Systems Analytics and Integration of Big Omics Data

A “genotype"" is essentially an organism's full hereditary information which is obtained from its parents. A ""phenotype"" is an organism's actual observed physical and behavioral properties. These may include traits such as morphology, size, height, eye color, metabolism, etc. One of the pressing challenges in computational and systems biology is genotype-to-phenotype prediction. This is challenging given the amount of data generated by modern Omics technologies. This “Big Data” is so large and complex that traditional data processing applications are not up to the task. Challenges arise in collection, analysis, mining, sharing, transfer, visualization, archiving, and integration of these data. In this Special Issue, there is a focus on the systems-level analysis of Omics data, recent developments in gene ontology annotation, and advances in biological pathways and network biology. The integration of Omics data with clinical and biomedical data using machine learning is explored. This Special Issue covers new methodologies in the context of gene–environment interactions, tissue-specific gene expression, and how external factors or host genetics impact the microbiome

DEVELOPING THERAPEUTIC BIOIMPLANTS FOR HEMOPHILIA A: BIOSAFETY EVALUATION OF TARGETED TRANSGENE INTEGRATION IN PRIMARY HUMAN UMBILICAL CORD-LINING CELLS USING PHIC31 INTEGRASE AND ZINC FINGER NUCLEASES

Ph.DDOCTOR OF PHILOSOPH

Translational software infrastructure for medical genetics

Diep in de kern van onze cellen zetelt het desoxyribonucleïnezuur (DNA) molecuul die bekend staat als het genoom.DNA codeert de informatie die het leven laat groeien, overleven, diversifiëren en evolueren.Helaas kunnen dezelfde mechanismes die ons laten aanpassen aan een veranderende omgeving ook genetische aandoeningen veroorzaken.Hoewel we in staat zijn een aantal van deze aandoeningen op te sporen door moderne technologische vorderingen, moet er nog veel ontdekt en begrepen worden.Dit proefschrift draagt software infrastructuur aan om de moleculaire oorzaak van genetische aandoeningen te onderzoeken, laat zien hoe nieuwe bevindingen vertaald worden van fundamenteel onderzoek naar nieuwe software voor genoom diagnostiek, en introduceert een raamwerk voor genetische analyses die de automatisering en validatie van nieuwe software ondersteunt voor toepassing in de patientenzorg.Eerst ontwikkelen we datamodellen en software die helpt te bepalen welke gebieden op het genoom verantwoordelijk zijn voor ziektes en andere fysieke kenmerken.Vervolgens trekken we deze principes door naar modelorganismen.Door moleculaire gelijkenissen te gebruiken, ontdekken we nieuwe manieren om nematodes in te zetten voor onderzoek naar menselijke ziektes.Daarnaast kunnen we onze kennis van het genoom en de evolutie gebruiken om te voorspellen hoe pathogeen nieuwe mutaties zijn.Het resultaat is een publieke website waar DNA snel en accuraat gescand kan worden op mogelijk ziekteverwekkende mutaties.Tenslotte presenteren we een compleet systeem voor geautomatiseerde DNA analyse, inclusief een protocol specifiek voor genoom diagnostiek om overzichtelijke patient rapportages te produceren voor medisch experts waarmee een diagnose sneller en makkelijker gesteld kan worden.Deep inside the core of our cells resides the deoxyribonucleic acid (DNA) molecule known as the genome.DNA encodes the information that allows life to grow, survive, diversify and evolve.Unfortunately, the same mechanisms that let us adapt to a changing environment can also cause genetic disorders.While we are able to diagnose a number of these disorders using modern technological advancements, much remains to be discovered and understood.This thesis presents software infrastructure for investigating the molecular etiology of genetic disease using data from model organisms, demonstrates how to translate findings from fundamental research into new software tools for genome diagnostics, and introduces a downstream genome analysis framework that assists the automation and validation of the latest tools for applied patient care.We first develop data models and software to help determine which region of the genome is responsible for diseases and other physical traits.We then extend these principles towards model organisms.By using molecular similarities, we discover new ways to use nematodes for research into human diseases.Additionally, we can use our knowledge of the genome and evolution to predict how pathogenic new mutations are.The result is a public website where DNA can be scanned quickly and accurately for probable pathogenic mutations.Finally, we present a complete system for automated DNA analysis, including a protocol specific for genome diagnostics to produce clear patient reports for medical experts with which a diagnosis is made faster and easier

Molecular genetic studies of inherited cystic kidney disease in Oman

Ph. D. ThesisInherited kidney diseases are fundamental causes of chronic kidney disease (CKD) and end stage kidney disease (ESKD); accounting for approximately 20% of all CKD cases and up to 10% of adults and over 70% of children reaching ESKD. Oman is the second largest country in the South East of Arabian Peninsula. Omani population is characterized by large family size, presence of tribal and geographical settlements and higher rates of consanguineous marriages, which facilitate the study of autosomal recessive disorders. Rare genetic disorders create considerable burden on healthcare system in Oman and are major causes of congenital abnormalities and perinatal deaths in hospitals. The prevalence of inherited kidney disease was estimated to be high, but there is a lack for a comprehensive data. Therefore, this study aimed to evaluate the magnitude of inherited kidney disease in this population and identify the molecular genetic causes of inherited cystic kidney diseases in Omani patients. First, I performed a population-based retrospective analysis of ESKD patients commencing RRT from 2001 to 2015 using the national renal replacement therapy (RRT) registry and evaluated the epidemiological and etiological causes of ESKD with focused attention on inherited kidney diseases. Second, I designed a targeted gene panel (49 genes) and used massive parallel sequencing technologies for the molecular genetic diagnosis of cystic kidney disease in 53 patients. An overall molecular genetic diagnostic yield of 75% was achieved; with 46% of detected causative variants were novel genetic findings. Third, I evaluated the utility of molecular genetic testing in patients with autosomal recessive polycystic kidney disease (ARPKD) and described the clinical and genetic profile of this cohort. Finally, whole exome sequencing (WES) was used to determine the genetic causes of CKD in 11 unrelated children suspected with recessively inherited kidney diseases. Definite genetic diagnosis was achieved in 54.5% of cases, reflecting the importance of genomic implications in those with uncertain aetiology causing CKD. This study creates a solid basis reflecting the genotype-phenotype of some inherited kidney diseases in Omani population and reveals the enormous diagnostic power of genomic technologies.The Research Council (TRC), Ministry of Health (MOH), Ministry of Higher Education (MOHE

Genetics of Hearing Impairment

The inner ear is a complex machinery at the cellular and molecular levels. Many different genes and proteins play roles in the development and maintenance of its structure and function, through participating in diverse molecular networks. A defect in any of these components can result in the loss of hearing. Consequently, hearing impairment encompasses a wide variety of disorders that are clinically and genetically heterogeneous. Understanding their genetic causes and their pathophysiological mechanisms, and characterizing the resulting phenotypes, are essential for developing novel therapies that target the specific defects. The articles and reviews in this book are representative of the many research lines that are currently active in the field, including recent advances in the genes and mutations involved in hearing impairment, the mechanisms through which mutations result in different syndromic or non-syndromic disorders, and the description of the associated phenotypes in humans and in animal models

A hybrid lentivirus-transposon vector for safer gene therapy

Gene therapy vectors based on the HIV-1 lentivirus are an attractive option for clinical applications because they enter a broad range of target cells efficiently and deliver stable gene expression through integration into host chromosomes. However, lentiviral vectors are known to integrate preferentially within actively transcribing genes. Leukaemia-like expansions observed in gene therapy trials using gammaretroviral vectors and are thought to have been caused by disruption of host proto-oncogenes at or close to the vector integration site, and the safety of these vectors may be related to the pattern of vector integration with respect to genes. The safety of integrating gammaretroviral and lentiviral vectors is therefore a significant concern with respect to their use in gene therapy. In this study, this problem was addressed by developing a novel hybrid vector which combines the efficient cell and nuclear entry properties of lentiviral vectors with chromosomal integration by the Sleeping Beauty transposase. Unlike the HIV-1 integrase enyme, Sleeping Beauty transposase does not exhibit a preference for integration within active genes. Nonintegrating lentiviral vectors were developed to carry Sleeping Beauty transposon and transposase expression cassettes. These were able to deliver transient transposase expression to target cells, and episomal lentiviral DNA was found to be a suitable substrate for integration by Sleeping Beauty transposase. Importantly, integration with this novel vector was found to occur significantly less frequently within active genes than a standard lentiviral vector. Finally, it was shown that the transposase protein can be incorporated into lentiviral vector particles in a manner analogous to HIV-1 integrase. The development of vectors with safer integration patterns may lead to better clinical outcomes for patients treated with gene therapy

PRELIMINARY FINDINGS OF A POTENZIATED PIEZOSURGERGICAL DEVICE AT THE RABBIT SKULL

The number of available ultrasonic osteotomes has remarkably increased. In vitro and in vivo studies have revealed differences between conventional osteotomes, such as rotating or sawing devices, and ultrasound-supported osteotomes (Piezosurgery®) regarding the micromorphology and roughness values of osteotomized bone surfaces. Objective: the present study compares the micro-morphologies and roughness values of osteotomized bone surfaces after the application of rotating and sawing devices, Piezosurgery Medical® and Piezosurgery Medical New Generation Powerful Handpiece. Methods: Fresh, standard-sized bony samples were taken from a rabbit skull using the following osteotomes: rotating and sawing devices, Piezosurgery Medical® and a Piezosurgery Medical New Generation Powerful Handpiece. The required duration of time for each osteotomy was recorded. Micromorphologies and roughness values to characterize the bone surfaces following the different osteotomy methods were described. The prepared surfaces were examined via light microscopy, environmental surface electron microscopy (ESEM), transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM) and atomic force microscopy. The selective cutting of mineralized tissues while preserving adjacent soft tissue (dura mater and nervous tissue) was studied. Bone necrosis of the osteotomy sites and the vitality of the osteocytes near the sectional plane were investigated, as well as the proportion of apoptosis or cell degeneration. Results and Conclusions: The potential positive effects on bone healing and reossification associated with different devices were evaluated and the comparative analysis among the different devices used was performed, in order to determine the best osteotomes to be employed during cranio-facial surgery

CD28 and TCR In-Situ Biophysical Analyses Inform T Cell Immunity Mechanisms

This work investigates two receptors on T lymphocytes that shape immunity, the T cell receptor (TCR) and cluster of differentiation 28 (CD28). T cells coordinate adaptive immunity, but how signaling via TCR and CD28 interactions with peptide-major compatibility complex (pMHC) and B7 family ligands on antigen presenting cells govern T cell function and differentiation remains poorly understood. In-situ biophysical measurements on live T cell surfaces suggest both B7 family ligands form monomeric bonds with CD28. This work demonstrated CD28 catch bonds with B7 family ligands. Catch bonds refer to a counter-intuitive phenomenon where force prolongs bond lifetime contrasted with the more intuitive slip bond where force shortens bond lifetime. Although TCR–pMHC catch bonds on splenic T cells characterize a well-established TCR mechanosensing mechanism, the same interaction on hepatic T cells showed slip bonds correlating with a more activated state among liver T cells. We also analyzed both short- and long-term memory effects from the same molecular interactions. Short-term (within seconds) memory analyses found that bond formation increased bond formation likelihood but not dissociation in the immediate future. Long-term (~5 minutes) memory analyses found that splenic T cells became more activated by repeated ligand engagement and receptor tension resulting in TCR–pMHC catch bond elimination. Our sensitive assay also revealed subtle T cell activation by piconewton-level T cell pushing and pulling forces as well as changes in short-term memory. This work suggests biophysical instrumentation employed in-situ can reveal information about dynamic processes mediating important immunological functions. The findings within this work provide insights into mechanistically how co-stimulation works at a single molecule level as well as how signaling overlap between TCR and CD28 influence receptor localization, mechanosensing, and triggering. These insights answer longstanding mechanistic questions about how T cells function and provide foundations for future investigations.Ph.D