Targeting mitochondria to prevent diclofenac-induced liver toxicity

The objective of the studies described in this thesis was to elucidate the mechanisms of diclofenac-induced toxicity. We focused on mitochondria as the principal target of reactive metabolites of diclofenac. In addition, we investigated several interventions aimed at protecting hepatocytes from diclofenac-induced toxicity, including the intracellular messenger cAMP, the glucose-lowering drug metformin and natural products derived from Opuntia cactus fruit, rich in polyphenols. In Chapter 1, we provide a general introduction to this thesis and an outline of the thesis. Chapter 2 investigates the role of cAMP and downstream cAMP targets like EPACs and protein kinase A in the protection against diclofenac-induced injury in rat hepatocytes. Chapter 3 investigates whether the glucose-lowering drug metformin protects against diclofenacinduced injury and whether any protective effect is related to EPAC. Chapter 4 explores the use of the cactus fruit Opuntia as a treatment for diclofenac induced toxicity, both in vivo and in vitro. Chapter 5 investigates whether steatosis, the accumulation of fat in hepatocytes, affects diclofenac metabolism and toxicity. Finally, Chapter 6 summarizes our results and provides a general discussion and perspective for future studies

Elevated cAMP Protects against Diclofenac-Induced Toxicity in Primary Rat Hepatocytes:A Protective Effect Mediated by the Exchange Protein Directly Activated by cAMP/cAMP-Regulated Guanine Nucleotide Exchange Factors

Background: Chronic consumption of the nonsteroidal anti-inflammatory drug diclofenac may induce drug-induced liver injury (DILI). The mechanism of diclofenac-induced liver injury is partially elucidated and involves mitochondrial damage. Elevated cAMP protects hepatocytes against bile acid-induced injury. However, it is unknown whether cAMP protects against DILI and, if so, which downstream targets of cAMP are implicated in the protective mechanism including the classical protein kinase A (PKA) pathway or alternative pathways like the exchange protein directly activated by cAMP (EPAC). Aim: Investigate whether cAMP and/or its downstream targets protect against diclofenac-induced injury in hepatocytes. Methods: Rat hepatocytes were exposed to 400 µmol/L diclofenac. Apoptosis and necrosis were measured by caspase-3 activity assay and Sytox green staining respectively. Mitochondrial membrane potential (MMP) was measured by JC-10 staining. mRNA and protein expression were assessed by qPCR and Western blot, respectively. The cAMP-elevating agent forskolin, the pan-phosphodiesterase inhibitor IBMX and EPAC inhibitors CE3F4 and ESI-O5 were used to assess the role of cAMP and its effectors, PKA or EPAC. Results: Diclofenac exposure induced apoptotic cell death and loss of MMP in hepatocytes. Both forskolin and IBMX prevented diclofenac-induced apoptosis. EPAC inhibition, but not PKA inhibition abolished the protective effect of forskolin and IBMX. Forskolin and IBMX preserved the MMP while both EPAC inhibitors diminished this effect. Both EPAC1 and EPAC2 were expressed in hepatocytes and localized in mitochondria. Conclusion: cAMP elevation protects hepatocytes against diclofenac-induced cell death, a process primarily involving EPACs. The cAMP/EPAC pathway may be a novel target for treatment of DILI. Significance Statement Our study shows two main highlights. First, elevated cAMP levels protect against diclofenac-induced apoptosis in primary hepatocytes via maintenance of mitochondrial integrity. In addition, we propose the existence of mitochondrial cAMP-EPAC microdomains in rat hepatocytes, opening new avenues for targeted therapy in DILI. Both EPAC1 and EPAC2, but not PKA, are responsible for this protective effect. Our findings present cAMP-EPAC as a potential target for the treatment of drug-induced liver injury (DILI) and liver injury involving mitochondrial dysfunction

Biocomponents from Opuntia robusta and Opuntia streptacantha fruits protect against diclofenac-induced acute liver damage in vivo and in vitro

This study aimed to investigate whether Opuntia spp-extracts protect against diclofenac (DF)-induced hepatotoxicity. Rats were pretreated with Opuntia extracts, betanin (Bet) and N-acetylcysteine (NAC) followed by a single challenge of diclofenac. Liver tissue was collected for biochemical and histological analysis. Primary rat hepatocytes were treated with diclofenac (400 mu mol/L) with and without pretreatment with Opuntia extract. Apoptosis was measured by caspase-3 activity and necrosis by Sytox green staining. RNA was isolated, and realtime qPCR was performed to assess mRNA levels of stress and apoptosis-related genes MnSOD (SOD2), GADD45B and P53. ROS production was measured using the fluorescent MitoSOX assay. Results demonstrated that Opuntia spp-extracts protect against DF-induced liver toxicity via reducing oxidative stress and the inhibition of P53

Burnout among surgeons before and during the SARS-CoV-2 pandemic: an international survey

Background: SARS-CoV-2 pandemic has had many significant impacts within the surgical realm, and surgeons have been obligated to reconsider almost every aspect of daily clinical practice. Methods: This is a cross-sectional study reported in compliance with the CHERRIES guidelines and conducted through an online platform from June 14th to July 15th, 2020. The primary outcome was the burden of burnout during the pandemic indicated by the validated Shirom-Melamed Burnout Measure. Results: Nine hundred fifty-four surgeons completed the survey. The median length of practice was 10 years; 78.2% included were male with a median age of 37 years old, 39.5% were consultants, 68.9% were general surgeons, and 55.7% were affiliated with an academic institution. Overall, there was a significant increase in the mean burnout score during the pandemic; longer years of practice and older age were significantly associated with less burnout. There were significant reductions in the median number of outpatient visits, operated cases, on-call hours, emergency visits, and research work, so, 48.2% of respondents felt that the training resources were insufficient. The majority (81.3%) of respondents reported that their hospitals were included in the management of COVID-19, 66.5% felt their roles had been minimized; 41% were asked to assist in non-surgical medical practices, and 37.6% of respondents were included in COVID-19 management. Conclusions: There was a significant burnout among trainees. Almost all aspects of clinical and research activities were affected with a significant reduction in the volume of research, outpatient clinic visits, surgical procedures, on-call hours, and emergency cases hindering the training. Trial registration: The study was registered on clicaltrials.gov "NCT04433286" on 16/06/2020

Combined searches for the production of supersymmetric top quark partners in proton-proton collisions at root s=13 TeV

A combination of searches for top squark pair production using proton-proton collision data at a center-of-mass energy of 13 TeV at the CERN LHC, corresponding to an integrated luminosity of 137 fb(-1) collected by the CMS experiment, is presented. Signatures with at least 2 jets and large missing transverse momentum are categorized into events with 0, 1, or 2 leptons. New results for regions of parameter space where the kinematical properties of top squark pair production and top quark pair production are very similar are presented. Depending on themodel, the combined result excludes a top squarkmass up to 1325 GeV for amassless neutralino, and a neutralinomass up to 700 GeV for a top squarkmass of 1150 GeV. Top squarks with masses from 145 to 295 GeV, for neutralino masses from 0 to 100 GeV, with a mass difference between the top squark and the neutralino in a window of 30 GeV around the mass of the top quark, are excluded for the first time with CMS data. The results of theses searches are also interpreted in an alternative signal model of dark matter production via a spin-0 mediator in association with a top quark pair. Upper limits are set on the cross section for mediator particle masses of up to 420 GeV

Search for new particles in events with energetic jets and large missing transverse momentum in proton-proton collisions at root s=13 TeV

A search is presented for new particles produced at the LHC in proton-proton collisions at root s = 13 TeV, using events with energetic jets and large missing transverse momentum. The analysis is based on a data sample corresponding to an integrated luminosity of 101 fb(-1), collected in 2017-2018 with the CMS detector. Machine learning techniques are used to define separate categories for events with narrow jets from initial-state radiation and events with large-radius jets consistent with a hadronic decay of a W or Z boson. A statistical combination is made with an earlier search based on a data sample of 36 fb(-1), collected in 2016. No significant excess of events is observed with respect to the standard model background expectation determined from control samples in data. The results are interpreted in terms of limits on the branching fraction of an invisible decay of the Higgs boson, as well as constraints on simplified models of dark matter, on first-generation scalar leptoquarks decaying to quarks and neutrinos, and on models with large extra dimensions. Several of the new limits, specifically for spin-1 dark matter mediators, pseudoscalar mediators, colored mediators, and leptoquarks, are the most restrictive to date.Peer reviewe

Probing effective field theory operators in the associated production of top quarks with a Z boson in multilepton final states at root s=13 TeV

Peer reviewe

Measurement of the top quark forward-backward production asymmetry and the anomalous chromoelectric and chromomagnetic moments in pp collisions at √s = 13 TeV

Abstract The parton-level top quark (t) forward-backward asymmetry and the anomalous chromoelectric (d̂ t) and chromomagnetic (μ̂ t) moments have been measured using LHC pp collisions at a center-of-mass energy of 13 TeV, collected in the CMS detector in a data sample corresponding to an integrated luminosity of 35.9 fb−1. The linearized variable AFB(1) is used to approximate the asymmetry. Candidate t t ¯ events decaying to a muon or electron and jets in final states with low and high Lorentz boosts are selected and reconstructed using a fit of the kinematic distributions of the decay products to those expected for t t ¯ final states. The values found for the parameters are AFB(1)=0.048−0.087+0.095(stat)−0.029+0.020(syst),μ̂t=−0.024−0.009+0.013(stat)−0.011+0.016(syst), and a limit is placed on the magnitude of | d̂ t| < 0.03 at 95% confidence level. [Figure not available: see fulltext.

Measurements of Higgs boson production cross sections and couplings in the diphoton decay channel at root s=13 TeV

Measurements of Higgs boson production cross sections and couplings in events where the Higgs boson decays into a pair of photons are reported. Events are selected from a sample of proton-proton collisions at root s = 13TeV collected by the CMS detector at the LHC from 2016 to 2018, corresponding to an integrated luminosity of 137 fb(-1). Analysis categories enriched in Higgs boson events produced via gluon fusion, vector boson fusion, vector boson associated production, and production associated with top quarks are constructed. The total Higgs boson signal strength, relative to the standard model (SM) prediction, is measured to be 1.12 +/- 0.09. Other properties of the Higgs boson are measured, including SM signal strength modifiers, production cross sections, and its couplings to other particles. These include the most precise measurements of gluon fusion and vector boson fusion Higgs boson production in several different kinematic regions, the first measurement of Higgs boson production in association with a top quark pair in five regions of the Higgs boson transverse momentum, and an upper limit on the rate of Higgs boson production in association with a single top quark. All results are found to be in agreement with the SM expectations.Peer reviewe

Observation of tW production in the single-lepton channel in pp collisions at root s=13 TeV

A measurement of the cross section of the associated production of a single top quark and a W boson in final states with a muon or electron and jets in proton-proton collisions at root s = 13 TeV is presented. The data correspond to an integrated luminosity of 36 fb(-1) collected with the CMS detector at the CERN LHC in 2016. A boosted decision tree is used to separate the tW signal from the dominant t (t) over bar background, whilst the subleading W+jets and multijet backgrounds are constrained using data-based estimates. This result is the first observation of the tW process in final states containing a muon or electron and jets, with a significance exceeding 5 standard deviations. The cross section is determined to be 89 +/- 4 (stat) +/- 12 (syst) pb, consistent with the standard model.Peer reviewe