The mechanism of allosteric coupling between the external ligand-binding domain and the internal signaling domain of bacterial chemoreceptors is poorly understood. Genetic, biochemical, and biophysical evidence suggests that transmembrane helix 2 (TM2) undergoes a piston-like displacement of approximately 1-3 Angstroms toward the cytoplasm upon the binding of aspartate to the Tar receptor. The signal is then transmitted to the cytoplasmic signaling domain via the HAMP domain, a conserved motif found in all methyl-accepting chemotaxis proteins (MCPs) and most histidine protein kinases (HPKs). HAMP forms a parallel four-helix bundle consisting of a dimer of two amphipathic helices (AS1 and AS2) connected by a flexible linker. The MLLT sequence between residues Arg-214, at the end of TM2, and the conserved residue Pro-219, at the beginning of AS1 of the HAMP domain (the TM2-HAMP junction), is predicted to be able to form a helical extension of TM2. We hypothesized that perturbing the native secondary structure and/or the length of the TM2-HAMP junction would disrupt the ability of HAMP to communicate the input signal from TM2 to the kinase-control domain. To test this hypothesis, we designed two experiments. First, constructs were made in which 1 to 3 Gly residues were inserted between T218 and P219. Second, Tar variants were constructed in which 1 to 9 Gly residues were inserted between R214 and P219. The results suggest that increasing the length and flexibility of the TM2-HAMP connection tends to uncouple signal propagation between the TM2 and the HAMP elements and suggests that HAMP alone causes an inhibitory effect on the cytoplasmic signaling domain. To determine whether the predicted helical register of the MLLT sequence is an important component of the propagation of the transmembrane signal from TM2 to the HAMP domain, we added and subtracted helical residues to the MLLT sequence. The results suggest that helical register and length of the TM2-HAMP junction are essential for optimal receptor function.

Within the context of the 2007/2008 subprime crisis, we examine the impact and relevance of previous ex ante residential mortgage research to prevent and mitigate mortgage default and develop a general taxonomy of (ex-ante) government intervention. We continue by investigating the various forms of (ex-post) default mitigation options prevalent in the United States and innovatively categorise them into one of three categories based on whether the mortgage contract has been renegotiated. We examine the strategic renegotiation option, alongside the more traditional ruthless or strategic default option, for a US owner-occupier residential mortgage holder and non-owner occupier residential mortgage holder uniquely deriving closed form solutions to calculate the optimal ex-ante LTV (Loan to Value) and ex-post exercise moment where a heterogeneous borrower exercises a renegotiation option. We finally relax the perpetual ability to pay assumption underlying strategic default and negotiation to investigate default and negotiation triggered by both an inability to pay and unwillingness to pay. We simulate the overall effect of (institutionalised) renegotiation under these two assumptions, comparing the effect of a stylised HAMP program on the overall default, foreclosure and prepayment probabilities of owner occupied residential homeowners in the absence of a HAMP program. We conclude that the traditional option theoretic assumption that homeowners "can always pay" is a very strong assumption the consequence of which could induce relevant policy makers to incorrectly interpret and act on conclusions and recommendations flowing from historical option theoretic mortgage research.

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Thesis (MSc (Genetics))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Oesophageal cancer (OC) is the sixth leading cause of cancer related deaths in the world with approximately 300 000 new cases reported each year. OC may be characterized into two forms with 90% of cases presenting as squamous-cell carcinoma (SCC) and the remaining 10% as adenocarcinoma (ADC). Several factors have been attributed to the development of OC, including oesphageal injury and/or irritation, chronic inflammation and excess iron associated with enhanced tumour growth. The HAMP gene codes for a 25 amino-acid protein found to be primarily expressed in the liver and crucial to regulation of bodily iron status. Defects occurring in the HAMP gene could therefore lead to the dysregulation of the gene, resulting in an iron overload status. Iron overload is a previously described risk factor in the development of various cancers, including OC, and therefore the aim of this study was to investigate whether dysregulation of the HAMP gene may be involved in the cancer phenotype exhibition. The study cohort comprised of 48 unrelated patients presenting with SCC and a control group of 51 healthy, unrelated population-matched individuals. Mutation detection techniques included polymerase chain reaction (PCR) amplification, heteroduplex single-stranded conformation polymorphism (HEX-SSCP) analysis and bi-directional semi-automated DNA sequencing analysis. Screening of the 5’ regulatory region (5’UTR) of the HAMP gene revealed one known (-582A/G) and two novel (-188C/T and -429G/T) variants with the -429G/T variant showing statistically significant reduction in expression in patients relative to controls. Iron parameters were correlated between patient and control cohorts, as well as for variant presence and absence within individuals. Luciferase reporter constructs were used to investigate the functional implications of the presence of a variant on HAMP gene expression, and how these results correlated to the iron parameter statistics obtained. Luciferase reporter assay results indicated the -188C/T and -429G/T variants to result in under-, and the -582A/G variant to result in over-expression at the basal level, relative to the respective wild-type sequence constructs. Correlation of the luciferase data with the iron parameter statistics, indicate the -429G/T variant to be coupled to significantly higher levels of ferritin and C-reactive protein (CRP) and significantly lower levels of serum-iron and transferrin when compared to individuals without the variant. Considering only the patient group, the presence of the -188C/T and -429G/T variants were coupled to significantly lower levels of transferrin in patients with either variant, compared to patients without. The variants found within the HAMP promoter region are therefore able to alter gene regulation to an extent where iron parameters deviate between healthy and OC afflicted individuals, and also between patients with and without a variant. This dysregulation in iron homeostasis may play a role in the development and/ or progression of OC. Characterisation of the 5’ UTR of the HAMP gene may contribute to linking iron regulation to the establishment of an effective screening program, facilitating the early detection of OC. / AFRIKAANSE OPSOMMING: Slukdermkanker (SK) is die sesde grootste oorsaak van kanker-verwante sterftes in die wêreld, met sowat 300 000 nuwe gevalle wat aangemeld word elke jaar. SK kan geklassifiseer word in twee vorme, waar 90% van die gevalle plaveisel-selkarsinoom (SSC) vorm en die oorblywende 10%, adenokarsinoom (ADC). Verskeie faktore word toegeskryf aan die ontwikkeling van SK, insluitend slukderm beserings en/ of irritasie, chroniese inflammasie en oormatige ystervlakke wat geassosieer word met verhoogde gewasgroei. Die HAMP geen kodeer vir 'n 25 aminosuur proteïen wat hoofsaaklik in die lewer uitgedruk word en noodsaaklik is vir die regulering van ystervlakke in die liggaam. Defekte wat in die HAMP geen voorkom kan dus die onreëlmatige regulering van die geen tot gevolg hê, wat lei tot yster-oorlading. Yster-oorlading is voorheen beskryf as ‘n risiko faktor in die ontwikkeling van verskillende vorme van kanker, insluitend SK en gevolglik was die doel van hierdie studie om te bepaal of die wanregulering van die HAMP geen betrokke mag wees by die uitdrukking van die kanker fenotipe. Die studiepopulasie het bestaan uit 48 onverwante pasiënte met SSC en ‘n kontrole-groep van 51 gesonde, onverwante soortgelyke individue. Die mutasie opsporingstegnieke wat gebruik is, het polimerase kettingreaksie (PKR) amplifisering, heterodupleks enkelstring-konformasie polimorfisme (HEX-SSCP) analise en bidireksionele semi-outomatiese DNS volgordebepaling-analise van die geïdentifiseerde variante ingesluit. Sifting van die 5’ regulerende area (5'UTR) van die HAMP geen het een bekende (-582A/G) en twee nuwe (-188C/T en -429G/T) variante opgelewer, met die -429G/T variant wat statisties beduidend onderdruk is in pasiënt uitdrukkings vlakke relatief tot 'n gesonde kontole-groep. Yster-parameters van alle pasiënt en kontole individue is gekorreleerd tussen pasiënt en kontrole groepe, sowel as vir teenwoordigheid of afwesigheid van variante in elke individu. Luciferase verklikker konstrukte is gebruik om die funksionele implikasies van die teenwoordigheid van ‘n variant op HAMP geenuitdrukking te ondersoek, en hierdie resultate te korreleer met yster-parameter statistieke wat verkry is. Luciferase verklikkertoetse dui aan dat die -188C/T en -429G/T variante tot verminderde, en die -582A/G variant lei tot die verhoogte uitdrukking op die basale vlak lei, relatief tot die onderskeie wilde-tipe konstukte. Korrelasie van die luciferase data met die yster-parameter statistieke, dui aan dat die -429G/T-variant gekoppel is aan aansienlik hoër vlakke van feritien en C-reaktiewe proteïen (CRP) en beduidend laer vlakke van serum-yster en transferrien in vergelyking is met individue sonder die variant. Met oorweging van slegs die pasiënt-groep, is die teenwoordigheid van die -188C/T en -429G/T variante beduidend gekoppel aan laer vlakke van transferrien in pasiënte met die variant, in vergelyking met pasiënte daarsonder. Variante binne die HAMP promotor is dus in staat om geenregulasie te verander tot so 'n mate dat die yster-parameters afwyk tussen gesonde en SK geaffekteerde individue, sowel as tussen pasiënte met en sonder ’n variant. Hierdie wanregulering in yster homeostase kan 'n rol speel in die ontwikkeling en/ of die progressie van SK. Karakterisering van die 5’ regulerende area van die HAMP geen kan grootliks bydra om ysterregulasie te verbind met die implementering van ‘n effektiewe siftingsprogram, en sodoende die vroeë opsporing van SK fasiliteer.

The photosensitive unit triggering the negative phototaxis in the haloarchaeum Natronomonas pharaonis consists of the receptor sensory rhodopsin II (NpSRII) and its cognate transducer (NpHtrII) in a 2:2 stoichiometry. Upon light excitation, a structural rearrangement in the receptor initiates a displacement/rotation of the transducer helix TM2, which can be considered as starting event for the signal transduction. This signal is further transmitted to the cytoplasmic signaling domain through the signal transduction unit comprising two HAMP domains.Structural information already exists for the transmembrane region of this complex (crystal structure) as well as for the rod shaped cytoplasmic part of NpHtrII due to its high homologies with chemoreceptors. Moreover, the solution NMR structure of the isolated HAMP domain from A. fulgidus recently obtained shows a homodimeric, four-helical, parallel coiled-coil with an unusual interhelical packing, that is thought to propagate a signal by virtue of concerted helix rotations. Here, an electron paramagnetic resonance (EPR) investigation of site-directed spin labeled transducers in the NpSRII/NpHtrII complex has been carried out for structural and functional elucidation of the N. pharaonis HAMP. For this purpose, cw as well as pulse EPR techniques have been used in terms of mobility, accessibility and intra-transducer dimer distance analyses. Conformational changes induced by environmental inputs, namely salt, temperature and pH, give insight into the two-state equilibrium existing between a highly dynamic (dHAMP) and a more compact (cHAMP) conformation of this linker region.