared microhistological and NGS diet analyses results for any wildlife species. My objectives were to compare diet composition results of both microhistological and NGS diet analyses through estimating diet richness, taxonomic resolution, percent diet, and frequency of occurrence of plant taxa across samples. Mule deer fecal samples were collected on the Navajo Nation from summer and winter ranges of two distinct mule deer populations, Chuska and Carrizo. I found far greater richness and resolution from NGS of plant taxa through the identification of a greater number of species and genera among all populations, within seasons. Upon testing both methods for both populations, no significant agreement was identified for percent of families identified in the diet with both methods, across all samples. I found trends of positive correlation in the occurrence of families between both methods for Carrizo summer diet, as well as among genera and families in Carrizo winter diet. Upon further statistical analyses, I found no significant positive correlation in the occurrence of genera and families identified with both methods among all samples. Genetic techniques may present innovative methods for determining mule deer diet in various ecosystems, and may also be applied to a broad range of herbivore diet studies.

Dezoito galinhas de uma população experimental utilizada em um cruzamento recíproco entre as linhagens de frangos de corte (TT) e de postura (CC) foram sequenciadas pela tecnologia de nova geração na plataforma Illumina com uma cobertura média de 10X. A descoberta de variantes genéticas foi realizada em uma região de locos de característica quantitativa (Quantitative Trait Locus, QTL), associado anteriormente com peso e percentagem de gordura abdominal no cromossomo 3 da galinha (GGA3), entre os marcadores microssatélites LEI0161 e ADL0371 (33,595,706-42,632,651 pb). O programa SAMtools foi utilizado na identificação de 136.054 SNPs únicos e 15.496 INDELs únicas nos 18 animais sequenciados e após a filtragem das mutações, 92.518 SNPs únicos e 9.298 INDELs únicas foram mantidas. Uma lista de 77 genes foi analisada buscando genes relacionados ao metabolismo de lipídios. Variantes localizadas na região codificante (386 SNPs e 15 INDELs) foram identificadas e associadas com vias metabólicas importantes. Variantes nos genes LOC771163, EGLN1, GNPAT, FAM120B, THBS2 e GGPS1 foram identificadas e podem ser responsáveis pela associação do QTL com a deposição de gordura na carcaça em galinhas. / Eighteen chickens from a parental generation used in a reciprocal cross with broiler and layer lines were sequenced by new generation technology with an average of 10-fold coverage. The DNA sequencing was performed by Illumina next generation platform. The genetic variants discovery was performed in a quantitative trait loci (QTL) region which was previously associated with abdominal fat weight and percentage in chicken chromosome 3 (GGA3) between the microsatellite markers LEI0161 and ADL0371 (33,595,706-42,632,651 bp). SAMtools software was used to detect 136,054 unique SNPs and 15,496 unique INDELs for the 18 chickens, and after quality filtration 92,518 unique SNPs and 9,298 unique INDELs were retained. One list of 77 genes was analised and genes related to lipid metabolism were searched. Variants located in coding region (386 SNPs and 15 INDELs) were identified and associated with important metabolic pathways. Loss of functional variants in the genes LOC771163, EGLN1, GNPAT, FAM120B, THBS2 and GGPS1 may be responsible for the QTL associated with fat deposition in chicken.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22002号 / 医博第4516号 / 新制||医||1038(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 滝田 順子, 教授 松田 文彦, 教授 山田 亮 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22011号 / 工博第4623号 / 新制||工||1720(附属図書館) / 京都大学大学院工学研究科都市環境工学専攻 / (主査)教授 酒井 伸一, 教授 高岡 昌輝, 准教授 平井 康宏 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13326号 / 論医博第2194号 / 新制||医||1043(附属図書館) / (主査)教授 小川 誠司, 教授 滝田 順子, 教授 河本 宏 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(社会健康医学) / 甲第22889号 / 社医博第113号 / 新制||社医||11(附属図書館) / 京都大学大学院医学研究科社会健康医学系専攻 / (主査)教授 川上 浩司, 教授 松田 文彦, 教授 中島 貴子 / 学位規則第4条第1項該当 / Doctor of Public Health / Kyoto University / DFAM