Functional Evaluation of Causal Mutations Identified in Human Genetic Studies

Human genetics has been experiencing a wave of genetic discoveries thanks to the development of several technologies, such as genome-wide association studies (GWAS), whole-exome sequencing, and whole genome sequencing. Despite the massive genetic discoveries of new variants associated with human diseases, several key challenges emerge following the genetic discovery. GWAS is known to be good at identifying the locus associated with the patient phenotype. However, the actually causal variants responsible for the phenotype are often elusive. Another challenge in human genetics is that even the causal mutations are already known, the underlying biological effect might remain largely ambiguous. Functional evaluation plays a key role to solve these key challenges in human genetics both to identify causal variants responsible for the phenotype, and to further develop the biological insights from the disease-causing mutations.

We adopted various methods to characterize the effects of variants identified in human genetic studies, including patient genetic and phenotypic data, RNA chemistry, molecular biology, virology, and multi-electrode array and primary neuronal culture systems. Chapter 1 is a broader introduction for the motivation and challenges for functional evaluation in human genetic studies, and the background of several genetics discoveries, such as hepatitis C treatment response, in which we performed functional characterization.

Chapter 2 focuses on the characterization of causal variants following the GWAS study for hepatitis C treatment response. We characterized a non-coding SNP (rs4803217) of IL28B (IFNL3) in high linkage disequilibrium (LD) with the discovery SNP identified in the GWAS. In this chapter, we used inter-disciplinary approaches to characterize rs4803217 on RNA structure, disease association, and protein translation.

Chapter 3 describes another avenue of functional characterization following GWAS focusing on the novel transcripts and proteins identified near the IL28B (IFNL3) locus. It has been recently speculated that this novel protein, which was named IFNL4, may affect the HCV treatment response and clearance. In this chapter, we used molecular biology, virology, and patient genetic and phenotypic data to further characterize and understand the biology of IFNL4. The efforts in chapter 2 and 3 provided new insights to the candidate causal variant(s) responsible for the GWAS for HCV treatment response, however, more evidence is still required to make claims for the exact causal roles of these variants for the GWAS association.

Chapter 4 aims to characterize a mutation already known to cause a disease (seizure) in a mouse model. We demonstrate the potential use of multi-electrode array (MEA) system for the functional characterization and drug testing on mutations found in neurological diseases, such as seizure. Functional characterization in neurological diseases is relatively challenging and available systematic tools are relatively limited. This chapter shows an exploratory research and example to establish a system for the broader use for functional characterization and translational opportunities for mutations found in neurological diseases.

Overall, this dissertation spans a range of challenges of functional evaluations in human genetics. It is expected that the functional characterization to understand human mutations will become more central in human genetics, because there are still many biological questions remaining to be answered after the explosion of human genetic discoveries. The recent advance in several technologies, including genome editing and pluripotent stem cells, is also expected to make new tools available for functional studies in human diseases.

Physical Insights, Steady Aerodynamic Effects, and a Design Tool for Low-Pressure Turbine Flutter

The successful, efficient, and safe turbine design requires a thorough understanding of the underlying physical phenomena. This research investigates the physical understanding and parameters highly correlated to flutter, an aeroelastic instability prevalent among low pressure turbine (LPT) blades in both aircraft engines and power turbines. The modern way of determining whether a certain cascade of LPT blades is susceptible to flutter is through time-expensive computational fluid dynamics (CFD) codes. These codes converge to solution satisfying the Eulerian conservation equations subject to the boundary conditions of a nodal domain consisting fluid and solid wall particles. Most detailed CFD codes are accompanied by cryptic turbulence models, meticulous grid constructions, and elegant boundary condition enforcements all with one goal in mind: determine the sign (and therefore stability) of the aerodynamic damping. The main question being asked by the aeroelastician, ``is it positive or negative?'' This type of thought-process eventually gives rise to a black-box effect, leaving physical understanding behind. Therefore, the first part of this research aims to understand and reveal the physics behind LPT flutter in addition to several related topics including acoustic resonance effects. A percentage of this initial numerical investigation is completed using an influence coefficient approach to study the variation the work-per-cycle contributions of neighboring cascade blades to a reference airfoil. The second part of this research introduces new discoveries regarding the relationship between steady aerodynamic loading and negative aerodynamic damping. Using validated CFD codes as computational wind tunnels, a multitude of low-pressure turbine flutter parameters, such as reduced frequency, mode shape, and interblade phase angle, will be scrutinized across various airfoil geometries and steady operating conditions to reach new design guidelines regarding the influence of steady aerodynamic loading and LPT flutter. Many pressing topics influencing LPT flutter including shocks, their nonlinearity, and three-dimensionality are also addressed along the way. The work is concluded by introducing a useful preliminary design tool that can estimate within seconds the entire aerodynamic damping versus nodal diameter curve for a given three-dimensional cascade.

Behavior Genetics and Post Genomics

The science of genetics is undergoing a paradigm shift. Recent discoveries, including the activity of retrotransposons, the extent of copy number variations, somatic and chromosomal mosaicism, and the nature of the epigenome as a regulator of DNA expressivity, are challenging a series of dogmas concerning the nature of the genome and the relationship between genotype and phenotype. DNA, once held to be the unchanging template of heredity, now appears subject to a good deal of environmental change; considered to be identical in all cells and tissues of the body, there is growing evidence that somatic mosaicism is the normal human condition; and treated as the sole biological agent of heritability, we now know that the epigenome, which regulates gene expressivity, can be inherited via the germline. These developments are particularly significant for behavior genetics for at least three reasons: First, these phenomena appear to be particularly prevalent in the human brain, and likely are involved in much of human behavior; second, they have important implications for the validity of heritability and gene association studies, the methodologies that largely define the discipline of behavior genetics; and third, they appear to play a critical role in development during the perinatal period, and in enabling phenotypic plasticity in offspring in particular. I examine one of the central claims to emerge from the use of heritability studies in the behavioral sciences, the principle of “minimal shared maternal effects,” in light of the growing awareness that the maternal perinatal environment is a critical venue for the exercise of adaptive phenotypic plasticity. This consideration has important implications for both developmental and evolutionary biology

A practical guide to photoacoustic tomography in the life sciences.

The life sciences can benefit greatly from imaging technologies that connect microscopic discoveries with macroscopic observations. One technology uniquely positioned to provide such benefits is photoacoustic tomography (PAT), a sensitive modality for imaging optical absorption contrast over a range of spatial scales at high speed. In PAT, endogenous contrast reveals a tissue's anatomical, functional, metabolic, and histologic properties, and exogenous contrast provides molecular and cellular specificity. The spatial scale of PAT covers organelles, cells, tissues, organs, and small animals. Consequently, PAT is complementary to other imaging modalities in contrast mechanism, penetration, spatial resolution, and temporal resolution. We review the fundamentals of PAT and provide practical guidelines for matching PAT systems with research needs. We also summarize the most promising biomedical applications of PAT, discuss related challenges, and envision PAT's potential to lead to further breakthroughs.

Photoacoustic tomography: principles and advances.

Photoacoustic tomography (PAT) is an emerging imaging modality that shows great potential for preclinical research and clinical practice. As a hybrid technique, PAT is based on the acoustic detection of optical absorption from either endogenous chromophores, such as oxy-hemoglobin and deoxy-hemoglobin, or exogenous contrast agents, such as organic dyes and nanoparticles. Because ultrasound scatters much less than light in tissue, PAT generates high-resolution images in both the optical ballistic and diffusive regimes. Over the past decade, the photoacoustic technique has been evolving rapidly, leading to a variety of exciting discoveries and applications. This review covers the basic principles of PAT and its different implementations. Strengths of PAT are highlighted, along with the most recent imaging results.

Commandeering Aesop’s Bamboo Canon: A 19th Century Confederacy of Creole Fugitive Fables

In my thesis, “Commandeering Aesop’s Bamboo Canon: A 19th Century Confederacy of Creole Fugitive Fables,” I ask and answer the ‘Who? What? Where? When? Why?” of Creole Literature using the 19th century production of Aesopian fables as clues to resolve a set of linguistic, historical, literary, and geographical enigmas pertaining the ‘birth-place(s)’ of Creolophone Literatures in the Caribbean Sea, North and South America, as well as the Indian Ocean. Focusing on the fables in Martinique (1846), Reunion Island (1826), and Mauritius (1822), my thesis should read be as an attempt capture the links between these islands through the creation of a particular archive defined as a cartulary-chronicle, a diplomatic codex, or simply a map in which I chart and trace the flight of the founding documents relating to the lives of the individual authors, editors, and printers in order to illustrate the articulation of a formal and informal confederation that enabled the global and local institutional promotion of Creole Literature. While I integrate various genres and multi-polar networks between the authors of this 19th century canon comprised of sacred and secular texts such as proclamations, catechisms, and proverbs, the principle literary genre charted in my thesis are collections of fables inspired by French 17th century French Classical fabulist, Jean de la Fontaine. Often described as the ‘matrix’ of Creolophone Literature, these blues and fables constitute the base of the canon, and are usually described as either ‘translated,’ ‘adapted,’ and even ‘cross-dressed’ into Creole in all of the French Creolophone spaces. My documentation of their transnational sprouting offers proof of an opaque canonical formation of Creole popular literature. By constituting this archive, I emphasize the fact that despite 200 years of critical reception and major developments and discoveries on behalf of Creole language pedagogues, literary scholars, linguists, historians, librarians, archivist, and museum curators, up until now not only have none have curated this literature as a formal canon. I also offer new empirical evidence in order to try and solve the enigma of “How?” the fables materially circulated between the islands, and seek to come to terms with the anonymous nature of the texts, some of which were published under pseudonyms. I argue that part of the confusion on the part of scholars has been the result of being willfully taken by surprise or defrauded by the authors, or ‘bamboozled’ as I put it. The major paradigmatic shift in my thesis is that while I acknowledge La Fontaine as the base of this literary canon, I ultimately bypass him to trace the ancient literary genealogy of fables to the infamous Aesop the Phrygian, whose biography – the first of a slave in the history of the world – and subsequent use of fables reflects a ‘hidden transcript’ of ‘masked political critique’ between ‘master and slave classes’ in the 4th Century B.C.E. Greece.

This archive draws on, connects and critiques the methodologies of several disciplinary fields. I use post-colonial literary studies to map the literary genealogies Aesop; use a comparative historical approach to the abolitions of slavery in both the 19th century Caribbean and the Indian Ocean; and chart the early appearance of folk music in early colonial societies through Musicology and Performance Studies. Through the use of Sociolinguistics and theories of language revival, ecology, and change, I develop an approach of ‘reflexive Creolistics’ that I ultimately hope will offer new educational opportunities to Creole speakers. While it is my desire that this archive serves linguists, book collectors, and historians for further scientific inquiry into the innate international nature of Creole language, I also hope that this innovative material defense and illustration of Creole Literature will transform the consciousness of Creolophones (native and non-native) who too remain ‘bamboozled’ by the archive. My goal is to erase the ‘unthinkability’ of the existence of this ancient maritime creole literary canon from the collective cultural imaginary of readers around the globe.

Opening Basement Membrane Gaps During C. elegans Uterine-Vulval Attachment

The basement membrane (BM) is a highly conserved form of extracellular matrix that underlies or surrounds and supports most animal tissues. BMs are crossed by cells during various remodeling events in development, immune surveillance, or during cancer metastasis. Because BMs are dense and not easily penetrable, most of these cells must open a gap in order to facilitate their migration. The mechanisms by which cells execute these changes are poorly understood. A developmental event that requires the opening of a BM gap is C. elegans uterine-vulval connection. The anchor cell (AC), a specialized uterine cell, creates a de novo BM gap. Subsequent widening of the BM gap involves the underlying vulval precursor cells (VPCs) and the π cells, uterine neighbors of the AC through non-proteolytic BM sliding. Using forward and reverse genetic screening, transcriptome profiling, and live-cell imaging, I investigated how the cells in these tissues accomplish BM gap formation. In Chapter 2, I identify two potentially novel regulators of BM breaching, isolated through a large-scale forward genetic screen and characterize the invasion defect in these mutants. In Chapter 3, I describe single-cell transcriptome sequencing of the invasive AC. In Chapter 4, I describe the role of the π cells in opening the nascent BM gap. A complete developmental pathway for this process has been elucidated: the AC induces the π fate through Notch signaling, after which the π cells upregulate the Sec14 family protein CTG-1, which in turn restricts the trafficking of DGN-1 (dystroglycan), a laminin receptor, allowing the BM to slide. Chapter 5 outlines the implications of these discoveries.