Proteins are among the most abundant biological macromolecules. The cellular machinery is coupled to exact structural shape and properties of the more than 100 000 different proteins. Still, proteins can sometimes completely change their character and as a result trigger neuro degenerative disease. Exactly what happens is yet poorly understood but misfolding and aggregation leading to toxic gain of function is probable causes, i.e. the protein adopts new noxious properties. In 1993 the protein superoxide dismutase (SOD) was found to be associated with the neuro degenerative disease ALS. Up to date more than 100 mutations in SOD have been associated with ALS. However, the mutations are scattered all over the structure and no common denominator for the disease mechanism has been found. This work has been focused on the molecular mechanism of the toxic gain-of - function of mutant SOD from the perspective of protein folding and structural stability. To facilitate the studies of SOD and its ALS associated mutations, an expression system resulting in increased copper content was developed. Coexpression with the copper chaperone for superoxide dismutase (yCCS) leads to increased expression levels, especially for the destabilised ALS mutants. Through thermodynamic studies, I show that with the exception of the most disruptive mutations the holo protein is only marginally destabilised, whereas all mutations show a pronounced destabilisation on the apo protein. Kinetic studies suggest further that the dimeric apoSOD folds via a three-state process where the dimerisation proceeds via a marginally stable monomer. The apoSOD monomer folds by a two-state process. The disulphide bond is not critical for the folding of the apoSOD monomer although it contributes significantly to its stability. Interestingly, in the absence of metals, reduction of the disulphide bond prevents the formation of the dimer. A mutation can affect the protein stability in various ways: either from destabilisation of the monomer (case 1), weakening of the dimer interface (case 2) or, in the worst case, from a combination of both (case 1+2). Thus, therapeutic strategies to prevent the noxious effects of mutant SOD must include both mechanisms. An important finding in this study is that we can see a correlation between the stability for each mutation and the mean survival time. This could be an opening in the development of therapeutic substances that counteract the defect in SOD upon mutation.

Deficient concrete structures are serious danger in seismic zones. In order to minimize economical and human loss, these structures should be retrofitted. Selecting suitable retrofitting schemes requires detailed investigation of these systems. Considering these facts, this study aims to calibrate analytical models of systems with chevron braces and internal steel frames / and evaluate their seismic performances. First, analytical models of the frames with braces and internal steel frames were prepared and then their responses were compared with cyclic responses of experimental studies. Results of these models were used to determine performance limits by the methods proposed by TEC2007 and ASCE/SEI-41. Then, calibrated models were employed for time history analyses with various scales of Duzce ground motion and analytical results were compared with experimental findings. Seismic performance of these systems was also evaluated by using aforementioned codes. Finally, evaluated retrofitting schemes were applied to a 4-story 3-bay reinforced concrete frame that was obtained from an existing deficient structure and effectiveness of applied retrofitting schemes was investigated in detail.

This thesis examines the seismic internal retrofitting of existing deficient reinforced concrete (RC) structures by using structural steel members. Both experimental and numerical studies were performed. The strengthening methods utilized with the scope of this work are chevron braces, internal steel frames (ISFs), X-braces and column with shear plate. For this purpose, thirteen strengthened and two as built reference one bay one story portal frame specimens having 1/3 scales were tested under constant gravity load and increasing cyclic lateral displacement excursions. In addition, two &frac12 / scaled three bay-two story frame specimens strengthened with chevron brace and ISF were tested by employing continuous pseudo dynamic testing methods. The test results indicated that the cyclic performance of the Xbrace and column with shear plate assemblage technique were unsatisfactory. On the other hand, both chevron brace and ISF had acceptable cyclic performance and these two techniques were found to be candidate solutions for seismic retrofitting of deficient RC structures. The numerical simulations by conducting nonlinear static and dynamic analysis were used to estimate performance limits of the RC frame and steel members. Suggested strengthening approaches, chevron brace and ISF, were also employed to an existing five story case study RC building to demonstrate the performance efficiency. Finally, design approaches by using existing strengthening guidelines in Turkish Earthquake Code and ASCE/SEI 41 (2007) documents were suggested.

Thesis submitted in partial fulfilment of the requirements for the degree Master of Technology: Chemical Engineering in the Faculty of Engineering at the Cape Peninsula University of Technology 2013 / In South Africa, the Department of Environmental Affairs (DEA) under the National Environmental Management Act, 1998 (Act 107 of 1998) (“NEMA”) sets out a series of environmental management principles that apply to the interpretation and application of all legislation that may affect the environment. Since 1998, various specific environmental statutes that fall under the NEMA framework have been promulgated, including the National Environmental Management: Air Quality Act, Act 39 of 2004 (NEM: AQA).NEM:AQA provides norms and standards for all technical aspects of air quality management. The National Framework (Sections 7 and 8 of NEM:AQA) must provide mechanisms, systems and procedures to promote holistic and integrated air quality management through pollution prevention and minimisation at source, and through impact management with respect to the receiving environment, from local scale to international issues. Among other measures, the NEM:AQA (Section 9) requires the establishment of Ambient Air Quality Standards and Emission Standards. These standards were promulgated in December 2009 and March 2010 respectively. Air quality monitoring stations, which sample and analyse pollutant concentrations continuously, are a common method of assessing air quality in a region. But a few continuous monitors located in source given region or airshed are inadequate for assessing compliance with ambient air quality standards – they are only able to monitor concentrations at a fixed site, not through the entire region of impact. In contrast, the ambient air quality standards are applicable everywhere. Air quality models estimate ground level ambient concentrations throughout the modelling domain, and in principle (subject to proper validation) provide better estimates of area-wide concentrations and hence the basis for assessing compliance with air quality standards. The United States Environmental Protection Agency (US EPA) approved atmospheric air dispersion models AERMOD and CALPUFF were used in this thesis to predict the ground level concentrations of SO2 emitted from Chevron Refinery (Cape Town), for the year 2010. The modelling is validated by comparing measured ambient concentrations with modelled concentrations. The results showed AERMOD-modelled annual average values for 2010, based on refinery emissions only, are in good agreement with monitored values at the Table View and Bothasig sites, predicting the monitored values by -11% and +17% respectively. The 24-hr average values similarly are in good agreement with monitored values, on average over-predicting by 9% at Table View, although the fit of the day-to-day modelled vs monitored values is comparatively poor (R2=0.32); at the Bothasig site the corresponding values are - 36% and R2= 0.089. The AERMOD-modelled isopleths imply that the 2010 annual average concentrations exceeded the South African Standard of 50 μg/m3 in a small area in the immediate vicinity of the refinery. The hourly and 24-hourly average standard concentrations of 350μg/m3 and 125μg/m3 respectively are exceeded in significantly larger areas. The allowable exceedences for hourly and 24-hourly averages are also exceeded, implying that the hourly and 24-hourly standards were exceeded. CALPUFF-modelled average values for 2010, based on refinery emissions only, are in comparatively poor agreement with monitored values at the Table View and Bothasig sites, under-predicting the monitored values by -20% and -61% respectively. Since the AERMOD-modelled concentrations are in far better agreement with monitored concentrations, only AERMOD was used for further analysis. The Emission Standards promulgated in March 2010 included emission limit values for sulphur dioxide emitted from oil refineries. If the actual 2010 emission rates were adjusted downwards to match the emission standards (to be complied with from 1 April 2015), AERMOD modelling indicates that the annual, 24-hourly and hourly Ambient Air Quality Standards would not be exceeded. Based on this case study, the current Emission Standard for SO2 emissions from existing crude oil refineries is therefore coherent with the Ambient Air Quality Standards. Regulatory air dispersion modelling practices in South Africa are being standardised for model applications regulatory purposes and to ensure that dispersion modelling practices are undertaken in a compatible form to ensure that results from one dispersion model study can be compared directly to those from another. In this study both AERMOD and CALPUFF modelling complied with the draft South African guidelines for Air Quality Modelling, yet the CALPUFF- modelled outputs differed significantly from the monitored values. This emphasizes the importance of the inclusion of modelling validation in guidelines for modelling for regulatory purposes. The 2012 draft regulation should be amended to make validation of regulatory dispersion modelling compulsory rather than optional as per the draft.

No description available.

The chevron skink (Oligosoma homalonotum) is one of the largest, yet least observed skink species in New Zealand. The species was thought to have once been widespread in Northern New Zealand, however currently it is only found on Great Barrier and Little Barrier Islands. Great Barrier Island is the apparent stronghold for the species although it appears to be in decline there, despite a net increase in habitat. Recent studies have increased the understanding of the general ecology of the species, however little is known about the threats to the survival of this species. This study had two main objectives; the first was to establish if rats are a threat to chevron skinks, and the second was to increase current knowledge of the species ecology. The research was undertaken in an area of extensive rodent control (Glenfern Sanctuary) and an adjacent unmanaged reserve in Port Fitzroy, on Great Barrier Island in 2008. The first objective of this study involved confirming that rat densities in the treatment (Glenfern Sanctuary) were sufficiently different to allow meaningful comparisons of chevron skink population characteristics between sites. This was achieved by determining absolute rat densities using Zippin’s removal method at four sites, and correlating these with a relative abundance measure (tracking rates) to give confidence in the observed trends. Rat densities were high (1.94 - 3.00 rats ha-1) in the control, and low (0.00 and 0.06 rats ha-1) in the treatment sites, and these correlated well with tracking rates. In light of these clear differences between the treatment and control, the population structure and condition of chevron skinks were compared between sites. The population structure showed erosion of juvenile and sub-adult size categories, which indicated differences in vulnerabilities between size categories. Physical evidence of failed rat predation was also observed in adult skinks in the unmanaged control, which confirmed that rats were interacting with chevron skinks. Although the adults survived the attacks they suffered injuries including eye damage, punctures, cuts and tail loss. Smaller skinks would be unlikely to survive such attacks due to the severity and scale of the injuries, supporting the assertions of the population structure that smaller skinks may be more vulnerable than adults. The extent of tail loss was converted to a condition index to determine if failed rat predation was more widespread in the population, than was observed by conspicuous injuries. This condition index (body-tail condition index) was stable through all size categories in the treatment, but significantly reduced in adults in the unmanaged sites. That there was no reduction in the condition of smaller skinks in the unmanaged control sites despite high rat densities suggests that interactions between rats and smaller skinks are fatal, and thus not represented in the data. Nine chevron skinks were radio-tracked to determine habitat use, home range and ranging behaviour. Habitat use of chevron skinks was similar to a previous study and demonstrated that trees, crevices and logs were important refuge sites. Chevron skinks were more likely to be found at sites with trees, crevices and debris dams. Chevron skink home ranges indicated that adults moved further away from streams than previously anticipated at this time of year, and skinks demonstrated site fidelity. There was also overlap in home ranges between individuals, and skinks with overlapping home ranges shared common refuges. During flooding events, chevron skinks exhibited an arboreal response that appears to be a behaviour specific to stream associated animals, which allows them to avoid being taken by floodwaters.

The chevron skink (Oligosoma homalonotum) is one of the largest, yet least observed skink species in New Zealand. The species was thought to have once been widespread in Northern New Zealand, however currently it is only found on Great Barrier and Little Barrier Islands. Great Barrier Island is the apparent stronghold for the species although it appears to be in decline there, despite a net increase in habitat. Recent studies have increased the understanding of the general ecology of the species, however little is known about the threats to the survival of this species. This study had two main objectives; the first was to establish if rats are a threat to chevron skinks, and the second was to increase current knowledge of the species ecology. The research was undertaken in an area of extensive rodent control (Glenfern Sanctuary) and an adjacent unmanaged reserve in Port Fitzroy, on Great Barrier Island in 2008. The first objective of this study involved confirming that rat densities in the treatment (Glenfern Sanctuary) were sufficiently different to allow meaningful comparisons of chevron skink population characteristics between sites. This was achieved by determining absolute rat densities using Zippin’s removal method at four sites, and correlating these with a relative abundance measure (tracking rates) to give confidence in the observed trends. Rat densities were high (1.94 - 3.00 rats ha-1) in the control, and low (0.00 and 0.06 rats ha-1) in the treatment sites, and these correlated well with tracking rates. In light of these clear differences between the treatment and control, the population structure and condition of chevron skinks were compared between sites. The population structure showed erosion of juvenile and sub-adult size categories, which indicated differences in vulnerabilities between size categories. Physical evidence of failed rat predation was also observed in adult skinks in the unmanaged control, which confirmed that rats were interacting with chevron skinks. Although the adults survived the attacks they suffered injuries including eye damage, punctures, cuts and tail loss. Smaller skinks would be unlikely to survive such attacks due to the severity and scale of the injuries, supporting the assertions of the population structure that smaller skinks may be more vulnerable than adults. The extent of tail loss was converted to a condition index to determine if failed rat predation was more widespread in the population, than was observed by conspicuous injuries. This condition index (body-tail condition index) was stable through all size categories in the treatment, but significantly reduced in adults in the unmanaged sites. That there was no reduction in the condition of smaller skinks in the unmanaged control sites despite high rat densities suggests that interactions between rats and smaller skinks are fatal, and thus not represented in the data. Nine chevron skinks were radio-tracked to determine habitat use, home range and ranging behaviour. Habitat use of chevron skinks was similar to a previous study and demonstrated that trees, crevices and logs were important refuge sites. Chevron skinks were more likely to be found at sites with trees, crevices and debris dams. Chevron skink home ranges indicated that adults moved further away from streams than previously anticipated at this time of year, and skinks demonstrated site fidelity. There was also overlap in home ranges between individuals, and skinks with overlapping home ranges shared common refuges. During flooding events, chevron skinks exhibited an arboreal response that appears to be a behaviour specific to stream associated animals, which allows them to avoid being taken by floodwaters.

<p>Proteins are among the most complex molecules in the cell and they play a major role in life itself. The complexity is not restricted to just structure and function, but also embraces the protein folding reaction. Within the field of protein folding, the focus of this thesis is on the features of the folding transition state in terms of growing contacts, common nucleation motifs and the contribution of charged residues to stability and folding kinetics. </p><p>During the resent decade, the importance of a certain residue in structure formation has been deduced from Φ-value analysis. As a complement to Φ-value analysis, I present how scatter in a Hammond plot is related to site-specific information of contact formation, Φ´(β^TS), and this new formalism was experimentally tested on the protein L23. The results show that the contacts with highest Φ growth at the barrier top were distributed like a second layer outside the folding nucleus. This contact layer is the critical interactions needed to be formed to overcome the entropic barrier. </p><p>Furthermore, the nature of the folding nucleus has been shown to be very similar among proteins with homologous structures and, in the split β-α-β family the proteins favour a two-strand-helix motif. Here I show that the two-strand-helix motif is also present in the ribosomal protein S6 from<i> A. aeolicus</i> even though the nucleation and core composition of this protein differ from other related structure-homologues. </p><p>In contrast to nucleation and contact growth, which are events driven by the hydrophobic effect, my most recent work is focused on electrostatic effects. By pH titration and protein engineering the charge content of S6 from <i>T. thermophilus</i> was altered and the results show that the charged groups at the protein surface might not be crucial for protein stability but, indeed, have impact on folding kinetics. Furthermore, by site-specific removal of all acidic groups the entire pH dependence of protein stability was depleted.</p>

Proteins are among the most complex molecules in the cell and they play a major role in life itself. The complexity is not restricted to just structure and function, but also embraces the protein folding reaction. Within the field of protein folding, the focus of this thesis is on the features of the folding transition state in terms of growing contacts, common nucleation motifs and the contribution of charged residues to stability and folding kinetics. During the resent decade, the importance of a certain residue in structure formation has been deduced from Φ-value analysis. As a complement to Φ-value analysis, I present how scatter in a Hammond plot is related to site-specific information of contact formation, Φ´(βTS), and this new formalism was experimentally tested on the protein L23. The results show that the contacts with highest Φ growth at the barrier top were distributed like a second layer outside the folding nucleus. This contact layer is the critical interactions needed to be formed to overcome the entropic barrier. Furthermore, the nature of the folding nucleus has been shown to be very similar among proteins with homologous structures and, in the split β-α-β family the proteins favour a two-strand-helix motif. Here I show that the two-strand-helix motif is also present in the ribosomal protein S6 from A. aeolicus even though the nucleation and core composition of this protein differ from other related structure-homologues. In contrast to nucleation and contact growth, which are events driven by the hydrophobic effect, my most recent work is focused on electrostatic effects. By pH titration and protein engineering the charge content of S6 from T. thermophilus was altered and the results show that the charged groups at the protein surface might not be crucial for protein stability but, indeed, have impact on folding kinetics. Furthermore, by site-specific removal of all acidic groups the entire pH dependence of protein stability was depleted.

To investigate how protein folding is affected by sequence connectivity five topological variants of the ribosomal protein S6 were constructed through circular permutation.  In these constructs, the chain connectivity (i.e. the order of secondary-structure elements) is changed without changing the native-state topology.  The effects of the permutations on the folding process were then characterised by φ-value analysis, which estimates the extent of contact formations in the transition-state ensemble.  The results show that the folding nuclei of the wild-type and permutant proteins comprises a common motif of one α-helix docking against two β-sheets, i.e. the minimal structure for folding.  However, this motif is recruited in different parts of the S6 structure depending on the permutation, either in the α1 or α2 half of the protein.  This minimal structure is not unique for S6 but can also be seen in other proteins.  As an effect of the dual nucleation possibilities, the transition-state changes describe a competition between two parallel pathways, which both include the central β-stand 1.  This strand constitutes thus a structural overlap between the two competing nuclei.  As similar overlap between competing nuclei is also seen in other proteins, I hypothesise that the coupling of several small nuclei into extended ‘super nuclei’ represents a general principle for propagating folding cooperativity across large structural distances.  Moreover, I demonstrate by NMR analysis that the existence of multiple folding nuclei renders the H/D-exchange kinetics independent of the folding pathway. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper IV: Manuscript