CRISPR-based functional genomics to study gene regulatory architecture and consequences of genetic variation

Thesis (Ph.D.)--University of Washington, 2023If we divide the human genome into its noncoding and coding parts, the noncoding portion takes up ~98-99% of the genome, and the coding portion takes up ~1-2% (ENCODE Project Consortium, 2012). The coding portion of the genome contains the genetic template for RNA transcription and protein translation, and translated proteins are the functional units of the cell that carry out biological function. Despite coding genes acting as templates for RNA transcription and protein translation, the noncoding genome contains the vast majority of sequences that regulate the expression of genes (some regulatory sequences reside in coding regions). The classes of sequences that make up regulatory sequences include promoters, enhancers, silencers, insulators, and repressors, amongst others. Proper regulation of genes at the right time and in the right cell types lies at the core of proper cellular function, and consequently, healthy cells, tissues, and organisms. Variants such as single base substitutions, deletions, and insertions, can occur in both the coding and noncoding portions of the genome. These variants are most often benign. However, some mutations are loss-of-function mutations, meaning that they interfere with and sometimes inhibit proper biological function. Understanding the functional consequences of these mutations is critical to understanding the genetic causes of diseases as well as to develop therapeutics to treat diseases that have a genetic cause. To study both coding and noncoding regions of the genome, novel methods and technologies are required. In particular, the development of methods that can perturb and assess genetic sequences at scale are needed to tackle the vast number of genes and regulatory sequences that the genome consists of, which is on the scale of tens to hundreds of thousands (ENCODE Project Consortium, 2012). In the first chapter, I introduce the field of DNA sequencing, genomics, and genomic technology development. I also discuss the various CRISPR/Cas9-based perturbation methods and how these methods can be utilized to develop novel screening methods, two of which I developed during my PhD. In the second chapter, I describe multiplex, single-cell CRISPR activation (CRISPRa) screening, a method I developed during my PhD and utilized to identify both proximal and distal cell type-specific regulatory elements that are capable of increasing target gene expression when activated via a specific CRISPRa perturbation. In the third chapter, I introduce a CRISPR prime editing based method that allows for the identification of drug resistance mutations in a multiplex and scaled manner. In the fourth and final chapter, I describe how I envision this work advancing further in order to gain a more comprehensive understanding of gene regulatory architecture and the functional consequences of genetic variation in all noncoding and coding sequences. I conclude by discussing how we can use this understanding to design and develop novel and effective therapies for the wide range of diseases that have genetic causes

Flow of ultra-hot orogens: A view from the Precambrian, clues for the Phanerozoic

International audienceThis contribution emphasizes first-order structural and metamorphic characters of Precambrian accretionary orogens to understand the kinematics and thermomechanical state of the continental lithosphere in convergent settings involving massive juvenile magmatism. We define a new class of orogens, called ultra-hot orogens (UHO), in which the weakest type of lithosphere on Earth is deformed. UHO are characterized by (1) distributed shortening and orogen-scale flow combining vertical and horizontal longitudinal advection, under long-lasting convergence, (2) homogeneous thickening by combined downward movements of supracrustal units and three-dimensional mass redistribution in the viscous lower crust, and (3) steady-state, negligible topography and relief leveled by syn-shortening erosion and near-field sedimentation. The flow analysis of UHO provides clues to understanding crustal kinematics beneath high plateaus and suggests that the seismic reflectivity pattern of hot orogens is an image of the layering produced by lateral flow of the lower crust and associated syn-kinematic plutonism. In between the UHO and the modern cold orogens (CO), developed by shortening of lithosphere bearing a stiff upper mantle, two classes of orogens are defined. Hot orogens (HO, representative of Cordilleran and wide mature collisional belts) share flow pattern characteristics with UHO, but involve a less intense magmatic activity and develop high topographies driving their collapse. Mixed-hot orogens (MHO, representative of magmatic arcs and Proterozoic collisional belts) are orogens made of UHO-type juvenile crust and display CO-like structure and kinematics. This classification points to the fundamental link between the presence of a stiff lithospheric mantle and strain localization along major thrusts in convergent settings. A high Moho temperature (> 900 °C), implying thinning of the lithospheric mantle, enhances three-dimensional flow of the lithosphere in response to convergence. Overall, this classification of orogens emphasizes the space and time variability of uppermost mantle temperature in controlling plate interactions and continental growth

Estimations de l’exposition foetale aux pyréthrinoïdes à partir de biomarqueurs urinaires maternels grâce à la modélisation p-PBPK (toxicocinétique basée sur la physiologie de la femme enceinte)

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Pregnancy specific biochemical and physiological processes integration in p-PBPK (pregnancy-Physiologically Based PharmacoKinetics) models

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An Integrated and Spatialized Approach to Identify Potentially Overexposed Population : A Case Study of Chlorpyrifos Contamination

Exposure to pesticides is characterized by the multiplicity of exposure routes (food, water, soil, air) related to their presence in all environmental media. First, for a fine characterization of the environmental exposure, it is necessary to gather within the same analysis system a dataset integrating the population behavior and the local contamination of the environmental media at fine resolutions and on large territories. This integration requires a sufficient toolchain to produce or process these data in a rigorous manner, characterize the entire continuum from the source of contamination, transport, and transfer of contaminants in environmental media, and estimate external and internal exposure doses for the general population. The aim of this work is to establish an integrated methodology for the mapping of exposure indicators at fine spatial and temporal resolutions (1 km² and week) directly comparable with individual biomonitoring measurements from French cohorts. To illustrate the approach, we consider the levels of chlorpyrifos among pregnant women in the Picardy region (France). Results provide insights into the identification of areas of potential overexposure and analysis of environmental determinants, elements useful for public health decision to reduce exposure and thus health and environmental impacts. However, the exposure characterization involves uncertainties regarding modeling assumptions and data measurements. Moreover, the efficiency of exposure modeling can be limited by calculation times. To optimize numerical resolution and reduce uncertainties related to knowledge limits and data representativeness, modeling assumptions and tools need to be reconsidered to improve the methodology and allow future developments

Phénomènes et unités : questions autour de la détection automatique des répétitions dans un corpus de langue parlée en interaction

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Les hétéro-répétitions dans une interaction orale : définition et conception d’un outil de détection automatique

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Other-repetition: displaying others' lexical choices as "commentable"

International audienceLexical other-repetition is a process that consists of repeating words that have been previously produced by another interactant. This leads to a lexical similarity of the participants' discourse. According to Tannen (2007), participants use lexical other-repetition to show their involvement in the interaction. She argues that repetition is useful at several levels: production (repetition facilitates encoding), understanding (repetition facilitates decoding), connection (it maintains cohesion in discourse), and interaction (repetition maintains the link between participants). We focus here on a subcategory of lexical other-repetitions: the phenomenon that we call "pinning" is a form of repair (Schegloff, 2007: 100-101), in so far as one element that has been uttered by one of the participants is afterward treated as a “trouble source”. It develops like a repair, and specifically like an other-repair: A turn that contains an element that will be taken as the source, "the repairable" B picks up this element in A's turn, repeats it and comments on it A/B various ways of responding to B's repair Two features characterize "repair": on the one hand, the fact that it consists in a process, and on the other hand that it is not inherently related to a real, an actual or an obvious problem. In the following definition, Schegloff insists on this last aspect: “Not only are "obvious" problems unaddressed; anything in the talk may be treated as in need of repair. Everything is, in that sense, a possible repairable or a possible trouble-source. It is overt efforts to deal with trouble-sources or repairables – marked off as distinct within the ongoing talk – that we are terming repair” (Schegloff, 2007: 100-101) For the cases we are dealing with, the repeat in the second position is neither uttered in order to clarify what has been said, nor in order to ask for an explanation, and even if it does, it is above all oriented towards underlining a more or less strange, unexpected or surprising feature of A's turn. Example 1: CLAPI. Corpus Grillage (Bert et al., 2010) A euh:::: un truc euh: bon y a personnage/ enfin tu vois y a trois: (0.6) trois trois symboles (.) et:: tu vois: (0.8) et puis sinon/ ben y a l` réglage des a- des asas euh: P des a[sas/ ((rire)) L [ouais A des asas et puis voilà In this case, P understands the element pronounced by A. Nevertheless he repeats it, with the same form as in a repair, but orienting it toward mockery, with a “savoring” function (Tannen, 2007). “Des asas” is extracted from the current discourse because it is “worthy” to be commented on. We could say that the repeated element is treated as a "commentable" (cf. "repairable") element and insofar initiates a specific form of repair. We propose to investigate whether the formal alignment (lexical similarity) due to repetition, and possibly leading to a humorous sequence, reveals convergence in interaction. During a previous collaboration in the SPIM project (Imitation in speech: From sensori-motor integration to the dynamics of conversational interaction), we gathered a large collection of examples of other-repetition, from a set of corpora of semi-spontaneous and spontaneous interactions, with various contexts and activities (conversation, storytelling) (Bertrand et al., 2008; Bert et al., 2010). Various aspects of "pinning" have been studied: - the process of extracting an element from the previous turn - the devices used to show that the repair is oriented towards mockery or humor, including the link between the first occurrence and the repeat, on the syntactic, phonetic, and interactional level - the different types of following turns (i.e., turns responding to the repeat) - the consequences for the degree of convergence within the sequence. In the cases we work on, a participant produces the first occurrence of a word or expression in the speech stream, and the other participant notes its incongruity by repeating it, eventually in a humorous perspective. The classical humorous schema consists of the presence of a connector (Greimas, 1966) and a disjunctor (Morin, 1966). In a “pinning”, A produces the connector, which allows two different interpretations (a logical one and an unexpected one), and B repeats it, adding a disjunctor, that actualizes the absurd interpretation. The gap between what A said, and what B interprets causes the incongruity, thereby creating humor. Example 2: Corpus of Interactional Data (Bertrand et al., 2008) LJ j'ai senti qu(e) ça s'adou-#cissait et bon après on a eu des rapports normaux AP mh mh LJ bon euh LJ euh mais au début putain j'é- j'étais mal quoi je euh AP mh mh AP normaux c'(es)t-à-dire euh hum LJ ((rires)) AP avec préservatif ou sans euh LJ oh putain ((rires)) ça y est t'es dedans là ((rires)) In example 2, LJ describes professional relations, and AP produces back-channel signals. Then AP repeats one word “normaux” (normal), treating it as ambiguous, which appears as a repair sequence. “Rapports” constitutes the connector, since it may be interpreted in different senses, and the repetition is already an attempt of humor. This makes LJ laugh. AP then explicitly actualizes a humorous sexual meaning, for which “préservatif” (condom) constitutes the disjunctor. In this case, there is no modification of the repeated element. In other cases from the corpora, we observe a slight phonetic modification, a syntactic restructuring, or a specific prosodic device, such as a prosodic matching (a type of prosodic orientation defined by Szcezpeck-Reed, 2006), that we will precisely analyze, in order to understand the cues given by the “repeater” to show the humorous or mockery dimension of the repetition (Bertrand & Priego-Valverde, 2011) If we consider our data, the process of “pinning” with a humorous purpose leads to three possible situations: 1/ Basic ratification: the humorous repetition receives indeed a basic ratification from A (laughter, “yes”…) but A quickly goes back to his narration. This humorous intervention of B constitutes a short digression from the current narration. 2/ Failure or non-continuation of the humorous mode of communication: the author of the first occurrence (the serious one) refuses to switch into a humorous theme, and continues his activity, whereas B develops, alone, a humorous continuation. The interactants develop parallel sequences, a humorous one and a serious one. They diverge on the kind of their activity at this point of the interaction, until B finally concedes and switches back to the serious modality. 3/ Joint fantasizing (Kotthoff, 2006): contrary to the previous situation, the humorous repetition is used as a starting point for a co-elaborated humorous sequence. In this case, A produces the first occurrence, and B repeats it (“pinning”), eventually with slight phonetic modifications, in order to switch to humor. Each participant overbids, and they co-elaborate a humoristic sequence. We consider these sequences as highly convergent moments in the interaction. Therefore, the same pattern: can be the starting point of a highly convergent sequence, or conversely, result in an interactional divergence. This is an evidence of the lack of equivalence between formal similarity (lexical repetition) on the one hand, and interactional convergence on the other

Caractérisation de l’exposition environnementale spatialisée à un pyréthrinoïde en Picardie

Pesticides are present in all areas of the environment and absorbed via many pathways (food, water, soil, and air). To characterize environmental exposures in detail, it is first necessary to combine within the same analysis data on the population’s lifestyles and on the local contamination of environmental media at appropriate resolutions and over large areas. As part of this pilot study,the objective of the CartoExpo project was to test the feasibility of an integrated methodology to map exposure indicators for small-area resolutions and short time intervals. To illustrate the approach, contamination of the general population was studied for a pyrethroid (cypermethrin) in the Picardy region (Northern France). For atmospheric dispersion, an innovative statistical meta-model method was developed, based on a machine learning technique that used a large database of simulations on a representative parcel. The meta-model was then applied to cypermethrin application at three-hourly intervals on all agricultural parcels in the Picardy region. Multimedia exposure models are needed to: 1) estimate emissions from soil and plant volatilization phenomena on agricultural land, 2) quantify contamination of local food products (excluding home vegetable gardens) due to proximity to agricultural land,a nd3)combine external exposures. Characterizing exposure involves numerous uncertainties, primarily about the assumptions underlying the different modeling approaches, and the representativeness and accuracy of the integrated data. Further measured and modeled data are therefore needed to validate the different approaches.Les expositions aux pesticides se caractérisent par la multiplicité des voies d’exposition (alimentation, eau, sol, air) liée à leur présence dans l’ensemble des milieux environnementaux. Pour une caractérisation fine des expositions environnementales, un premier verrou réside dans la capacité à rassembler au sein d’un même système d’analyse un ensemble de données combinant le mode de vie des populations et la contamination locale des milieux environnementaux sur des résolutions spatiales appropriées et des territoires étendus (échelles régionales ou nationales). Dans le cadre de cette étude pilote, l’objectif du projet CartoExpo était de tester la faisabilité d’une méthodologie intégrée pour la cartographie d’indicateurs d’exposition sur des résolutions spatiales et temporelles fines. Pour illustrer la démarche, la contamination de la population générale est étudiée pour un pyréthrinoïde (la cyperméthrine) sur la Picardie en considérant les voies d’ingestion (eau, sol, aliments) et d’inhalation (gaz et aérosol). Pour la dispersion atmosphérique, une méthode innovante de métamodèle statistique a été développée. Elle repose sur une technique d’apprentissage automatique à partir d’une vaste base de données de simulations sur une parcelle élémentaire. Le métamodèle est dans un deuxième temps appliqué aux épandages relevés à une fréquence tri-horaire sur l’ensemble des parcelles agricoles en région Picardie. Des modèles d’exposition multimédia sont nécessaires pour 1) estimer les flux d’émission liés aux phénomènes de volatilisation du sol et des plantes au niveau des parcelles agricoles, 2) quantifier la contamination des produits alimentaires locaux (hors champ : jardin potager) liée à la proximité des parcelles agricoles et 3) combiner l’exposition externe. L’exercice de caractérisation de l’exposition comporte cependant de nombreux manques et incertitudes dont l’impact, dans un sens ou dans l’autre, est à ce stade difficile à quantifier. Le besoin de valider les approches mises en œuvre nécessite de mobiliser des données (mesurées ou modélisées) supplémentaires

Impact of oocytes with CLCG on ICSI outcomes and their potential relation to pesticide exposure

Abstract Background Oocyte quality is a key limiting factor in female fertility which is primarily reflected in morphological features. Centrally located cytoplasm granulation (CLCG) is one type of cytoplasmic dimorphism exhibited by oocytes that could be linked to pesticide exposure with a significant risk of decreased ICSI outcomes. Methods This retrospective study included 633 women who were part of an intracytoplasmic spermatozoa injection (ICSI) program between 2009 and 2011. The participants lived in the Picardy region of France and had been exposed to pesticides. The participants were divided in two groups based on prevalence of oocytes with CLCG (LCLCG [n = 83]: low prevalence of oocytes with CLCG under 25%. HCLCG [n = 68]: high prevalence of CLCG over 75%). The embryological and clinical outcomes were analysed for both groups and were calculated using the difference between the two values. Results Results for couples with HCLCG compared to LCLCG showed a decrease in embryo cleavage, ongoing pregnancy, and live birth rates (82%, 14%, 13% vs 99%, 32%, 30%, respectively).The early miscarriage rate was increased (47% vs 11%), with an OR of 3.1 (95%CI [2.1–4.1]). Due to high pesticide exposure (over 3000 g/ha), there is a higher risk of a resulting disturbed oocyte cohort with a high prevalence of CLCG over 75%. Conclusion The high prevalence of oocytes with CLCG over 75% has a negative effect on embryos and the general ICSI clinical outcomes. Furthermore, a putative association between pesticide exposure and risk of CLCG was identified, justifying the need for further research and a potential need to find alternative assisted reproductive technologies for these couples. Trial registration Tabacfertimasc. ID number: ID2011-A00634–37 ; registered 2011/2/