Charting PARP-1 dependent mechanisms for DNA double-strand break resection

L'intégrité de l'ADN génomique humain est maintenue par des systèmes de réparation de l'ADN qui protègent les cellules des dommages causés par des agents environnementaux ou des lésions spontanées de l'ADN. Chaque cellule peut subir jusqu'à 10⁵ lésions par jour, y compris les cassures double-brin de l'ADN (CDB). La poly(ADPribosyl)ation (PARylation) est l'un des premiers événements de signalisation moléculaire survenant aux CDBs. Il est catalysé par les poly(ADP-ribose)polymérases (PARP) qui sont directement activées par ces lésions d'ADN. Le fait de ne pas générer de poly(ADP)ribosyl (pADPr) en réponse à des dommages à l'ADN par une inhibition chimique ou par l'absence de PARP-1 augmente la sensibilité cellulaire au stress génotoxique, indiquant que la pADPr elle-même est une molécule clé de signalisation des dommages à l'ADN. L'inhibition de l'enzyme de signalisation des dommages à l'ADN, la poly(ADP-ribose) polymérase-1 (PARP-1) est l'une des nouvelles thérapies les plus prometteuses contre le cancer. Les inhibiteurs de PARP sensibilisent les cellules cancéreuses aux agents endommageant l'ADN et tuent efficacement les cellules cancéreuses du sein, des ovaires et du pancréas déficientes en BRCA1 (Breast Cancer gene 1) et BRCA2 (Breast Cancer gene 2), ce qui suggère que les cellules déficientes en réparation des CDBs sont extrêmement sensibles à l'inhibition de PARP. Pourtant, les mécanismes sous-jacents à cette létalité synthétique entre le déficit de réparation du CDB et l'inhibition de PARP restent mal définis. Il y a un débat considérable sur le mécanisme par lequel l'inhibition de PARP tue les cellules déficientes en réparation de l'ADN, et le plein potentiel des inhibiteurs de PARP dans le traitement du cancer ne peut être obtenu que par une compréhension claire des voies de réponse aux dommages de l'ADN (DDR) aux CDB et comment ils sont affectés par les inhibiteurs de PARP. L'objectif général de ma thèse est d'étudier le rôle de PARP-1 dans la réparation DSB et d'identifier les interacteurs de PARP-1 qui jouent également un rôle dans ce processus. Les cellules eucaryotes réparent les CDBs par deux voies principales, la jonction d'extrémité non homologue (NHEJ) et la recombinaison homologue (HR). La HR est initiée par la liaison des CDBs par BRCA1 et le complexe MRE11-RAD50-NBS1 et des nucléases EXO1/DNA2 pour générer de l'ADN simple-brin, qui est ensuite utilisé par la recombinase RAD51 et le complexe BRCA1-PALB2-BRCA2. Une question clé dans notre domaine concerne les facteurs critiques pour réguler le choix de la voie CDB. HR est initiée à partir d'extrémités DSB hautement résectées, tandis que dans le NHEJ, la résection est empêchée par des facteurs de réparation clés incluant RIF1 et 53BP1. En utilisant des cellules déficientes en PARP-1, nous avons observé que deux inhibiteurs de la résection de l'ADN et des régulateurs de choix de voie, RIF1 et 53BP1, la formation de foyers induits par des dommages à l'ADN sont fortement altérés. Cela confirme notre hypothèse selon laquelle PARP-1 participe à la réparation du DSB en influençant la résection de l'ADN. Afin de mieux comprendre le mécanisme de résection et le rôle que PARP-1 y joue, nous avons identifié d'autres protéines qui interagissent avec PARP-1 et modulent ce processus. Pour ce faire, nous avons utilisé des données sur les protéines de liaison au pADPr générées à la fois dans notre laboratoire et celui de notre collaborateur Ted Dawson de Johns Hopkins. Les candidats sélectionnés à partir de ces listes ont été criblés pour identifier une seule cible qui démontrerait un phénotype similaire à la perte de PARP-1. Deux cibles initiales ont été explorées et finalement une seule protéine à doigt de zinc a été choisie comme cible principale. Nous devons relever la fonction de ce doigt de zinc en HR, dans l'espoir qu'il permettra de découvrir davantage les mécanismes de PARP-1 en résection. En résumé, cette thèse élucide le rôle de PARP-1 dans la résection de l'ADN et identifie une protéine à doigt de zinc non étudiée auparavant qui interagit avec PARP-1 et partage une fonction similaire à PARP-1 dans la résection de l'ADN.The integrity of human genomic DNA is maintained by DNA repair systems that will protect cells from damage by environmental agents or spontaneous DNA lesions. Each cell can experience up to 10⁵ lesions daily, including DNA double-strand breaks (DSB)s. Poly(ADP-ribosyl)ation (PARylation) is one of the earliest molecular signalling events occurring at DNA DSBs. It is catalysed by poly(ADP-ribose) polymerases (PARPs) that are directly activated by those DNA lesions. Failure to generate pADPr in response to DNA damage by either chemical inhibition or absence of PARP-1 increases the cellular sensitivity to genotoxic stress, indicating that pADPr itself is a key DNA damage signalling molecule. Inhibition of the DNA damage signalling enzyme poly(ADP-ribose) polymerase-1 (PARP-1) is among the most promising new therapies in cancer. PARP inhibitors sensitize cancer cells to DNA damaging agents and efficiently kill BRCA1- and BRCA2-deficient breast, ovarian and pancreatic cancer cells, suggesting that cells deficient in DSB repair are exquisitely sensitive to PARP inhibition. Yet, the mechanisms underlying this synthetic lethality between DSB repair deficiency and PARP inhibition remain poorly defined. There is considerable debate about the mechanism through which PARP inhibition kills DNA repair-deficient cells, and the full benefit of PARP inhibitors in cancer therapy can only be achieved by a clear understanding of the DNA damage response (DDR) pathways to DSBs and how these are affected by PARP inhibitors. The overall aim of my PhD is to investigate the role of PARP-1 in DSB repair and identify interactors of PARP-1 which also play a role in this process. Eukaryotic cells repair DSBs by two major pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR). HR is initiated by the binding of DSB by BRCA1 and the end resection of the DSB by MRE11 (and the associated NBS1, RAD50, CtIP, and EXO1) to generate single-stranded DNA, which is further processed by RAD51 and BRCA1-PALB2-BRCA2. A key question in our field regards which factors are critical for regulating the DSB pathway choice. HR is initiated from highly resected DSB ends, whereas in NHEJ, resection is prevented by key repair factors that include RIF1 and 53BP1. Using PARP-1-deficient cells, we have observed that two inhibitors of DNA resection and regulators of pathway choice, RIF1 and 53BP1, are strongly impaired in forming DNA damage-induced foci. This supports our hypothesis that PARP-1 participates in DSB repair by influencing DNA resection. In order to further understand the mechanism of resection and the role that PARP-1 plays in it we also aim to identify other proteins which interact with PARP-1 and modulate this process. To accomplish this, we made use of data on PAR binding proteins generated both in our lab and that of our collaborator Ted Dawson. The candidates selected from these lists were screened to identify a single target that would demonstrate a similar phenotype to PARP-1 loss. Two initial targets were further explored and finally a single zinc finger protein was selected as our primary target. We aim to characterize the function of this zinc finger in HR, in the hopes that it will further uncover the mechanisms of PARP-1 in resection. In summary this thesis elucidates the role of PARP-1 in DNA resection and identifies a previously unstudied zinc finger protein which interacts with PARP-1 and shares a similar function to PARP-1 in DNA resection

Misreading the marshes: past and present perceptions of the East Anglian Fens, UK

This paper aims to demonstrate the arbitrariness of the structural opposition between wet and dryland landscapes and people. This opposition, and an accompanying negative attitude to wetlands, is often held by ‘outsiders’, those who do not regularly interact with wetland environments. The outsider view of wetlands can be traced throughout history and is problematic as it often unwittingly influences our own understanding of past wetland(er)s. To address this issue, an alternative, ‘insiders’ perception of wetlands should be considered. This paper will do so by re-evaluating historical accounts written by outsiders and integrating these with information from the rich archaeological record of the East Anglian Fens. Doing so reveals several modes of human-wetland interaction in the (pre)historic Fens which reflect the different ways in which this wetland was perceived by various people through time. Considering multiple voices and integrating various categories of evidence from both within and outside wetlands provides us with a more accurate understanding of the dynamics of past life in and around these landscapes and thus helps us break down modern dichotomies which create artificial boundaries between wet and dryland(er)s

Investigating the Impact of the Covid-19 Pandemic on Undergraduate Business Education: Using Learning Gain as a Measure to Compare Two Cohorts of Marketing Students

This paper considers management education and specifically how student learning has been impacted by the online replacement teaching offered by universities during the COVID-19 pandemic. The study utilizes a learning gain model which considers the students’ own perception of their learning, and separates the provision of theoretical explicit knowledge (distance travelled) from that of practical tacit understanding (journey travelled). In 2019, data were collected from a cohort of marketing students studying for an undergraduate UK business studies degree. In 2021, data were again collected from a new cohort of students studying the same business studies degree course, but this time during the COVID-19 period. A comparison was undertaken to identify any differences in learning gain. Overall, a drop in perceived learning was reported, although in a few areas, an indication of stronger learning was identified. Interestingly, female students reported the most significant drop in their learning related to journey travelled, whereas for male students, it related to distance travelled

Comparative genetic and physiological characterisation of Pectinatus species reveals shared tolerance to beer-associated stressors but halotolerance specific to pickle-associated strains

This work was supported by Heineken Supply Chain B.V. PL, SD & HJF receive support from the Scottish Government Rural and Environment Sciences and Analytical Services (RESAS). We would like to express thanks to Nicola Smith for her help formatting Fig. 1, Dr Paul Sheridan for bioinformatics-based advice and help with troubleshooting scripts, Dr Kenneth Forbes for his advice on core and pan genome analysis, Sandro Cocuzza for providing the hop extract and Marine Freyereisen for supplying Lactobacillus brevis MB521.Peer reviewedPostprin

FIREBall-2: advancing TRL while doing proof-of-concept astrophysics on a suborbital platform

Here we discuss advances in UV technology over the last decade, with an emphasis on photon counting, low noise, high efficiency detectors in sub-orbital programs. We focus on the use of innovative UV detectors in a NASA astrophysics balloon telescope, FIREBall-2, which successfully flew in the Fall of 2018. The FIREBall-2 telescope is designed to make observations of distant galaxies to understand more about how they evolve by looking for diffuse hydrogen in the galactic halo. The payload utilizes a 1.0-meter class telescope with an ultraviolet multi-object spectrograph and is a joint collaboration between Caltech, JPL, LAM, CNES, Columbia, the University of Arizona, and NASA. The improved detector technology that was tested on FIREBall-2 can be applied to any UV mission. We discuss the results of the flight and detector performance. We will also discuss the utility of sub-orbital platforms (both balloon payloads and rockets) for testing new technologies and proof-of-concept scientific ideasComment: Submitted to the Proceedings of SPIE, Defense + Commercial Sensing (SI19

Multifocal demyelinating motor neuropathy and hamartoma syndrome associated with a de novo PTEN mutation.

OBJECTIVE: To describe a patient with a multifocal demyelinating motor neuropathy with onset in childhood and a mutation in phosphatase and tensin homolog (PTEN), a tumor suppressor gene associated with inherited tumor susceptibility conditions, macrocephaly, autism, ataxia, tremor, and epilepsy. Functional implications of this protein have been investigated in Parkinson and Alzheimer diseases. METHODS: We performed whole-exome sequencing in the patient's genomic DNA validated by Sanger sequencing. Immunoblotting, in vitro enzymatic assay, and label-free shotgun proteomic profiling were performed in the patient's fibroblasts. RESULTS: The predominant clinical presentation of the patient was a childhood onset, asymmetric progressive multifocal motor neuropathy. In addition, he presented with macrocephaly, autism spectrum disorder, and skin hamartomas, considered as clinical criteria for PTEN-related hamartoma tumor syndrome. Extensive tumor screening did not detect any malignancies. We detected a novel de novo heterozygous c.269T>C, p.(Phe90Ser) PTEN variant, which was absent in both parents. The pathogenicity of the variant is supported by altered expression of several PTEN-associated proteins involved in tumorigenesis. Moreover, fibroblasts showed a defect in catalytic activity of PTEN against the secondary substrate, phosphatidylinositol 3,4-trisphosphate. In support of our findings, focal hypermyelination leading to peripheral neuropathy has been reported in PTEN-deficient mice. CONCLUSION: We describe a novel phenotype, PTEN-associated multifocal demyelinating motor neuropathy with a skin hamartoma syndrome. A similar mechanism may potentially underlie other forms of Charcot-Marie-Tooth disease with involvement of the phosphatidylinositol pathway

The burden of diabetes mellitus during pregnancy in low- and middle-income countries : a systematic review

Peer reviewedPublisher PD

Scaling carbon fluxes from eddy covariance sites to globe: Synthesis and evaluation of the FLUXCOM approach

FLUXNET assembles globally-distributed eddy covariance-based estimates of carbon fluxes between the biosphere and the atmosphere. Since eddy covariance flux towers have a relatively small footprint and are distributed unevenly across the world, upscaling the observations is necessary in order to obtain global-scale estimates of biosphere-atmosphere exchange from the flux tower network. Based on cross-consistency checks with atmospheric inversions, sun-induced fluorescence (SIF) and dynamic global vegetation models (DGVM), we provide here a systematic assessment of the latest upscaling efforts for gross primary production (GPP) and net ecosystem exchange (NEE) of the FLUXCOM initiative, where different machine learning methods, forcing datasets, and sets of predictor variables were employed. Spatial patterns of mean GPP are consistent among FLUXCOM and DGVM ensembles (R2 > 0.94 at 1° spatial resolution) while the majority of DGVMs are outside the FLUXCOM range for 70 % of the land surface. Global mean GPP magnitudes for 2008–2010 from FLUXCOM members vary within 106 and 130 PgC yr−1 with the largest uncertainty in the tropics. Seasonal variations of independent SIF estimates agree better with FLUXCOM GPP (mean global pixel-wise R2 ~ 0.75) than with GPP from DGVMs (mean global pixel wise R2 ~ 0.6). Seasonal variations of FLUXCOM NEE show good consistency with atmospheric inversion-based net land carbon fluxes, particularly for temperate and boreal regions (R2 > 0.92). Interannual variability of global NEE in FLUXCOM is underestimated compared to inversions and DGVMs. The FLUXCOM version which uses also meteorological inputs shows a strong co-variation of interannual patterns with inversions (R2 = 0.88 for 2001–2010). Mean regional NEE from FLUXCOM shows larger uptake than inversion and DGVM-based estimates, particularly in the tropics with discrepancies of up to several hundred gC m2 yr−1. These discrepancies can only partly be reconciled by carbon loss pathways that are implicit in inversions but not captured by the flux tower measurements such as carbon emissions from fires and water bodies. We hypothesize that a combination of systematic biases in the underlying eddy covariance data, in particular in tall tropical forests, and a lack of site-history effects on NEE in FLUXCOM are likely responsible for the too strong tropical carbon sink estimated by FLUXCOM. Furthermore, as FLUXCOM does not account for CO2 fertilization effects carbon flux trends are not realistic. Overall, current FLUXCOM estimates of mean annual and seasonal cycles of GPP as well as seasonal NEE variations provide useful constraints of global carbon cycling, while interannual variability patterns from FLUXCOM are valuable but require cautious interpretation. Exploring the diversity of Earth Observation data and of machine learning concepts along with improved quality and quantity of flux tower measurements will facilitate further improvements of the FLUXCOM approach overall

FIREBall-2: The Faint Intergalactic Medium Redshifted Emission Balloon Telescope

The Faint Intergalactic Medium Redshifted Emission Balloon (FIREBall) is a mission designed to observe faint emission from the circumgalactic medium of moderate redshift (z~0.7) galaxies for the first time. FIREBall observes a component of galaxies that plays a key role in how galaxies form and evolve, likely contains a significant amount of baryons, and has only recently been observed at higher redshifts in the visible. Here we report on the 2018 flight of the FIREBall-2 Balloon telescope, which occurred on September 22nd, 2018 from Fort Sumner, New Mexico. The flight was the culmination of a complete redesign of the spectrograph from the original FIREBall fiber-fed IFU to a wide-field multi-object spectrograph. The flight was terminated early due to a hole in the balloon, and our original science objectives were not achieved. The overall sensitivity of the instrument and telescope was 90,000 LU, due primarily to increased noise from stray light. We discuss the design of the FIREBall-2 spectrograph, modifications from the original FIREBall payload, and provide an overview of the performance of all systems. We were able to successfully flight test a new pointing control system, a UV-optimized, delta-doped and coated EMCCD, and an aspheric grating. The FIREBall-2 team is rebuilding the payload for another flight attempt in the Fall of 2021, delayed from 2020 due to COVID-19.Comment: 23 Pages, 14 Figures, Accepted for Publication in Ap

Achievable agricultural soil carbon sequestration across Europe from country-specific estimates

Publication history: Accepted - 9 September 2021; Published online - 20 September 2021.The role of soils in the global carbon cycle and in reducing GHG emissions from agriculture has been increasingly acknowledged. The ‘4 per 1000’ (4p1000) initiative has become a prominent action plan for climate change mitigation and achieve food security through an annual increase in soil organic carbon (SOC) stocks by 0.4%, (i.e. 4‰ per year). However, the feasibility of the 4p1000 scenario and, more generally, the capacity of individual countries to implement soil carbon sequestration (SCS) measures remain highly uncertain. Here, we evaluated country-specific SCS potentials of agricultural land for 24 countries in Europe. Based on a detailed survey of available literature, we estimate that between 0.1% and 27% of the agricultural greenhouse gas (GHG) emissions can potentially be compensated by SCS annually within the next decades. Measures varied widely across countries, indicating differences in country-specific environmental conditions and agricultural practices. None of the countries' SCS potential reached the aspirational goal of the 4p1000 initiative, suggesting that in order to achieve this goal, a wider range of measures and implementation pathways need to be explored. Yet, SCS potentials exceeded those from previous pan-European modelling scenarios, underpinning the general need to include national/regional knowledge and expertise to improve estimates of SCS potentials. The complexity of the chosen SCS measurement approaches between countries ranked from tier 1 to tier 3 and included the effect of different controlling factors, suggesting that methodological improvements and standardization of SCS accounting are urgently required. Standardization should include the assessment of key controlling factors such as realistic areas, technical and practical feasibility, trade-offs with other GHG and climate change. Our analysis suggests that country-specific knowledge and SCS estimates together with improved data sharing and harmonization are crucial to better quantify the role of soils in offsetting anthropogenic GHG emissions at global level.This study has been funded and supported by the Horizon 2020 European Joint Programme SOIL (EJP-SOIL), grant agreement: 862695; Funding source: H2020-SFS-2018-2020/H2020-SFS-2019-1