Thesis (MScMedSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Posttraumatic stress disorder (PTSD) is a multifactorial disorder, with substantial evidence for a genetic contribution. Although genetic association studies have been conducted to identify vulnerability factors in PTSD, the results remain largely inconsistent. Identifying ligands of proteins that are involved in the aetiology of PTSD represents a means of delineating the network of interactions that may play a role in the development of the disorder. Numerous animal studies have identified the Wolframin protein (WFS1) as a putative biomarker for the development of PTSD. However, the function of WFS1 has not yet been fully elucidated. The aim of the present investigation was to identify proteins that interact with the N-terminal domain of WFS1, in order to possibly elucidate the function of the protein, and to subsequently hypothesise on the role that WFS1 may play in the development of PTSD. Yeast two-hybrid (Y2H) methodology was used to identify putative ligands of the N-terminal domain of WFS1 (amino acids 1-300) by screening a human adult brain complementary DNA (cDNA) library. Successive selection stages reduced the number of putative WFS1 N-terminal ligand-containing colonies (preys) from 878 to three. Putative ligands were sequenced and indentified by BLAST-search. Four preys were excluded because they were either out of frame with the vector or the protein they encoded occurred in a subcellular location that was not compatible with the location of the N-terminal domain of WFS1. An interesting putative ligand was identified as carboxypeptidase E (CPE). Colocalisation analyses verified that CPE colocalises with WFS1 in rat hypothalamic GT1-7 cells. Coimmunoprecipitation (Co-IP) further verified a direct interaction between WFS1 and CPE in rat hypothalamic GT1-7 cells, providing conclusive evidence that WFS1 and CPE interact. Both WFS1 and CPE are upregulated in response to fear and both are localised to the secretory granules of the regulated secretory pathway. WFS1 has been detected in both the ER and secretory granules it seems to play an important role in protein biosynthesis, modification, folding, trafficking and the regulation of calcium homeostasis. CPE is involved in neuropeptide processing and trafficking of secreted proteins. The interaction between CPE and WFS1 may thus serve to facilitate an optimal environment in which neuropeptides can be processed and secreted. / AFRIKAANSE OPSOMMING: Posttraumatiese stresversteuring (PTSV) is 'n multifaktoriese siekte, met aansienlike bewyse vir 'n genetiese bydrae. Hoewel genetiese assosiasie-studies uitgevoer word om kwesbaarheidsfaktore in PTSV te identifiseer, is die resultate grootliks teenstrydig. Identifiseering van ligande van proteїene wat betrokke is in die etiologie van PTSV dien as middel om die netwerk van interaksies wat ń moontlike rol in die ontwikkeling van die versteuring kan speel, te oudersoek talle diere studies het die Wolframin proteien (WFS1) geїdentifiseer as 'n moontlike biomerker vir die ontwikkeling van PTSV. Die funksie van WFS1 is egter nog nie ten volle beskryf nie. Die doel van die huidige studie was om proteїene wat interaksie met die N-terminale domein van WFS1 her te identifiseer, om sodoende die funksie van die proteїen uit te lig, en daardeur die rol wat WFS1 kan speel in die ontwikkeling van PTSV te bepaal. Die gis twee-hibried metodologie is gebruik om moontlike ligande van die N-terminale domein van WFS1 te identifiseer, deur die sifting van 'n mens volwasse brein komplementêre DNS biblioteek. Opeenvolgende seleksie stappe het die aantal moontlike WFS1 N-terminale ligand wat moontlike prooi kolonies bevat van 878 tot en met ses verminder. Die DNS volgorde van die moontlike prooi-plasmiede is bepaal en geїdentifiseer deur die BLAST soek-engin. Vier prooi-plasmiede is uitgesluit omdat hulle of nie in die korrekte lees-raam in die vektor was nie of die subsellulêre ligging van die proteїen wat uitgedrukword is nie versoenbaar met die N-terminale domein van WFS1. 'n Interessante moontlike ligand is geїdentifiseer as Karboxypeptidase E (CPE). Ko-lokalisering ontleding bevestig dat CPE ko-lokaliseer met WFS1 in rot hipotalamiese selle (GT1-7). Ko-immunopresipitasie (Ko-IP) toon verder 'n direkte interaksie tussen WFS1 and CPE in rot GT1-7 selle. Wat dus bewys dat WFS1 en CPE wel met mekaar 'n interaksie het. Beide WFS1 en CPE toon 'n verhoogde uitdrukking in respons tot ń vrees-situasie. Beide van hierdie proteїene kom voor in die sekretoriese korrels van die gereguleerde sekretoriese pad. Die WFS1 proteien word bevind in die endoplasmiese retikulum (ER) van die sel, waar dit verantwoordelik is vir proteien biosintese, modifikasie, vouing, vervoer en die reguleering van kalsium homeostase. Die CPE proteїen is verantwoordelik vir die proseseering van neuropeptiede en die vervoer van uitgeskiede proteїene. Dus kan die interaksie tussen CPE en WFS1 dien om 'n optimale omgewing te skep waarin neuropeptiede geproseseer en uitgeskei kan word. / The National Research Foundation (NRF), the Harry Crossley Foundation and the Medical Research Council (MRC)