8,012 research outputs found

    Complementarities in human capital production:The Importance of Gene-Environment Interactions

    Get PDF

    Contribution of Relatedness and Genetic Factors to the Clinical Picture of Coeliac Disease

    Get PDF
    Keliakia on yleinen autoimmuunisairaus, jossa tärkein altistava ympäristötekijä on ravinnon sisältämä gluteeni. Keliakiaa esiintyy maailmanlaajuisesti, mutta sen esiintyvyys Suomessa on erityisen korkea, vaikkakin laajalti alidiagnosoitu. Osasyy tälle on keliakian kirjava taudinkuva. Ruuansulatuskanavan oireet muodostivat pitkään keliakialle tyypillisen oirekuvan, mutta se on vähitellen korvautunut lievemmillä, usein ruuansulatuskanavan ulkopuolisilla oireilla. Nykyään keliakia voi ilmentyä myös täysin oireettomana. Oireiden lisäksi niiden puhkeamisessa, voimakkuudessa sekä kehittymisessä on paljon yksilöllisiä eroja. Myös keliakiapotilaiden vaste gluteenittomaan ruokavaliohoitoon vaihtelee. Syitä tai tekijöitä tämän potilaskohtaisesti vaihtelevan taudinkuvan taustalla ei kuitenkaan vielä tunneta. Nykyisin moni keliaakikko on osannut aiemmin epäillä kohdallaan keliakiaa ja hakeutunut lääkäriin, koska sairaus on ollut tuttu keliakiaa sairastavien sukulaisten tai perheenjäsenten kautta. Nämä ns. familiaaliset keliaakikot ovat enemmistö, mutta keliakiaa esiintyy myös ns. sporadisesti, jolloin potilaalla ei ole tiedettyä sukutaustaa keliakialle. Tunnetuin keliakialle altistava perintötekijä on HLA-DQ2.5-haplotyyppi, mutta muidenkin, HLA-alueen ulkopuolisten, geenialueiden on todettu assosioituvan keliakiaan. Tässä väitöskirjatutkimuksessa selvitettiin familiaalisen ja sporadisen keliakian eroavaisuuksia perintötekijöiden ja taudinkuvan suhteen. Lisäksi tutkittiin, onko HLA-DQ2.5-kuormalla (eli sillä, onko potilas HLA-DQ2.5-negatiivinen, - heterotsygootti vai -homotsygootti) ja HLA:n ulkopuolisilla geneettisillä varianteilla, mukaan lukien variantit kandidaattigeeneissä CCR9 ja CCL25, vaikutusta keliakian kliiniseen taudinkuvaan. Tämän väitöskirjan tulokset paljastivat, että sporadinen keliakia omana erillisenä tautimuotonaan eroaa HLA-DQ-genotyypiltään familiaalisesta tautimuodosta, vaikka useita eroavaisuuksia kliinisen taudinkuvan suhteen ei löydettykään. Sporadisten potilaiden taudinkuva diagnoosihetkellä oli kuitenkin vakavampi ja yleinen terveydentila seurantahetkellä, gluteenittomalla dieetillä, huonompi kuin familiaalisilla potilailla. Tutkimukset myös osoittivat, että HLA-DQ2.5-negatiivisia potilaita leimasi HLA-DQ2.5-heterotsygootteja ja -homotsygootteja useammin klassinen oirekuva diagnoosihetkellä sekä pitkittyneet oireet seurantahetkellä. Nämä tutkimuslöydökset toivottavasti rohkaisevat lääkäreitä sekä muuta hoitohenkilökuntaa kiinnittämään erityistä huomiota edellä mainittuihin potilasryhmiin. Lisäksi väitöstutkimuksen geneettisten analyysien tulokset osoittivat neljän HLA-alueen ulkopuolisen variantin assosioituvan familiaaliseen keliakiaan. Näiden assosiaatioiden merkitys tulee kuitenkin vahvistaa jatkotutkimuksilla. HLA- DQ2.5-kuorman tai CCR9- ja CCL25-kandidaattigeenialueiden varianttien vaikutus keliakian taudinkuvaan oli tämän väitöskirjatutkimuksen perusteella vähäinen.Coeliac disease, a systemic autoimmune disorder induced by dietary gluten, is widespread globally, but in Finland even particularly prevalent although still heavily underdiagnosed. One reason contributing to this is the remarkably multifaceted clinical picture of coeliac disease. The originally typical gastrointestinal symptoms are currently being increasingly replaced by a milder presentation with extraintestinal symptoms and even totally asymptomatic presentation is no longer abnormal. Nevertheless, coeliac disease patients do not differ from each other only in terms of symptoms. There is a wide individual variation concerning the onset, severity and progression of symptoms as well as response to dietary treatment i.e., gluten-free diet (GFD). The reasons and contributary factors underlying this wide clinical heterogeneity remain obscure. For many people nowadays, suspicion of coeliac disease is closely connected to known familial history of the disease. These familial cases are in the majority, but not every patient has affected relatives and such patients are considered sporadic. Where coeliac disease heredity is concerned, HLA-DQ2.5 is the best-known genetic component. However, numerous loci outside the HLA region have also been associated with the disease. The studies presented in this dissertation focused in investigating whether there are genetic and/or phenotypic differences between familial and sporadic coeliac disease and whether the dose of HLA-DQ2.5 or the presence of genetic variants outside HLA region, including the ones within candidate genes CCR9 and CCL25, contributes to the clinical picture of the disease. The findings of this dissertation managed to describe sporadic coeliac disease as an independent entity with a distinct HLA-DQ genotype even though not many significant phenotypic differences were observed between familial and sporadic coeliac disease. Nevertheless, the sporadic cases had more severe clinical phenotype at diagnosis as well as poorer overall health even after dietary treatment. Moreover, HLA-DQ2.5-negative coeliac disease patients were observed to present with classical phenotype at diagnosis as well as with persistent symptoms after dietary treatment more often than patients heterozygous or homozygous for high-risk HLA- DQ2.5. These findings will hopefully encourage physicians to pay special attention to both these patient groups. Four distinct non-HLA variants were associated with increased risk for familial coeliac disease, but the associations need to be confirmed in future studies. The contribution of HLA-DQ2.5 dose as well as non-HLA single nucleotide polymorphisms (SNPs) within CCR9 and CCL25 to the clinical picture of coeliac disease was found to be only modest

    Using machine learning to predict pathogenicity of genomic variants throughout the human genome

    Get PDF
    Geschätzt mehr als 6.000 Erkrankungen werden durch Veränderungen im Genom verursacht. Ursachen gibt es viele: Eine genomische Variante kann die Translation eines Proteins stoppen, die Genregulation stören oder das Spleißen der mRNA in eine andere Isoform begünstigen. All diese Prozesse müssen überprüft werden, um die zum beschriebenen Phänotyp passende Variante zu ermitteln. Eine Automatisierung dieses Prozesses sind Varianteneffektmodelle. Mittels maschinellem Lernen und Annotationen aus verschiedenen Quellen bewerten diese Modelle genomische Varianten hinsichtlich ihrer Pathogenität. Die Entwicklung eines Varianteneffektmodells erfordert eine Reihe von Schritten: Annotation der Trainingsdaten, Auswahl von Features, Training verschiedener Modelle und Selektion eines Modells. Hier präsentiere ich ein allgemeines Workflow dieses Prozesses. Dieses ermöglicht es den Prozess zu konfigurieren, Modellmerkmale zu bearbeiten, und verschiedene Annotationen zu testen. Der Workflow umfasst außerdem die Optimierung von Hyperparametern, Validierung und letztlich die Anwendung des Modells durch genomweites Berechnen von Varianten-Scores. Der Workflow wird in der Entwicklung von Combined Annotation Dependent Depletion (CADD), einem Varianteneffektmodell zur genomweiten Bewertung von SNVs und InDels, verwendet. Durch Etablierung des ersten Varianteneffektmodells für das humane Referenzgenome GRCh38 demonstriere ich die gewonnenen Möglichkeiten Annotationen aufzugreifen und neue Modelle zu trainieren. Außerdem zeige ich, wie Deep-Learning-Scores als Feature in einem CADD-Modell die Vorhersage von RNA-Spleißing verbessern. Außerdem werden Varianteneffektmodelle aufgrund eines neuen, auf Allelhäufigkeit basierten, Trainingsdatensatz entwickelt. Diese Ergebnisse zeigen, dass der entwickelte Workflow eine skalierbare und flexible Möglichkeit ist, um Varianteneffektmodelle zu entwickeln. Alle entstandenen Scores sind unter cadd.gs.washington.edu und cadd.bihealth.org frei verfügbar.More than 6,000 diseases are estimated to be caused by genomic variants. This can happen in many possible ways: a variant may stop the translation of a protein, interfere with gene regulation, or alter splicing of the transcribed mRNA into an unwanted isoform. It is necessary to investigate all of these processes in order to evaluate which variant may be causal for the deleterious phenotype. A great help in this regard are variant effect scores. Implemented as machine learning classifiers, they integrate annotations from different resources to rank genomic variants in terms of pathogenicity. Developing a variant effect score requires multiple steps: annotation of the training data, feature selection, model training, benchmarking, and finally deployment for the model's application. Here, I present a generalized workflow of this process. It makes it simple to configure how information is converted into model features, enabling the rapid exploration of different annotations. The workflow further implements hyperparameter optimization, model validation and ultimately deployment of a selected model via genome-wide scoring of genomic variants. The workflow is applied to train Combined Annotation Dependent Depletion (CADD), a variant effect model that is scoring SNVs and InDels genome-wide. I show that the workflow can be quickly adapted to novel annotations by porting CADD to the genome reference GRCh38. Further, I demonstrate the integration of deep-neural network scores as features into a new CADD model, improving the annotation of RNA splicing events. Finally, I apply the workflow to train multiple variant effect models from training data that is based on variants selected by allele frequency. In conclusion, the developed workflow presents a flexible and scalable method to train variant effect scores. All software and developed scores are freely available from cadd.gs.washington.edu and cadd.bihealth.org

    On farm and off-farm feed utilization and improved management options: A Synthesis

    Get PDF
    The challenges identified in the proposal of the CGIAR Research Program (CRP) on Livestock, for enhancing livestock production and productivity, have been the shortage of quality feeds, inefficient utilization of feed resources, degradation of rangelands, and lack of knowledge and skills of stakeholders in the feed production and utilization value chains. To address these challenges, the research and development work, innovations developed and tested, results-dissemination and concerted efforts towards capacity building have been presented in the following three main sections. 1) Enlarging the feed quality resource base 2) Efficient use of feed resources 3) Rehabilitation of degraded rangeland

    Rational development of stabilized cyclic disulfide redox probes and bioreductive prodrugs to target dithiol oxidoreductases

    Get PDF
    Countless biological processes allow cells to develop, survive, and proliferate. Among these, tightly balanced regulatory enzymatic pathways that can respond rapidly to external impacts maintain dynamic physiological homeostasis. More specifically, redox homeostasis broadly affects cellular metabolism and proliferation, with major contributions by thiol/disulfide oxidoreductase systems, in particular, the Thioredoxin Reductase Thioredoxin (TrxR/Trx) and the Glutathione Reductase-Glutathione-Glutaredoxin (GR/GSH/Grx) systems. These cascades drive vital cellular functions in many ways through signaling, regulating other proteins' activity by redox switches, and by stoichiometric reductant transfers in metabolism and antioxidant systems. Increasing evidence argues that there is a persistent alteration of the redox environment in certain pathological states, such as cancer, that heavily involve the Trx system: upregulation and/or overactivity of the Trx system may support or drive cancer progression, making both TrxR and Trx promising targets for anti-cancer drug development. Understanding the biochemical mechanisms and connections between certain redox cascades requires research tools that interact with them. The state-of-the-art genetic tools are mostly ratiometric reporters that measure reduced:oxidized ratios of selected redox pairs or the general thiol pool. However, the precise cellular roles of the central oxidoreductase systems, including TrxR and Trx, remain inaccessible due to the lack of probes to selectively measure turnover by either of these proteins. However, such probes would allow measuring their effective reductive activity apart from expression levels in native systems, including in cells, animals, or patient samples. They are also of high interest to identify chemical inhibitors for TrxR/Trx in cells and to validate their potential use as anti-cancer agents (to date, there is no selective cellular Trx inhibitor, and most known TrxR inhibitors were not comprehensively evaluated considering selectivity and potential off-targets). However, small molecule redox imaging tools are underdeveloped: their protein specificity, spectral properties, and applicability remain poorly precedented. This work aimed to address this opportunity gap and develop novel, small molecule diagnostic and therapeutic tools to selectively target the Trx system based on a modular trigger cargo design: artificial cyclic disulfide substrates (trigger) for oxidoreductases are tethered to molecular agents (cargo) such that the cargo’s activity is masked and is re-established only through reduction by a target protein. The rational design of these novel reduction sensors to target the cell's strongest disulfide-reducing enzymes was driven by the following principles: (i) cyclic disulfide triggers with stabilized ring systems were used to gain low reduction potentials that should resist reduction except by the strongest cellular reductases, such as Trx; and (ii) the cyclic topology also offers the potential for kinetic reversibility that should select for dithiol-type redox proteins over the cellular monothiol background. Creating imaging agents based on such two-component designs to selectively measure redox protein activity in native cells required to combine the correct trigger reducibility, probe activation kinetics, and imaging modalities and to consider the overall molecular architecture. The major prior art in this field has applied cyclic 5-membered disulfides (1,2 dithiolanes) as substrates for TrxR in a similar way to create such tools. However, this motif was described elsewhere as thermodynamically instable and was due to widely used for dynamic covalent cascade reactions. By comparing a novel 1,2 dithiolane-based probe to the state-of-the-art probes, including commercial TrxR sensors, by screening a conclusive assay panel of cellular TrxR modulations, I clarified that 1,2 dithiolanes are not selective substrates for TrxR in biological settings (Nat Commun 2022). Instead, aiming for more stable ring systems and thus more robust redox probes, during this work, I developed bicyclic 6 membered disulfides (piperidine fused 1,2 dithianes) with remarkably low reduction potentials. I showed that molecular probes using them as reduction sensors can be mostly processed by thioredoxins while being stable against reduction by GSH. The thermodynamically stabilized decalin like topology of the cis-annelated 1,2 dithianes requires particularly strong reductants to be cleaved. They also select for dithiol type redox proteins, like Trx, based on kinetic reversibility and offer fast cyclization due to the preorganization by annelation (JACS 2021). This work further expanded the system’s modularity with structural cores based on piperazine-fused 1,2 dithianes with the two amines allowing independent derivatization. Diagnostic tools using them as reduction sensors proved equally robust but with highly improved activation kinetics and were thus cellularly activated. Cellular studies evolved that they are substrates for both Trxs and their protein cousins Grxs, so measuring the cellular dithiol protein pool rather than solely Trx activity (preprint 2023). Finally, a trigger based on a slightly adapted reduction sensor, a desymmetrized 1,2 thiaselenane, was designed for selective reduction by TrxR’s selenol/thiol active site, then combined with a precipitating large Stokes’ shift fluorophore and a solubilizing group, to evolve the first selective probe RX1 to measure cellular TrxR activity, which even allowed high throughput inhibitor screening (Chem 2022). The central principle of this work was further advanced to therapeutic prodrugs based on the duocarmycin cargo (CBI) with tunable potency (JACS Au 2022) that can be used to create off-to-on therapeutic prodrugs. Such CBI prodrugs employing stabilized 1,2 dichalcogenide triggers proved to be cytotoxins that depend on Trx system activity in cells. They could further be exploited for cell-line dependent reductase activity profiling by screening their redox activation indices, the reduction-dependent part of total prodrug activation, in 177 cell lines. Beyond that, these prodrugs were well-tolerated in animals and showed anti-cancer efficacy in vivo in two distinct mouse tumor models (preprint 2022). Taken together, I introduced unique monothiol-resistant reducible motifs to target the cellular Trx system with chemocompatible units for each for TrxR and Trx/Grx, where the cyclic nature of the dichalcogenides avoids activation by GSH. By using them with distinct molecular cargos, I developed novel selective fluorescent reporter probes; and introduced a new class of bioreductive therapeutic constructs based on a common modular design. These were either applied to selectively measure cellular reductase activity or to deliver cytotoxic anti cancer agents in vivo. Ongoing work aims to differentiate between the two major redox effector proteins Trx and Grx, requiring additional layers of selectivity that may be addressed by tuned molecular recognition. The flexible use of various molecular cargos allows harnessing the same cellular redox machinery by either probes or prodrugs. This allows predictive conclusions from diagnostics to be directly translated into therapy and offers great potential for future adaptation to other enzyme classes and therapeutic venues.Die zelluläre Redox-Homöostase hängt von Thiol/Disulfid-Oxidoreduktasen ab, die den Stoffwechsel, die Proliferation und die antioxidative Antwort von Zellen beeinflussen. Die wichtigsten Netzwerke sind die Thioredoxin Reduktase-Thioredoxin (TrxR/Trx) und Glutathion Reduktase-Glutathion-Glutaredoxin (GR/GSH/Grx) Systeme, die über Redox-Schalter in Substratproteinen lebenswichtige zelluläre Funktionen steuern und so an der Redox-Regulation und -Signalübertragung beteiligt sind. Persistente Veränderungen des Redoxmilieus in pathologischen Zuständen, wie z. B. bei Krebs, sind in hohem Maße mit dem Trx-System verbunden. Eine Hochregulierung und/oder Überaktivität des Trx-Systems, die bei vielen Krebsarten auftreten, unterstützt zudem das Fortschreiten des Krebswachstums, was TrxR/Trx zu vielversprechenden Zielproteinen für die Entwicklung neuer Krebsmedikamente macht. Um die biochemischen Prozesse dahinter zu erforschen, sind spezielle Techniken zur Visualisierung und Messung enzymatischer Aktivität nötig. Die hierzu geeigneten, meist genetischen Sensoren messen ratiometrisch das Verhältnis reduzierter/oxidierter Spezies in zellulärem Umfeld oder spezifisch ausgewählte Redoxpaare. Die weitere Erforschung der exakten Funktion von TrxR/Trx und deren Substrate ist jedoch durch mangelnde Nachweismethoden limitiert. Diese sind außerdem zur Validierung chemischer Hemmstoffe für TrxR/Trx in Zellen und deren potenziellen Verwendung als Krebsmittel von großem Interesse. Bislang gibt es keinen selektiven zellulären Trx-Inhibitor und potenzielle Off-Target-Effekte der bekannten TrxR-Inhibitoren wurden nicht abschließend bewertet. Ziel dieser Arbeit ist die Entwicklung niedermolekularer, diagnostischer und therapeutischer Werkzeuge, die selektiv auf das Trx-System abzielen und auf einem modularen Trigger-Cargo Design basieren. Hierzu werden zyklische Disulfid-Substrate (Trigger) für Oxidoreduktasen so mit molekularen Wirkstoffen (Cargo) verknüpft, dass dabei die Wirkstoffaktivität maskiert, und erst nach Reduktion durch ein Zielprotein wiederhergestellt wird. Diese neuartigen, synthetischen Reduktionssensoren basieren auf den folgenden Grundprinzipien: (i) Zyklische Disulfide sind thermodynamisch stabilisiert und können nur durch die stärksten Reduktasen gespalten werden; und (ii) die zyklische Topologie ermöglicht die kinetische Reversibilität der zwei Thiol-Disulfid-Austauschreaktionen, die eine erste Reaktion mit Monothiolen, wie z. B. GSH, sofort umkehrt und so eine vollständige Reduktion verhindert. Die meisten früheren Arbeiten auf diesem Gebiet verwendeten ein zyklisches, fünfgliedriges Disulfid (1,2 Dithiolan) als Substrat für TrxR. Das gleiche Strukturmotiv wurde jedoch an anderer Stelle als thermodynamisch instabil beschrieben und aufgrund dieser Eigenschaft explizit für dynamische Kaskadenreaktionen verwendet. Deshalb vergleicht diese Arbeit zu Beginn einen neuen 1,2 Dithiolan basierten fluorogenen Indikator mit bestehenden, z. T. kommerziellen, Redox Sonden für TrxR in einer Reihe von Zellkultur-Experimenten unter Modulation der zellulären TrxR Aktivität und stellt so einen Widerspruch in der Literatur klar: 1,2 Dithiolane eignen sich nicht als selektive Substrate für TrxR, da sie labil sowohl gegen die Reduktion durch andere Redoxproteine, als auch gegen den Monothiol Hintergrund in Zellen sind (Nat. Commun. 2022). Als alternatives Strukturmotiv wird in dieser Arbeit ein bizyklisches sechsgliedriges Disulfid (anneliertes 1,2 Dithian) etabliert. Durch sein niedriges Reduktionspotenzial, also seine hohe Resistenz gegen Reduktion, werden molekulare Sonden basierend auf diesem 1,2 Dithian als Reduktionssensor fast ausschließlich von Trx aktiviert, nicht aber von TrxR oder GSH (JACS 2021). Dieses Kernmotiv bestimmt dabei die Reduzierbarkeit, und damit die Enzymspezifität, durch seine zyklische Natur und die Annelierung, auch unter Verwendung unterschiedlicher Farb-/Wirkstoffe. Auf dieser Grundlage konnte die molekulare Struktur durch einen weiteren Modifikationspunkt für die flexible Verwendung weiterer funktioneller Einheiten ergänzt werden. Obwohl zelluläre Studien ergaben, dass diese neuartigen 1,2 Dithian Einheiten in Zellen sowohl Trx als auch das strukturell verwandte Grx adressieren, sind die daraus resultierenden diagnostischen Moleküle wertvoll, um den katalytischen Umsatz zellulärer Dithiol-Reduktasen, der sogenannten Trx Superfamilie, selektiv anzuzeigen (Preprint 2023). Begünstigt durch das modulare Moleküldesign stellt diese Arbeit zudem das erste Reportersystem RX1 zum selektiven Nachweis der TrxR-Aktivität in Zellen vor. Es basiert auf der Verwendung eines zyklischen, unsymmetrischen Selenenylsulfid-Sensors (1,2 Thiaselenan), der selektiv von dem einzigartigen Selenolat der TrxR angegriffen wird, und dadurch letztlich nur von TrxR reduziert werden kann. RX1 eignete sich zudem für eine Hochdurchsatz-Validierung bestehender TrxR Inhibitoren und unterstreicht dadurch den kommerziellen Nutzen derartiger Diagnostika (Chem 2022). Das zentrale Trigger-Cargo Konzept dieser Arbeit wurde für therapeutische Zwecke weiterentwickelt und nutzt dabei den einzigartigen Wirkmechanismus der Duocarmycin-Naturstoffklasse (CBI) (JACS Au 2022) zur Entwicklung reduktiv aktivierbarer Therapeutika. CBI Prodrugs basierend auf stabilisierten Redox-Schaltern (1,2 Dithiane für Trx; 1,2 Thiaselenan für TrxR) reagierten signifikant auf TrxR-Modulation in Zellen. Sie wurden darüber hinaus durch das Referenzieren ihrer Aktivität gegenüber nicht-reduzierbaren Kontrollmoleküle für die Erstellung zelllinienabhängiger Profile der Reduktaseaktivität in 177 Zelllinien genutzt. Schließlich waren diese neuen Krebsmittel im Tiermodell gut verträglich und zeigten in zwei verschiedenen Mausmodellen eine krebshemmende Wirkung (Preprint 2022b). Zusammenfassend präsentiert diese Dissertation monothiol-resistente reduzierbare Trigger-Einheiten für das zelluläre Trx-System zur Entwicklung neuartiger, selektiver Reporter-Sonden, sowie eine neue Klasse reduktiv aktivierbarer Krebsmittel auf Basis eines adaptierbaren Trigger-Cargo Designs. Diese fanden entweder zur selektiven Messung zellulärer Proteinaktivität oder zum Einsatz als Antikrebsmittel Verwendung. Es wurden chemokompatible Motive sowohl für TrxR als auch für Trx/Grx identifiziert, wobei deren zyklische Natur eine Aktivierung durch GSH verhindert. Eine weitere Differenzierung zwischen den beiden Redox-Proteinen Trx und Grx und anderen Proteinen der Trx-Superfamilie erfordert eine zusätzliche Ebene der Selektierung, z. B. durch molekulare Erkennung, und ist Gegenstand laufender Arbeiten. Die flexible Verwendung verschiedener molekularer Wirkstoffe ermöglicht dabei die „Pipeline-Entwicklung“ von Diagnostika und Therapeutika, die von der zellulären Redox-Maschinerie analog umgesetzt werden, und dadurch Schlussfolgerungen aus der Diagnostik direkt auf eine Therapie übertragbar machen. Dies birgt großes Potenzial für künftige Entwicklungen bei einer potenziellen Übertragung des modularen Konzepts auf andere Enzymklassen und therapeutische Einsatzgebiete

    PROBABILISTIC MODELING OF CHROMATIN INTERACTIONS

    Get PDF
    Higher-order chromatin architecture plays an important role in mammalian transcriptional regulation. However, understanding the mechanisms and impact of complex chromatin contacts remain challenging. In the past decade, breakthroughs in experimental techniques like Chromatin Conformation Capture (3C) assays enable genome-wide detection of chromatin interactions at high resolution. This provides great opportunities of computational modeling to predict functional interactions and identify target genes of disease variants. In this thesis, I started with critical assessment of an existing method for predicting enhancer-promoter interactions. I reported severe overfitting issues of it resulting from improper machine learning experimental design. I also found the limitation of resolution in their training datasets hinder accurate assignment of single regulatory element at interaction boundaries. In the second part, I developed a novel mathematical model to predict CTCF-mediated chromatin loops, which is the most prominent class of chromatin interactions, based on the biological hypothesis of loop extrusion. I showed that this model is capable of predicting CTCF loops measured by CTCF ChIA-PET data with high accuracy, using CTCF ChIP-seq alone as input. Furthermore, this model consistently predicts chromatin interaction frequency due to changes of CTCF binding site by genetic perturbation and looping-related protein factor degradation events. In the last part, I applied this computational framework to a greater set of ChIA-PET data. The analysis result inspired the development of a simple and interpretable for predicting enhancer-promoter interactions. I showed that this model outperforms existing methods on predicting CRISPRi hits that regulated gene expression. Overall, these approaches are applicable to diverse datasets to advance our understanding of chromatin interaction mechanisms as well as their implication in gene regulation and diseases

    Vitalism and Its Legacy in Twentieth Century Life Sciences and Philosophy

    Get PDF
    This Open Access book combines philosophical and historical analysis of various forms of alternatives to mechanism and mechanistic explanation, focusing on the 19th century to the present. It addresses vitalism, organicism and responses to materialism and its relevance to current biological science. In doing so, it promotes dialogue and discussion about the historical and philosophical importance of vitalism and other non-mechanistic conceptions of life. It points towards the integration of genomic science into the broader history of biology. It details a broad engagement with a variety of nineteenth, twentieth and twenty-first century vitalisms and conceptions of life. In addition, it discusses important threads in the history of concepts in the United States and Europe, including charting new reception histories in eastern and south-eastern Europe. While vitalism, organicism and similar epistemologies are often the concern of specialists in the history and philosophy of biology and of historians of ideas, the range of the contributions as well as the geographical and temporal scope of the volume allows for it to appeal to the historian of science and the historian of biology generally

    Science and Innovations for Food Systems Transformation

    Get PDF
    This Open Access book compiles the findings of the Scientific Group of the United Nations Food Systems Summit 2021 and its research partners. The Scientific Group was an independent group of 28 food systems scientists from all over the world with a mandate from the Deputy Secretary-General of the United Nations. The chapters provide science- and research-based, state-of-the-art, solution-oriented knowledge and evidence to inform the transformation of contemporary food systems in order to achieve more sustainable, equitable and resilient systems

    Novel tools for identification of oncogenic driver mutations

    Get PDF
    Genetic alterations contribute to the development and pathogenesis of several human cancers. These mutations accumulate in a cancer tissue over the course of time due to the instability of the cancer genome. Large-scale sequencing efforts have enabled identification of an abundance of these somatic mutations, and the amount of data is constantly increasing due to the improved accessibility of next-generation sequencing technologies. From this multitude of cancer-associated somatic mutations, a large majority are predicted to be inconsequential “passenger” mutations, (i.e., mutations which do not confer a selective growth advantage to the cancer cells); and only a handful have been validated as “driver” mutations (i.e., mutations playing a critical role in the development or maintenance of cancer). These driver mutations also function as predictive markers for survival, therapeutic efficacy, and often make the cancer cells susceptible to therapeutic intervention. Identification of driver mutations is an integral part of biomarker discovery in cancer research, and my thesis aimed to address this by developing a screening platform and a database. The in vitro Screen for Activating Mutations (iSCREAM) is a high-throughput screening workflow which was established with Epidermal Growth Factor Receptor (EGFR) as a model. The screen was validated by detection of known activating mutations like EGFR L858R. A previously known EGFR variant of unknown significance (VUS), EGFR A702V, was discovered in the screen and was functionally characterized to be an activating mutation. The iSCREAM screening methodology was further used to systematically study ERBB4, another gene in the EGFR family of receptor tyrosine kinases. We detected ERBB4 VUS R687K, and E715K in the screen and identify them as activating mutations. The ERBB4 mutations were characterized for their effect on ERBB4 phosphorylation, their sensitivity to various tyrosine kinase inhibitors, and their tumorigenicity was evaluated with in vivo allografts. The Database Of Recurrent Mutations (DORM), was prepared by analyzing a public registry of somatic mutations and preparing a catalog of the mutations identified from genome-wide studies to recapitulate the “real-world” frequency of all the recurrent (n > 1) somatic mutations. DORM allows limiting the scope of search to 38 tissue types and supports advanced queries using regular expressions. The easy-to-use database and its backend are written to be very responsive and fast in comparison to contemporary public cancer databases. Taken together, the findings and resources presented in this thesis establish grounds for further studies with other tyrosine kinases and potentially enable diversification into new niches.Uusia työkaluja syöpää aiheuttavien mutaatioiden tunnistamiseksi Geneettiset muutokset vaikuttavat useiden ihmisen syöpien syntyyn ja kehittymiseen. Syöpäkudokseen geenimutaatioita kertyy yhä enemmän ajan kuluessa syövän genomisen instabiliteetin vuoksi. Laajamittaisten sekvensointihankkeiden avulla on pystytty tunnistamaan paljon erilaisia somaattisia eli hankinnallisia mutaatioita ja sekvensointitulosten määrä kasvaa jatkuvasti uuden sukupolven sekvensointitekniikoiden (engl. next generation sequencing, NGS) paremman saatavuuden ansiosta. Näistä lukuisista syöpään liittyvistä somaattisista mutaatioista suurin osa on potilaan ennusteen kannalta merkityksettömiä "matkustajamutaatioita" (engl. passenger mutation) eli mutaatioita, jotka eivät anna valikoivaa kasvuetua syöpäsoluille. Vain muutamia somaattisia mutaatioita on validoitu "ajajamutaatioiksi" (engl. driver mutation) eli mutaatioiksi, joilla on kriittinen rooli syövän kehittymisessä tai ylläpitämisessä. Nämä ajajamutaatiot toimivat usein eloonjäämisen sekä hoidon tehon ennusteellisina markkereina ja usein myös herkistävät syöpäsoluja hoidoille. Ajajamutaatioiden tunnistaminen on olennainen osa syövän biomarkkereiden tutkimusta. Väitöskirjatyöni tavoitteena oli kehittää ajajamutaatioiden seulonta-alusta ja tietokanta. Aktivoivien mutaatioiden in vitro -seulonta (engl. in vitro Screen for Activating Mutations, iSCREAM) on tehoseulontamenetelmä, jonka kehittämistyössä käytettiin mallina epidermaalista kasvutekijäreseptoria (EGFR) koodaavaa geeniä iSCREAM-seulonnalla tunnistettiin jo tunnettuja aktivoivia EGFR-mutaatioita, kuten L858R, mikä validoi menetelmän toimivuuden. Seulontamenetelmällä tunnistettiin ja karakterisoitiin myös uusi EGFR-geenin aktivoiva mutaatio, A702V, jonka oletettu toimintamekanismi selvitettiin. iSCREAM-seulontamenetelmää hyödynnettiin tässä työssä myös EGFR-reseptorityrosiinikinaasiperheen toisen geenin, ERBB4-geenin, systemaattiseen tutkimiseen, jonka avulla löydettiin uusina aktivoivina mutaatioina ERBB4 R687K ja E715K. Näiden ERBB4-mutaatioiden vaikutusta ERBB4:n fosforylaatioon ja lääkeherkkyyteen erilaisille tyrosiinikinaasiestäjille karakterisoitiin, ja niiden tuumorigeenisyys validoitiin in vivo -allografteissa. Toistuvien mutaatioiden tietokanta (engl. Database Of Recurrent Mutations, DORM) luotiin analysoimalla somaattisten mutaatioiden julkista rekisteriä ja laatimalla luettelo genominlaajuisissa tutkimuksissa tunnistetuista mutaatioista, jotta kaikkien toistuvien (n > 1) somaattisten mutaatioiden "todellinen" esiintymistiheys voitaisiin laskea. DORM mahdollistaa haun rajoittamisen 38:aan kudostyyppiin ja tukee edistyneempiä kyselyjä säännöllisten lausekkeiden (engl. regular expression) avulla. Helppokäyttöinen tietokanta ja sen taustajärjestelmä kehitettiin hyvin reagoivaksi ja nopeaksi nykyisiin julkisiin syöpätietokantoihin verrattuna. Tässä työssä esitetyt havainnot ja resurssit luovat yhdessä perustan jatkotutkimuksille muilla tyrosiinikinaaseilla ja ovat mahdollisesti laajennettavissa muillekin tutkimusalueille
    corecore