The Development, Validation and Implementation of a Broad-Based ADME Genotyping Assay into Research and Clinical Trials

Afin d’adresser la variabilité interindividuelle observée dans la réponse pharmacocinétique à de nombreux médicaments, nous avons créé un panel de génotypage personnalisée en utilisant des méthodes de conception et d’élaboration d’essais uniques. Celles-ci ont pour but premier de capturer les variations génétiques présentent dans les gènes clés impliqués dans les processus d'absorption, de distribution, de métabolisme et d’excrétion (ADME) de nombreux agents thérapeutiques. Bien que ces gènes et voies de signalement sont impliqués dans plusieurs mécanismes pharmacocinétiques qui sont bien connues, il y a eu jusqu’à présent peu d'efforts envers l’évaluation simultanée d’un grand nombre de ces gènes moyennant un seul outil expérimental. La recherche pharmacogénomique peut être réalisée en utilisant deux approches: 1) les marqueurs fonctionnels peuvent être utilisés pour présélectionner ou stratifier les populations de patients en se basant sur des états métaboliques connus; 2) les marqueurs Tag peuvent être utilisés pour découvrir de nouvelles corrélations génotype-phénotype. Présentement, il existe un besoin pour un outil de recherche qui englobe un grand nombre de gènes ADME et variantes et dont le contenu est applicable à ces deux modèles d'étude. Dans le cadre de cette thèse, nous avons développé un panel d’essais de génotypage de 3,000 marqueurs génétiques ADME qui peuvent satisfaire ce besoin. Dans le cadre de ce projet, les gènes et marqueurs associés avec la famille ADME ont été sélectionnés en collaboration avec plusieurs groupes du milieu universitaire et de l'industrie pharmaceutique. Pendant trois phases de développement de cet essai de génotypage, le taux de conversion pour 3,000 marqueurs a été amélioré de 83% à 97,4% grâce à l'incorporation de nouvelles stratégies ayant pour but de surmonter les zones d'interférence génomiques comprenant entre autres les régions homologues et les polymorphismes sous-jacent les régions d’intérêt. La précision du panel de génotypage a été validée par l’évaluation de plus de 200 échantillons pour lesquelles les génotypes sont connus pour lesquels nous avons obtenu une concordance > 98%. De plus, une comparaison croisée entre nos données provenant de cet essai et des données obtenues par différentes plateformes technologiques déjà disponibles sur le marché a révélé une concordance globale de > 99,5%. L'efficacité de notre stratégie de conception ont été démontrées par l'utilisation réussie de cet essai dans le cadre de plusieurs projets de recherche où plus de 1,000 échantillons ont été testés. Nous avons entre autre évalué avec succès 150 échantillons hépatiques qui ont été largement caractérisés pour plusieurs phénotypes. Dans ces échantillons, nous avons pu valider 13 gènes ADME avec cis-eQTL précédemment rapportés et de découvrir et de 13 autres gènes ADME avec cis eQTLs qui n'avaient pas été observés en utilisant des méthodes standard. Enfin, à l'appui de ce travail, un outil logiciel a été développé, Opitimus Primer, pour aider pour aider au développement du test. Le logiciel a également été utilisé pour aider à l'enrichissement de cibles génomiques pour d'expériences séquençage. Le contenu ainsi que la conception, l’optimisation et la validation de notre panel le distingue largement de l’ensemble des essais commerciaux couramment disponibles sur le marché qui comprennent soit des marqueurs fonctionnels pour seulement un petit nombre de gènes, ou alors n’offre pas une couverture adéquate pour les gènes connus d’ADME. Nous pouvons ainsi conclure que l’essai que nous avons développé est et continuera certainement d’être un outil d’une grande utilité pour les futures études et essais cliniques dans le domaine de la pharmacocinétique, qui bénéficieraient de l'évaluation d'une longue liste complète de gènes d’ADME.In order to better assess the inter-individual variability observed in a patient’s pharmacokinetic response to many medications, we have created a custom genotyping panel that uses unique assay designs to analyze variation present in key genes involved in the absorption, distribution, metabolism and excretion (ADME) of many therapeutic agents. These genes and pathways involved in most pharmacokinetic mechanisms are well known. However, as yet, there has been little effort to develop tools that can interrogate a large number of variations in most known drug metabolizing genes simultaneously within a single experimental tool. Pharmacogenomic research has historically been conducted using two approaches: targeted studies that screen a small number of specific functional markers to identify known metabolic status phenotypes, and genome-wide studies that identify novel genetic correlations with drug response phenotypes. Thus, a gap currently exists for a targeted ADME research tool that can evaluate a large number of key ADME genes and variants in a format that can be applicable to both types of study designs. As part of this thesis, we have developed a 3000 SNP broad based ADME genotyping panel that can address this need. Genes and markers for the genotyping panel were selected in collaboration with many groups from both academia and the pharmaceutical industry in an effort to capture all pertinent genes and metabolic pathways that have been implicated in drug metabolism. The final assay design was composed of over 3000 markers in 181 genes. Over three phases of iterative development, the assay conversion rate for the 3000 markers was improved from 83.0% to 97.4% through the incorporation of novel design strategies to overcome areas of genomic interference such as regions of homology and underlying polymorphisms. Accuracy of the assay was validated by screening more than 200 samples of known genotype with a concordance of 99%. Additionally, data from the assay has also been compared to data from different technological platforms and has an overall concordance of 99.5%. The effectiveness of the design strategy was demonstrated in the successful utilization of the assay in the screening of over 1000 samples which identified several novel pharmacogenetic associations between ADME variations and adverse drug reactions in children. Another goal of this thesis was to demonstrate what added benefit/utility the 3000 SNP ADME panel would have when compared to currently available genotyping assays. Using 150 extensively investigated liver samples, the broad based assay was not only able to detect and validate 13 previously reported cis eQTLs in ADME genes but further identified an additional 13 novel ADME cis eQTLs that had never been observed before, doubling the number previously identified using standard methods on the same samples. Finally, in support of this work, a number of bioinformatic tools had to be developed to help expedite this research. These tools have been further refined and are currently being used to assist with enrichment of genomic targets for next generation sequencing experiments. In conclusion, this work has led to a better understanding of ADME genetics and the nuances of assaying ADME genes. The content and designs of the developed assay sets it apart from currently available commercial assays that contain only functional markers in a small number of genes or do not have adequate coverage across ADME genes. The assay has the ability to play a significant role in pharmacogenomic studies to identify known and novel pharmacogenomic biomarkers. These will lead to improved biomarkers that will help better stratify pharmaceutical clinical trial populations or assist physicians to select better, more personalized, efficacious and safer therapies for their patients

Pure O-sequences and matroid h-vectors

We study Stanley's long-standing conjecture that the h-vectors of matroid simplicial complexes are pure O-sequences. Our method consists of a new and more abstract approach, which shifts the focus from working on constructing suitable artinian level monomial ideals, as often done in the past, to the study of properties of pure O-sequences. We propose a conjecture on pure O-sequences and settle it in small socle degrees. This allows us to prove Stanley's conjecture for all matroids of rank 3. At the end of the paper, using our method, we discuss a first possible approach to Stanley's conjecture in full generality. Our technical work on pure O-sequences also uses very recent results of the third author and collaborators.Comment: Contains several changes/updates with respect to the previous version. In particular, a discussion of a possible approach to the general case is included at the end. 13 pages. To appear in the Annals of Combinatoric

Feedback from users of energy efficiency information on the Internet: Analysis of the US CADDET home page

This paper describes the US experience to date with providing energy efficiency information from the Center for the Analysis and Dissemination of Demonstrated Energy Technologies (CADDET) on the Internet. The paper begins by describing the way that information is displayed in the US CADDET home page system. Statistics are then provided on numbers and types of users of the home page. Next we describe the frequency with which different types of CADDET information have been accessed and summarize the feedback provided by users. Drawing on this experience with the US CADDET home page system, the authors conclude that energy efficiency information systems on the World Wide Web can contribute significantly to the goals of CADDET and other information outreach programs. However, to reach a wider range of audiences, Internet systems need to be supplemented by other dissemination efforts aimed at reaching individuals in countries and organizations that are not currently using Internet services. In addition, more personal and customized information sources are needed to provide users with the types of assistance guidance that may be required to translate knowledge of a technology`s technical financial performance, into a decision to adopt the technology

The State of Self-Organized Criticality of the Sun During the Last Three Solar Cycles. II. Theoretical Model

The observed powerlaw distributions of solar flare parameters can be interpreted in terms of a nonlinear dissipative system in the state of self-organized criticality (SOC). We present a universal analytical model of a SOC process that is governed by three conditions: (i) a multiplicative or exponential growth phase, (ii) a randomly interrupted termination of the growth phase, and (iii) a linear decay phase. This basic concept approximately reproduces the observed frequency distributions. We generalize it to a randomized exponential-growth model, which includes also a (log-normal) distribution of threshold energies before the instability starts, as well as randomized decay times, which can reproduce both the observed occurrence frequency distributions and the scatter of correlated parametyers more realistically. With this analytical model we can efficiently perform Monte-Carlo simulations of frequency distributions and parameter correlations of SOC processes, which are simpler and faster than the iterative simulations of cellular automaton models. Solar cycle modulations of the powerlaw slopes of flare frequency distributions can be used to diagnose the thresholds and growth rates of magnetic instabilities responsible for solar flares.Comment: Part II of Paper I: The State of Self-Organized Criticality of the Sun During the Last Three Solar Cycles. I. Observation

Sedimentary ancient DNA from Lake Skartjorna, Svalbard: assessing the resilience of arctic flora to Holocene climate change

Reconstructing past vegetation and species diversity from arctic lake sediments can be challenging because of low pollen and plant macrofossil concentrations. Information may be enhanced by metabarcoding of sedimentary ancient DNA (sedaDNA). We developed a Holocene record from Lake Skartjørna, Svalbard, using sedaDNA, plant macrofossils and sediment properties, and compared it with published records. All but two genera of vascular plants identified as macrofossils in this or a previous study were identified with sedaDNA. Six additional vascular taxa were found, plus two algal and 12 bryophyte taxa, by sedaDNA analysis, which also detected more species per sample than macrofossil analysis. A shift from Salix polaris-dominated vegetation, with Koenigia islandica, Ranunculaceae and the relatively thermophilic species Arabis alpina and Betula, to Dryas octopetala-dominated vegetation ~6600–5500 cal. BP suggests a transition from moist conditions 1–2°C warmer than today to colder/drier conditions. This coincides with a decrease in runoff, inferred from core lithology, and an independent record of declining lacustrine productivity. This mid-Holocene change in terrestrial vegetation is broadly coincident with changes in records from marine sediments off the west coast of Svalbard. Over the Holocene sedaDNA records little floristic change, and it clearly shows species persisted near the lake during time intervals when they are not detected as macrofossils. The flora has shown resilience in the presence of a changing climate, and, if future warming is limited to 2°C or less, we might expect only minor floristic changes in this region. However, the Holocene record provides no analogues for greater warming

Maternal cardiovascular adaptation to twin pregnancy: A population-based prospective cohort study

Background: In women with singleton pregnancies, maternal adaptation is considered a stress test for later life cardiovascular disease. The aim of this study was to assess maternal adaptation in women with twin pregnancies compared to women carrying singletons during and after pregnancy. Methods: This was a population based prospective cohort study of 91 women with twin pregnancies and 8107 women carrying singletons. The association of twin pregnancy and maternal adaptation was examined using regression analyses. In pregnancy, we measured soluble fms-like tyrosine kinase-1 (sFLT-1), placental growth (PGF) factor, systolic (SBP) and diastolic blood pressure (DBP), and the occurrence of pre-eclampsia (PE). After pregnancy, measurements were obtained on SBP and DBP, cardiac function, retinal calibres, intima media thickness and distensibility of the common carotid artery. Results: sFLT-1 and PGF concentrations were higher in early (13.4 weeks) and mid-pregnancy (20.4 weeks) in women with twin pregnancies compared to women with singleton pregnancies. Women with twin pregnancies had a different DBP pattern in pregnancy. Women with twin pregnancies were more likely to have PE (odds ratio 3.63; 95% CI [1.76 to 7.48]). Six and ten years after pregnancy, no differences in maternal adaptation were observed. Conclusions: Women with twin pregnancies show an altered adaptation during pregnancy compared to women with singleton pregnancies. This is associated with a substantially increased incidence of PE, but does not lead to persistent altered maternal adaptation years after pregnancy

Septation and valvar formation in the outflow tract of the embryonic chick heart

There is no agreement, in the chick, about the number of the endocardial cushions within the outflow tract or their pattern of fusion. Also, little is known of their relative contributions to the formation of the arterial valves, the subpulmonary infundibulum, and the arterial valvar sinuses. As the chick heart is an important model for studying septation of the outflow tract, our objective was to clarify these issues. Normal septation of the outflow tract was studied in a series of 60 staged chick hearts, by using stained whole-mount preparations, serial sections, and scanning electron microscopy. A further six hearts were examined subsequent to hatching. At stage 21, two pairs of endocardial cushions were seen within the developing outflow tract. One pair was positioned proximally, with the other pair located distally. By stage 25, a third distal cushion had developed. This finding was before the appearance of two further, intercalated, endocardial cushions, also distally positioned, which were first seen at stage 29. In the arterial segment, the aortic and pulmonary channels were separated by the structure known as the aortopulmonary septum. The dorsal limb of this septum penetrated the distal dorsal cushion, whereas the ventral limb grew between the remaining two distal cushions, both of which were positioned ventrally. The three distal endocardial cushions, and the two intercalated endocardial cushions, contributed to the formation of the leaflets and sinuses of the arterial roots. The two proximal cushions gave rise to a transient septum, which later became transformed into the muscular component of the subpulmonary infundibulum. Concomitant with these changes, an extracardiac tissue plane was formed which separated this newly formed structure from the sinuses of the aortic root. Our study confirms that three endocardial cushions are positioned distally, and two proximally, within the developing outflow tract of the chick. The pattern of the distal cushions, and the position of the ventral limb of the aortopulmonary septum, differs significantly from that seen in mammals. Anat Rec 264:273-283, 2001. (C) 2001 Wiley-Liss, Inc.Animal science

Simultaneous Softening of sigma and rho Mesons associated with Chiral Restoration

Complex poles of the unitarized pi-pi scattering amplitude in nuclear matter are studied. Partial restoration of chiral symmetry is modeled by the decrease of in-medium pion decay constant f*_{pi}. For large chiral restoration (f*_{pi}/f_{pi} << 1), 2nd sheet poles in the scalar (sigma) and the vector (rho) mesons are both dictated by the Lambert W function and show universal softening as f*_{pi} decreases. In-medium pi-pi cross section receives substantial contribution from the soft mode and exhibits a large enhancement in low-energy region. Fate of this universality for small chiral restoration (f*_{pi}/f_{pi} ~ 1) is also discussed.Comment: 5 pages, 4-eps figures, version accepted by Phys. Rev. C (R) with minor modification

Spatial Degrees of Freedom in Everett Quantum Mechanics

Stapp claims that, when spatial degrees of freedom are taken into account, Everett quantum mechanics is ambiguous due to a "core basis problem." To examine an aspect of this claim I generalize the ideal measurement model to include translational degrees of freedom for both the measured system and the measuring apparatus. Analysis of this generalized model using the Everett interpretation in the Heisenberg picture shows that it makes unambiguous predictions for the possible results of measurements and their respective probabilities. The presence of translational degrees of freedom for the measuring apparatus affects the probabilities of measurement outcomes in the same way that a mixed state for the measured system would. Examination of a measurement scenario involving several observers illustrates the consistency of the model with perceived spatial localization of the measuring apparatus.Comment: 34 pp., no figs. Introduction, discussion revised. Material tangential to main point remove

BPS-Saturated Walls in Supersymmetric Theories

Domain-wall solutions in four-dimensional supersymmetric field theories with distinct discrete vacuum states lead to the spontaneous breaking of supersymmetry, either completely or partially. We consider in detail the case when the domain walls are the BPS-saturated states, and 1/2 of supersymmetry is preserved. Several useful criteria that relate the preservation of 1/2 of supersymmetry on the domain walls to the central extension appearing in the N=1 superalgebras are established. We explain how the central extension can appear in N=1 supersymmetry and explicitly obtain the central charge in various models: the generalized Wess-Zumino models, and supersymmetric Yang-Mills theories with or without matter. The BPS-saturated domain walls satisfy the first-order differential equations which we call the creek equations, since they formally coincide with the (complexified) equations of motion of an analog high-viscosity fluid on a profile which is given by the superpotential of the original problem. Some possible applications are considered.Comment: Several equations are corrected, the discussion of the two-dimensional soliton in Section 6 is modified, references are updated and expande