Using a systems approach to identify the mechanism of action of correctors

Tese de doutoramento, Biologia (Biologia de Sistemas), Universidade de Lisboa, Faculdade de Ciências, 2018Cystic Fibrosis (CF), the most common life-shortening genetic disorder among Caucasians, is caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, an ion channel expressed at the apical membrane of epithelial cells. High-throughput screens (HTS) identified several novel molecules potentially targeting the underlying CFTR defect but only for some patients: potentiator VX-770 (Ivacaftor/Kalydeco), for subjects bearing G551D and other gating mutations, the combination corrector/potentiator VX-809 (Lumacaftor)/VX-770 (Orkambi) for F508del-homozygous patients and another similar combination VX-661 (Tezacaftor)/VX-770 is under approval. The main objective of this PhD work was to study new compounds that correct the basic CF defect, by rescuing CFTR protein traffic and function, focusing both on individual responses of CF patients with different CFTR mutations to these new drugs, and their mechanism of action. Chapter 1 focusses on the measurement of functional responses on human bronchial epithelial cells (HBE’s) derived from CF lung explants bearing different CFTR mutations to VX-809 namely: A561E, N1303K, G542X and Y1092X. Our data showed a positive response of A561E/A561E to VX-809 and F508del/Y1092X but not F508del/G542X. In Chapter 2, we evaluated the efficacy of CFTR modulators (correctors/potentiators) in physiologically relevant tissues, namely rectal biopsies, intestinal organoids, (HBE’s) and human nasal epithelial cells (HNE’s), from CF patients with rare CFTR mutations. Data obtained here showed that neither R560S nor H1079P-could not be rescued by any of the CFTR modulators, but 3849+10kbC>T and R334W and c.120del23-CFTR were rescued by VX-770 alone or with VX-809. In Chapter 3 we evaluated the efficacy of two novel CFTR correctors (B9, E12) in primary HBE cells, and three novel compounds E-act mimics (C2, C5, and C7) as enhancers of alternative Cl- channel TMEM16A in human intestinal organoids. In Chapter 4 (final) we assessed the effect of CFTR modulators and their possible additivity with F508del-CFTR genetic revertants 4RK, R1070W, and G550E to understand the mechanism of action of small molecule correctors and another variant diacidic ER exit code DD/AA in CFBE mCherry cells expressing these varinats by Ussing chamber analysis with or without CFTR modulators. Our data show that C18 and VX-661 and low temperature (But not VX-809) rescued DD/AA to the cell surface and genetic revertants restore the channel function without any CFTR modulator. Altogether, results from this work bring new insights into how the CFTR genotype may influence CFTR function and response to CFTR modulators and how each patient should be assessed individually for the responsiveness to the CFTR modulators towards personalized therapeutics

RG Flow from ϕ4\phi^4 Theory to the 2D Ising Model

We study 1+1 dimensional $\phi^4$ theory using the recently proposed method of conformal truncation. Starting in the UV CFT of free field theory, we construct a complete basis of states with definite conformal Casimir, $\mathcal{C}$. We use these states to express the Hamiltonian of the full interacting theory in lightcone quantization. After truncating to states with $\mathcal{C} \leq \mathcal{C}_{\max}$, we numerically diagonalize the Hamiltonian at strong coupling and study the resulting IR dynamics. We compute non-perturbative spectral densities of several local operators, which are equivalent to real-time, infinite-volume correlation functions. These spectral densities, which include the Zamolodchikov $C$-function along the full RG flow, are calculable at any value of the coupling. Near criticality, our numerical results reproduce correlation functions in the 2D Ising model.Comment: 31+12 page

Rete: Learning Namespace Representation for Program Repair

A key challenge of automated program repair is finding correct patches in the vast search space of candidate patches. Real-world programs define large namespaces of variables that considerably contributes to the search space explosion. Existing program repair approaches neglect information about the program namespace, which makes them inefficient and increases the chance of test-overfitting. We propose Rete, a new program repair technique, that learns project-independent information about program namespace and uses it to navigate the search space of patches. Rete uses a neural network to extract project-independent information about variable CDU chains, def-use chains augmented with control flow. Then, it ranks patches by jointly ranking variables and the patch templates into which the variables are inserted. We evaluated Rete on 142 bugs extracted from two datasets, ManyBugs and BugsInPy. Our experiments demonstrate that ReTe generates six new correct patches that fix bugs that previous tools did not repair, an improvement of 31% and 59% over the existing state of the art

Measurement of risk and stressors of post-traumatic stress disorder during the first lockdown period of COVID-19 among Industrial workers, Hyderabad, India

Introduction: Post-traumatic stress disorder usually occurs after a traumatic event and this COVID-19 pandemic has affected mental health to a great extent. While there is extensive research being conducted about this aspect among the general population, the most neglected are the Industrial workers during the first lockdown period. The aim of the study was to assess the risk of post-traumatic stress disorder symptoms using the impact of event scale-revised (IES-R), their main stressors and coping measurements among the industrial workers of Hyderabad. Methods:  A telephonic interview schedule was adopted to fill the online questionnaire among Industrial workers of age group 20-55 years during the lockdown period. Hundred and seventy-four participants were included in our study and data was collected using standardized tools IES-R and brief resilient coping scale. Various stressors and also their coping strategies were elucidated. Results:  At the risk of post-traumatic stress disorder symptoms were found among 20.6% and at risk of suppressed immune system functioning which is presumed to have its effect for the next 10 years was 13.8%. The main stressors of COVID-19 with increased IES-R score were not staying with family members, influence of social media, Any acquaintance with COVID-19 infected person effect on daily life and white-collar group. Coping measurements with a brief resilient coping scale found 17.4% as low resilient copers. Conclusion: This identification of high-risk post-traumatic stress disorder symptoms needs to be addressed earlier to bring up preventive strategies such as counseling services in the occupational health care centers of our industries

Effect of Surgical Fusion on Volitional Weight-Shifting in Individuals With Adolescent Idiopathic Scoliosis

Study Design Prospective. Objectives The goals of this study were to (1) evaluate the differences in weightbearing symmetry between individuals with adolescent idiopathic scoliosis (AIS) and typically developing controls; (2) observe the effect of posterior spinal fusion and instrumentation (PSFI) on volitional weight-shifting at 1 and 2 years postoperatively; and (3) evaluate whether lowest instrumented fusion level (ie, lowest instrumented vertebra [LIV]) in PSFI has an effect on volitional weight-shifting. Summary of Background Data Previous studies have conflicting findings with regard to the effect of scoliosis on postural control tasks as well as the effect of surgery. They have also noted an inconsistent effect of PSFI at different LIVs, with more distal LIVs exhibiting greater reductions in postoperative range of motion. Methods The study was designed with an AIS group of 41 patients (8 males and 33 females) with AIS who underwent PSFI, along with a Control Group of 24 age-matched typically developing participants (12 male and 12 female). Both groups performed postural control tasks (static balance and volitional weight-shifting), with the AIS group repeating the tasks at 1 and 2 years postoperatively. Results At baseline, the AIS group showed increased weightbearing asymmetry than the Control Group (p = .01). The AIS group showed improvements in volitional weight-shifting at 2 years over baseline (p \u3c .01). There was no effect of LIV on volitional weight-shifting by the second postoperative year. Conclusions Individuals with AIS have greater weightbearing asymmetry but improved volitional weight-shifting over typically developing controls. PSFI improves volitional weight-shifting beyond preoperative baseline but does not differ significantly by LIV

Effect of Surgical Fusion on Volitional Weight-Shifting in Individuals With Adolescent Idiopathic Scoliosis

Study Design Prospective. Objectives The goals of this study were to (1) evaluate the differences in weightbearing symmetry between individuals with adolescent idiopathic scoliosis (AIS) and typically developing controls; (2) observe the effect of posterior spinal fusion and instrumentation (PSFI) on volitional weight-shifting at 1 and 2 years postoperatively; and (3) evaluate whether lowest instrumented fusion level (ie, lowest instrumented vertebra [LIV]) in PSFI has an effect on volitional weight-shifting. Summary of Background Data Previous studies have conflicting findings with regard to the effect of scoliosis on postural control tasks as well as the effect of surgery. They have also noted an inconsistent effect of PSFI at different LIVs, with more distal LIVs exhibiting greater reductions in postoperative range of motion. Methods The study was designed with an AIS group of 41 patients (8 males and 33 females) with AIS who underwent PSFI, along with a Control Group of 24 age-matched typically developing participants (12 male and 12 female). Both groups performed postural control tasks (static balance and volitional weight-shifting), with the AIS group repeating the tasks at 1 and 2 years postoperatively. Results At baseline, the AIS group showed increased weightbearing asymmetry than the Control Group (p = .01). The AIS group showed improvements in volitional weight-shifting at 2 years over baseline (p \u3c .01). There was no effect of LIV on volitional weight-shifting by the second postoperative year. Conclusions Individuals with AIS have greater weightbearing asymmetry but improved volitional weight-shifting over typically developing controls. PSFI improves volitional weight-shifting beyond preoperative baseline but does not differ significantly by LIV

Bonding of 304L Stainless Steel to Cast Iron by Selective Laser Melting

While cast iron is widely used in industry, a major limitation is the weldability of a dissimilar material onto cast iron due to hot cracking as a result of lack of ductility from graphite flakes. Consequently, a significant amount of preheat is often employed to reduce the cooling rate in the fusion zone, which, however, may lead to distortion of the welded parts. A potential remedy could be the Selective Laser Melting (SLM) process, where only small melt pools are created and thus the overall energy input is reduced. The present paper describes an investigation of the SLM process to join 304L stainless steel with cast iron. In this study, 304L stainless steel particles ranging from 15-45 μm in size were melted on a grey cast iron substrate by the SLM process. Multiple sets of parameter values were chosen to test different energy densities on the tensile strength of the bond created. Subsequent characterization of the bonded area included energy dispersive spectroscopy (EDS) mapping for obtaining insight into the elemental diffusion, and metallography for visualization of the microstructure. A range of energy densities was identified for purposes of eliminating bond delamination and maximizing mechanical strength