Modelling of TLR4 and JAK/STAT Signalling Pathways and Protein-Protein Interaction Studies to Examine the Pathophysiology of Sepsis

In this thesis, the medical condition of sepsis is considered at molecular level (signallingpathways)usingcomputationalsystems-levelapproachesinordertogetinsightsintothemechanismofdiseaseprogression.Sepsisisthepathologicalcondition provokedduetothepresenceofbacterialendotoxininthebloodstream.Subsequently Toll like receptors (TLR)4 and JAK/STAT signalling pathways attempt to reduce the pathogen burden by inducing pro- and anti-inflammatory innate immune responses. However, in some instances, an overwhelmed immune system could not properly regulate the balance between infection and inflammation that may ultimately lead to organdamageandconsequentlytodeath.Inrecentyears,therehasbeenanincreasing amount of literature on the pattern of pro- and anti-inflammatory response elicited during sepsis, though; there has been a little agreement on the roles of pro- and antiinflammatory cytokines (AiCyts) in sepsis. This study mainly aims to address the controversy behind roles of pro- and anti-inflammatory cytokines in sepsis by modellingthesignallingpathwayofTLR4andoneoftheconnectedsignallingcascadesof JAK/STAT using qualitative modelling approach introduced by René Thomas'. The possible system dynamics of TLR4-JAK/STAT signalling pathways are produced for two medical conditions i.e. non-sepsis (type of infections that generally do not cause sepsis) and sepsis along with perturbations in these two cases. As a result, recurrent induction and inactivation of pro-inflammatory cytokines is found as the basic featureassociatedwithsepsis.BesidesAiCyts,IFN-? andSOCS-1arefoundtomediate down-regulationofpro-inflammatorycytokinesatdifferentstagesofsignallingwhich cause variation of pro-inflammatory cytokines levels. It is observed that intervention in IFN-? mediated down-regulation of pro-inflammatory cytokines at earlier stages of system dynamics,while intervening the SOCS-1 mediated down-regulation of proinflammatory cytokines at later stages ensures hyper-inflammatory condition. On the other hand, interventions in TLR4, NF?B (transcription factor involved in the TLR4\ud signalling pathway) and JAK/STAT signalling are good choices for supporting the anti-inflammatoryimmuneresponses.Thusonlypossibleprotein-proteininteractions involvedintheinitialdownstreaminteractionsofTLR4signallingarestudiedinorder to predict a more appropriate target in these interactions. Previous studies indicated thatMyD88adaptor-likeprotein(MAL)isanendogenousadaptorproteinrecognized as an important protein involved in the induction of TLR4 mediated downstream signalling pathway. Moreover, it has also been demonstrated that BTK and PKC? phosphorylate MAL (positions Tyr86 and Tyr106) which ultimately activates MAL. ThusthemodellingofPKC? andprotein-proteininteractionsofbothBTKandPKC? with MAL is performed in order to explore their competitive interaction. Molecular docking and physicochemical analysis reveals that PKC? may phosphorylate only Tyr106ofMAL,whileBTKmayphosphorylateMALatbothpositions.Interestingly, the charge and hydrophobicity at interfaces of PKC? and BTK are found different in nature yet well-compatible with the individual positions of Tyr86 and Tyr106 of MAL. The mostprominentfindings emergedfromthis analysis isthatposition Tyr86 of MAL may explicitly be phosphorylated by BTK, while position Tyr106 of MAL may be phosphorylated by the competing interest of both PKC? and BTK. In conclusion, this thesis will enhance our understanding about the signalling and protein-protein interactions involved in sepsis which will contribute to the development of drugs and vaccines against the medical condition of sepsis