Indiana University-Purdue University Indianapolis (IUPUI) / Voltage-gated sodium channels (VGSCs) are transmembrane proteins responsible for the initiation of action potentials in excitable tissues by selectively allowing Na+ to flow through the cell membrane. VGSC subtype Nav1.7 is highly expressed in nociceptive (pain-sensing) neurons. It has recently been shown that individuals lacking the Nav1.7 subtype do not experience pain but otherwise function normally. In addition, dysfunction of Nav1.7 caused by point mutations in the channel is involved in two inherited pain disorders, primary erythromelalgia (PE) and paroxysmal extreme pain disorder (PEPD). This indicates Nav1.7 is a very important component in nociception. The aims of this dissertation were to 1) investigate if the antipsychotic drug, trifluoperazine (TFP), could modulate Nav1.7 current; 2) examine changes in Nav1.7 properties produced by the PE mutation N395K including sensitivity to the local anesthetic (LA), lidocaine; and 3) determine how different inactivated conformations of Nav1.7 affect lidocaine inhibition on the channel using PEPD mutations (I1461T and T1464I) that alter transitions between the different inactivated configurations of Nav1.7. Standard whole-cell electrophysiology was used to determine electrophysiological and pharmacological changes in WT and mutant sodium currents. Results from this dissertation demonstrate 1) TFP inhibits Nav1.7 channels through the LA interaction site; 2) the N395K mutation alters electrophysiological properties of Nav1.7 and decreases channel sensitivity to the local anesthetic lidocaine; and 3) lidocaine stabilizes Nav1.7 in a configuration that decreases transition to the slow inactivated state of the channel. Overall, this dissertation answers important questions regarding the pharmacology of Nav1.7 and provides insight into the changes in Nav1.7 channel properties caused by point mutations that may contribute to abnormal pain sensations. The results of this dissertation on the function and pharmacology of the Nav1.7 channel are crucial to the understanding of pain pathophysiology and will provide insight for the advancement of pain management therapies.

Indiana University-Purdue University Indianapolis (IUPUI) / The  cardiac  voltage-­gated  sodium  channels  (Nav1.5)  play  a  specific  and   critical  role  in  regulating  cardiac  electrical  activity  by  initiating  and  propagating   action  potentials  in  the  heart.  The  association  between  Nav1.5  dysfunctions  and   generation  of  various  types  of  cardiac  arrhythmia  disease,  including  long-­QT3   and  Brugada  syndrome,  is  well  established.  Many  types  of  post-­translational   modifications  have  been  shown  to  regulate  Nav1.5  biophysical  properties,   including  phosphorylation,  glycosylation  and  ubiquitination.  However,  our   understanding  about  how  post-­translational  lipid  modification  affects  sodium   channel  function  and  cellular  excitability,  is  still  lacking.  The  goal  of  this   dissertation  is  to  characterize  Nav1.5  palmitoylation,  one  of  the  most  common   post-­translational  lipid  modification  and  its  role  in  regulating  Nav1.5  function  and   cardiac  excitability.     In  our  studies,  three  lines  of  biochemistry  evidence  were  shown  to  confirm   Nav1.5  palmitoylation  in  both  native  expression  background  and  heterologous   expression  system.  Moreover,  palmitoylation  of  Nav1.5  can  be  bidirectionally   regulated  using  2-­Br-­palmitate  and  palmitic  acid.  Our  results  also  demonstrated   that  enhanced  palmitoylation  in  both  cardiomyocytes  and  HEK293  cells   increases  sodium  channel  availability  and  late  sodium  current  activity,  leading  to   enhanced  cardiac  excitability  and  prolonged  action  potential  duration.  In  contrast,   blocking  palmitoylation  by  2-­Br-­palmitiate  increases  closed-­state  channel inactivation  and  reduces  myocyte  excitability.  Our  computer  simulation  results   confirmed  that  the  observed  modification  in  Nav1.5  gating  properties  by  protein   palmitoylation  are  adequate  for  the  alterations  in  cardiac  excitability.  Mutations  of   potential  palmitoylation  sites  predicted  by  CSS-­Palm  bioinformatics  tool  were   introduced  into  wild-­type  Nav1.5  constructs  using  site-­directed  mutagenesis.   Further  studies  revealed  four  cysteines  (C981,  C1176,  C1178,  C1179)  as   possible  Nav1.5  palmitoylation  sites.  In  particular,  a  mutation  of  one  of  these   sites(C981)  is  associated  with  cardiac  arrhythmia  disease.  Cysteine  to   phenylalanine  mutation  at  this  site  largely  enhances  of  channel  closed-­state   inactivation  and  ablates  sensitivity  to  depalmitoylation.  Therefore,  C981  might  be   the  most  important  site  that  regulates  Nav1.5  palmitoylation.  In  summary,  this   dissertation  research  identified  novel  post-­translational  modification  on  Nav1.5   and  revealed  important  details  behind  this  process.  Our  data  provides  new   insights  on  how  post-­translational  lipid  modification  alters  cardiomyocyte   excitability  and  its  potential  role  in  arrhythmogenesis.

Indiana University-Purdue University Indianapolis (IUPUI) / The voltage-gated sodium channels (VGSCs) are critical determinants of neuronal excitability. They set the threshold for action potential generation. The VGSC isoform Nav1.6 participates in various physiological processes and contributes to distinct pathological conditions, but how Nav1.6 function is differentially regulated in different cell types and subcellular locations is not clearly understood. Some VGSC isoforms are subject to S-palmitoylation and exhibit altered functional properties in different S-palmitoylation states. This dissertation investigates the role of S-palmitoylation in Nav1.6 regulation and explores the consequences of S-palmitoylation in modulating neuronal excitability in physiological and pathological conditions. The aims of this dissertation were to 1) provide biochemical and electrophysiological evidence of Nav1.6 regulation by S-palmitoylation and identify specific S-palmitoylation sites in Nav1.6 that are important for excitability modulation, 2) determine the biophysical consequences of epilepsy-associated mutations in Nav1.6 and employ computational models for excitability prediction and 3) test the modulating effects of S-palmitoylation on aberrant Nav1.6 activity incurred by epilepsy mutations. To address these aims, an acyl-biotin exchange assay was used for Spalmitoylation detection and whole-cell electrophysiology was used for channel characterization and excitability examination. The results demonstrate that 1) Nav1.6 is biochemically modified and functionally regulated by S-palmitoylation in an isoform- and site-specific manner and altered S-palmitoylation status of the channel results in substantial changes of neuronal excitability, 2) epilepsy associated Nav1.6 mutations affect different aspects of channel function, but may all converge to gain-of-function alterations with enhanced resurgent currents and increased neuronal excitability and 3) S-palmitoylation can target specific Nav1.6 properties and could possibly be used to rescue abnormal channel function in diseased conditions. Overall, this dissertation reveals S-palmitoylation as a new mechanism for Nav1.6 regulation. This knowledge is critical for understanding the potential role of S-palmitoylation in isoform-specific regulation for VGSCs and providing potential targets for the modulation of excitability disorders. / 2022-05-06

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第18584号 / 工博第3945号 / 新制||工||1606(附属図書館) / 31484 / 京都大学大学院工学研究科建築学専攻 / (主査)教授 神吉 紀世子, 教授 門内 輝行, 教授 髙田 光雄 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

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This research report is submitted to the Faculty of Humanities, University of the Witwatersrand, Johannesburg in partial fulfilment of the degree of Masters of Arts in Social and Psychological Research, 2017 / South Africa has undergone a number of social and geographical changes since the end of apartheid. This has drastically changed urban spaces, where we have seen the proliferation of Gated Communities (GCs). These spaces have come to signify how inequality has evolved from being an issue exclusively bound to race to one that now occurs within races and between classes. Therefore, in order to better understand inequality the current study considered the individuals who occupy spaces of privilege, but are not necessarily part of those spaces – such as security guards. It attempts to provide descriptions of inequality, moving away from a traditional macroeconomic understanding. This is done through a thematic analysis of interviews conducted with security guards from GCs. The analysis outlines the descriptions given by the security guards on their experience of working at GCs. Four superordinate themes were derived: Professionalism; Education and Knowledge; Commodification of Life; and Violence. From these themes it is clear that we cannot only interpret inequality from an income perspective as there are a number of psychosocial factors that are integrated into the construct of inequality. / XL2018

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