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Unbiased Analysis of the Functions and Interactions of Human Inflammasome Mutations

Spiker, Lindsey (2021) Unbiased Analysis of the Functions and Interactions of Human Inflammasome Mutations. Master's Thesis, University of Pittsburgh. (Unpublished)

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Abstract

Background: Gain-of-Function (GOF) inflammasome mutations helped define the field of autoinflammation. Genotype/phenotype correlations both found within and between mutated genes that cause inflammasomopathies. We sought to identify features unique to NLRC4 GOF mutations and to develop a system for the unbiased assessment of Protein-Protein Interactions (PPi) made/lost by introduction of NLRC4 or NLRP3 inflammasome mutations in a myelomonocyte cell line.
Methods: Part 1: Transduction of cell lines with WT or Mutant NLRC4 alone was preformed, followed by messenger RNA-sequencing and analysis. Part 2: Transduction of cell lines with WT or various of mutated forms of NLRP3 and NLRC4 was preformed to represent a diversity of clinical autoinflammatory phenotypes directly conjugated to the BirA biotinylase. Several experiments were conducted to assess biotinylation and confirm proper transduction. Interactome was established through the use of mass spectrometry.
Results: Part 1: The most differentially expressed genes were evaluated. Unbiased GSEA analysis identified a strong IFN-induced gene signature among the most differentially expressed genes. Part 2: Inborn errors of immunity may be an initiating factor in the hyperactivation of the inflammasome.
Public Health Relevance: The assessment of various mutations within the same gene or different genes thought to have similar functions may help to diagnose and investigate phenotypic outcomes of disease.
Conclusion: NLRC4 may directly induce IFN production, potentially through a non-canonical pathway. Screening for interacting partners may identify the mechanism by which NLRP3 causes most IL-1β mediated disease and NLRC4 drives both Interleukin-1β (IL-1β) and Interleukin-18 (IL-18) production.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Spiker, Lindseylspiker@pitt.edulspiker
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairCanna, Scottcannas@chop.edu
Committee MemberDemirci, F. Yesimfyd1@pitt.edu
Committee MemberUrban, Zsolturbanz@pitt.edu
Date: 12 May 2021
Date Type: Publication
Defense Date: 26 March 2021
Approval Date: 12 May 2021
Submission Date: 28 April 2021
Access Restriction: 1 year -- Restrict access to University of Pittsburgh for a period of 1 year.
Number of Pages: 47
Institution: University of Pittsburgh
Schools and Programs: Graduate School of Public Health > Human Genetics
Degree: MS - Master of Science
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: Monogenic autoinflammatory diseases (AIDs); pathogen-associated molecular patterns (PAMPs); damage-associated molecular patterns (DAMPs); Pattern Recognition Receptors (PRRs); Toll-like Receptor (TLR); Lipopolysaccharide (LPS); Gasdermin-D (GSDMD); Interleukin-1β (IL-1β); Interleukin-18 (IL-18); wildtype (WT); RNA-sequencing (RNA-seq); phorbol myristate acetate (PMA); gain-of-function (GOF); nucleotide-binding domain, leucine-rich repeat containing (NLR); NLR family CARD domain-containing protein 4 (NLRC4); NLR family pyrin domain containing 3 (NLRP3); Familial Cold-Induced Autoinflammatory Syndrome (FCAS); Macrophage Activation Syndrome (MAS); mitochondrial reactive oxygen species (mtROS); NLR family, apoptosis inhibitory proteins (NAIPs); Muckle Wells Syndrome (MWS); autoinflammation with infantile enterocolitis (AIEFC); neonatal onset multisystem inflammatory disease (NOMID); proximity labeling (PL); BirA miniTurbo (BirAMT); Human acute monocytic leukemia cell line (THP-1); protein-protein interactions (PPI); co-immunoprecipitation (Co-IP); Gene Set Enrichment Analysis (GSEA); transcripts per kilobase million (TPM); interferon-response genes (IRG); SV40 large T antigen (HEK293T); interferon-α (IFNα); nominal molecular weight limit (NMWL); principal component analysis (PCA); empty vector (EV); Brilliant Violet 421 (BV421-A)
Related URLs:
Date Deposited: 12 May 2021 19:35
Last Modified: 12 May 2021 19:35
URI: http://d-scholarship.pitt.edu/id/eprint/40847

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