In this paper commentary is offered on the role of architecture in a democratic society, as manifested in its use as a tool in the creation of spaces through which the public has equal opportunities for expression and interaction, at both the micro and macro scales. How the public connects to the city is questioned, and the value of politicized South African architectural symbols to the current political environment, in which a higher level of transparency is being sought, is considered. The value of a localised platform acting as a mediator between architecture, the public and the political powers is argued for. Consideration is given to the greater continuum of architectural thinking by discussing modern examples of political ‘democratic’ architecture and comparing them to those seen in the South African context. Furthermore, an understanding of democracy in both the social and spatial realms becomes an important informant in establishing value to society. The intention with the paper is to contribute to a way of thinking when designing within the confines of buildings that have past or present political value, such as the Pretoria City Hall, by addressing the question of how politics shapes architecture and how architecture, through politics, shapes the political environment. The focus is on how architecture is able to change meaning by inverting past symbols, so that the existing is enabled to become more representative of and responsive to the current socio-political environment. / In hierdie referaat word kommentaar gelewer op die rol van argitektuur in ‘n demokratiese samelewing, waar dit gebruik kan word as ‘n instrument vir die skep van ruimtes wat aan die publiek gelyke geleenthede vir uitdrukking en interaksie verskaf, op beide die mikro- en makroskale. Hoe die publiek met die stad konnekteer word bevraagteken, en die waarde van verpolitiseerde Suid-Afrikaanse argitektoaniese simbole vir die huidige politiese omgewing, waarin ‘n hoër vlak van deursigtigheid nagestreef word, word oorweeg. Daar word ten gunste van die waarde van ‘n gelokaliseerde platform wat as bemiddelaar tussen argitektuur, die publiek en die politieke magte kan optree, geargumenteer. Die groter kontinuum van argitektoniese denke word oorweeg deur moderne voorbeelde van politiese ‘demokratiese’ argitektuur te bespreek en met dié binne Suid-Afrikaanse konteks te vergelyk. Verder word ‘n begrip van demokrasie op beide die sosiale en ruimtelike terreine ‘n belangrike informant in die vaslegging van waarde vir die samelewing. Met die referaat word daar beoog om by te dra tot ‘n manier van dink wanneer daar ontwerp word binne die grense van geboue met teenswoordige of geskiedkundige politieke waarde, soos die Pretoria Stadsaal, deur die vraag aan te spreek oor hoe politiek die argitektuur vorm en hoe argitektuur, deur politiek, die politiese omgewing vorm. Die fokus word geplaas op hoe betekenis deur middel van argitektuur verander kan word deur simbole van die verlede om te keer, sodat die bestaande meer verteenwoordigend van en responsief tot die huidige sosio-politiese omgewing kan word. / Mini Dissertation MArch(Prof)--University of Pretoria, 2018. / Architecture / MArch (Prof) / Unrestricted

Barriers to interbreeding limit gene flow between sister taxa, leading to reproductive isolation and the maintenance of distinct species. These barriers come in many forms, and can act at different stages in the reproductive process. Pre-copulatory barriers may be due to individuals discriminating against heterospecifics in mate choice decisions. These decisions may be informed through a range of sensory modalities. If a female is mated and inseminated, then there may be multiple postmating-prezygotic barriers that affect the success of heterospecific sperm in attaining fertilisations. Post-zygotic barriers can be very early acting, resulting in embryonic fatality, or may be later acting, affecting the fitness of hybrid offspring. In this thesis I investigate potential reproductive barriers between the interbreeding field crickets Gryllus bimaculatus and G. campestris. I find that females of both species show only weak preference for conspecific calling song, and may even respond phonotactically to songs typical of heterospecific males. Female G. bimaculatus are repeatable in their preferences and strength of response. G. bimaculatus females presented with synthetic songs prefer those with longer inter-pulse intervals, whereas G. campestris show no discrimination between these songs. Upon meeting, G. campestris females strongly discriminate against heterospecific males, behaving aggressively towards them. This is likely driven by females responding to close range species recognition cues, including chemoreception. The species differ in their cuticular hydrocarbon profiles, and females that are no longer able to use their antennae to receive chemosensory information reduced their aggressive behaviour towards heterospecific males. G. bimaculatus females will mate with heterospecific males, though less readily than to conspecifics. When sequentially mated to both conspecific and heterospecific males, these females will preferentially take up and store sperm from the conspecific male, and sperm from conspecific males is more likely to sire offspring than would be predicted from the proportion of sperm in storage. Eggs from inter-species mating pairs are less likely to begin embryogenesis, and are more likely to suffer developmental arrest during the early stages of embryogenesis. However hybrid embryos that survive to later stages of development have hatching success similar to that of pure-bred embryos. After mating, phonotaxis of G. bimaculatus females towards male songs follows a pattern of suppression and subsequent recovery, likely triggered through detection of seminal proteins transferred in the male ejaculate, or detection of mechanical filling of the spermatheca. This pattern of suppression and recovery of phonotaxis does not differ between females mated to conspecific or heterospecific males. Females that lay few or no eggs do not experience a refractory period.

Europe’s largest population of wild red deer (Cervus elaphus) resides in the British Isles and has been present since the end of the last ice age, c. 11,000BP. Since the mid-19th century, multiple introductions of Japanese sika (Cervus nippon) and wapiti (Cervus canadensis) have taken place across the British Isles. While wapiti introductions have generally gone extinct, sika have thrived and expanded and now often live in sympatry with red deer. Hybridisation between these species has been demonstrated in captivity and in the wild. This study sought to determine the extent of hybridisation and introgression between red and sika across large parts of the British Isles and elucidate some of its potential consequences. Chapter 2 addresses the extent of hybridisation and introgression across Scotland and NW England. A total of 2984 samples from the North Highlands, the central Highlands, the Hebrides, Kintyre and the English Lake District were genotyped at 22 microsatellite loci, which are highly diagnostic for red and sika and strongly diagnostic for red and wapiti and a mitochondrial marker that is diagnostic for red and sika, alongside 49 wapiti samples from Canada. Microsatellite data was analysed using the Bayesian clustering program Structure 2.3 to determine the extent of admixture between species. There was some evidence for very low-level introgression by wapiti into a small number of Scottish red deer (<0.2% of total). Only two areas (both in Kintyre, Argyll) showed extensive introgression with collapse of assortative mating between red and sika (50.4% and 61.8% of sampled individuals were hybrid in West Loch Awe and South Kintyre, respectively). However, rare and widely scattered individuals with low-level sika introgression or cytonuclear disequilibrium suggest hybridisation has occurred in several other places in mainland Scotland and Cumbria in the past without subsequent loss of assortative mating. Chapter 3 addresses the extent of hybridisation in Ireland. There are now an estimated 4,000 red deer in Ireland and their numbers are increasing. It has recently been determined that the red deer in Killarney, County Kerry are descended from an ancient (c. 5,000BP) introduction and therefore merit genetic conservation. Introduction of exotic species, including Japanese sika and North America wapiti, since the 19th century have primarily occurred via the now defunct Powerscourt Park, County Wicklow, which was the source of many translocations to the rest of Ireland as well as to the UK. 374 deer samples from across Ireland were analysed as in Chapter 2. Wapiti introgression was again very low, with trace amounts of introgression detected in a small proportion of samples (0.53%), whilst 41% of 197 deer sampled in Co. Wicklow and 47% of 15 deer sampled in Co. Cork were red-sika hybrids according to either their nuclear genome or mitochondrial haplotype. No pure red deer were detected in Co. Wicklow, suggesting that in this region the red deer has disappeared following hybridisation. Whilst no hybrids were detected among 37 red samples and 77 sika samples in Co. Kerry, the Co. Cork hybrids pose a threat to the Killarney populations due to their proximity. Chapter 4 investigates population genetic structure within red and sika populations across the British Isles and investigates whether low-level introgression by the other species influences the resolved population structure. Structure analysis was conducted separately using 2307 ‘pure’ red deer individuals and 752 ‘pure’ sika animals from the British Isles (defined as Q > 0.95 for red and Q < 0.05 for sika) and then on reduced sample sizes using more stringent purity criteria (Q ≥ 0.99 and Q ≤ 0.01). As might be predicted, the more stringent criteria removed individuals in areas known to contain advanced backcrosses. In red deer, there was some evidence for a loss of within-species population structure under the more stringent criteria, while for sika there was not. Datasets were also analysed using Discriminate Analysis of Principal Components; a multivariate method designed to infer and describe genetic population structure. In red deer, both analytical approaches confirmed the strong separation of the deer on Harris and Lewis from others, and there is support for clusters typified by the other Hebridean islands, Kintyre, central and North Scotland and the English sites. Among sika, both approaches supported the likelihood of three clusters which are presumably the result of bottleneck events as each introduction was made. Chapter 5 investigates the phenotypic consequences of hybridisation by three approaches. Firstly, carcass weight was regressed against genetically-determined hybrid scores (at two stringency levels, see Chapter 4) and heterozygosity (in terms of red and sika alleles). Among hybrids, carcass weight is linearly related to hybrid score (Q) and there is some evidence for a positive relationship with heterozygosity. This suggests that additive genetic variation explains variation in carcass weight to a greater extent than heterosis. Secondly, analysis of five case studies representing individual putative hybrids submitted by stalkers from areas without known hybridisation, two proved to be hybrids, while the other three were pure sika. Lastly, in regions known to contain hybrids, the accuracy of ranger-assigned phenotype averaged 78% and revealed that in Scotland accuracy tends to decline as an individual becomes more genetically intermediate; whilst in Co. Wicklow it is the identification of pure parental animals that is more challenging. In conclusion, the existence of rare and widely scattered advanced red-sika backcrosses with low-level nuclear introgression and/or mitochondrial introgression (e.g. in North of Scotland, Cumbria) highlight that some hybridisation events are followed by extensive backcrossing without the breakdown of assortative mating, while others are followed by the generation of a hybrid swarm (e.g. in South Kintyre, West Loch Awe, Co. Wicklow, Co. Cork). Phenotypic traits can become intermediate due to hybridisation and this may facilitate further gene flow and hybridisation. New molecular tools including next generation sequencing (NGS) will enable better understanding the hybridisation process and its phenotypic consequences in this and other systems.

This thesis outlines a study of the physical changes that hybridisation imposes on single-stranded DNA (ssDNA) immobilised by one end to a substrate, and of how such physical changes can be exploited to detect specific sequences of DNA in a target solution. The system studied was composed of a mixed monolayer of 20mer ssDNA with C6 alkanethiolate modifications on their 3??? ends and short-chain hydroxyterminated alkanethiolates, on a gold substrate. It was prepared using the self-assembly properties of alkanethiols on gold. Atomic force microscopy images showed that the end-immobilised ssDNA is flexible enough to lie over the diluent hydroxy-terminated self-assembled monolayer (SAM). Hybridisation was shown to cause the DNA to become more rigid and stand up off the substrate due to an increase in persistence length. Such physical changes of the DNA upon hybridisation were significant enough to be exploited in the development of a DNA recognition interface. The recognition interface was designed with the view of keeping it both simple to make and simple to use, and was coupled with electrochemical transduction. A label-free recognition interface was developed that relied on the oxidation of the sulfur head group of the alkanethiolate SAM to detect hybridisation (firstly air oxidation and then electrochemical oxidation). It produced a positive signal upon hybridisation with complementary target DNA. Improvements in the reliability and robustness of the recognition interface were made using a labelled approach. The labelled version employed electroactive molecules as labels on the 5??? ends of the probe DNA strands. Two labels were investigated ??? anthraquinone and ferrocene. The flexibility of the ssDNA ensured that the redox labels were able to directly access the underlying gold electrode. Hybridisation was expected to remove the labels from the electrode due to an increase in the DNA???s persistence length, and thus perturb the electrochemical signal. The use of ferrocene as a label provided a ???proof-of-concept??? for the system. The labelled recognition interface provides a foundation for the future development of a simple, reliable, and selective DNA hybridisation biosensor.

Field pea (Pisum sativum L.) is highly susceptible to ascochyta blight, primarily caused by the pathogen Mycosphaerella pinodes (Berk. & Blox.) Vestergr. Grasspea (Lathyrus sativus L.) has been reported to possess a moderate level of resistance to ascochyta blight caused by M. pinodes. The work reported in this thesis aimed to develop the various techniques that would be required to transfer ascochyta blight resistance from grasspea into field pea via somatic hybridisation. This thesis also assesses the feasibility of achieving this goal. Field pea shoot cultures were established on hormone-free MS medium, and a protoplast isolation protocol developed for both species. Grasspea shoot cultures were established on both RL and SSB8 medium. Friable grasspea callus was achieved on media supplemented with 2,4-D in the range 4.523 µM, whereas kinetin tested at any concentration did not appear to influence callus growth. A suspension culture of grasspea was developed for the first time, in B5 medium supplemented with 4.5 µM 2,4-D and 0.5 µM kinetin. Grasspea protoplasts were isolated from both in vitro seedlings and suspension cultures. Protocols for hybrid shoot culture on KM8p medium were developed via organogenesis and somatic embryogenesis.

Field pea (Pisum sativum L.) is highly susceptible to ascochyta blight, primarily caused by the pathogen Mycosphaerella pinodes (Berk. & Blox.) Vestergr. Grasspea (Lathyrus sativus L.) has been reported to possess a moderate level of resistance to ascochyta blight caused by M. pinodes. The work reported in this thesis aimed to develop the various techniques that would be required to transfer ascochyta blight resistance from grasspea into field pea via somatic hybridisation. This thesis also assesses the feasibility of achieving this goal. Field pea shoot cultures were established on hormone-free MS medium, and a protoplast isolation protocol developed for both species. Grasspea shoot cultures were established on both RL and SSB8 medium. Friable grasspea callus was achieved on media supplemented with 2,4-D in the range 4.523 µM, whereas kinetin tested at any concentration did not appear to influence callus growth. A suspension culture of grasspea was developed for the first time, in B5 medium supplemented with 4.5 µM 2,4-D and 0.5 µM kinetin. Grasspea protoplasts were isolated from both in vitro seedlings and suspension cultures. Protocols for hybrid shoot culture on KM8p medium were developed via organogenesis and somatic embryogenesis.

The studies described in this thesis investigate the role of indirect effects in invasion biology. The Introduction provides a brief overview of indirect effects and an outline of the thesis structure. The role of indirect effects in the context of invasion biology is addressed in an in-depth published literature review that comprises the second chapter, providing a theoretical background for the subsequent empirical studies. Chapters Three to Six are comprised of manuscripts that have been published or are under review or in press, which describe studies that investigate the importance of indirect effects in invasion biology using a model system consisting of the alien Asteraceae Senecio madagascariensis, a closelyrelated native, Senecio pinnatifolius, and the insect species with which they interact. Senecio madagascariensis and S. pinnatifolius occur in a similar geographic range in eastern Australia and these studies were conducted in mixed and pure populations of the two species. The herbivore and floral visitor assemblages of the two Senecio species at seven field sites in South-east Queensland were compared using sweep-net sampling, manual searching and floral visitor observation techniques. The floral visitor assemblages were similar between the two species, comprised largely of species of Syrphidae and the European honeybee, Apis mellifera. Herbivore assemblages, however, were highly variable both between species and between sites, with greater herbivore abundance and diversity recorded on the native S. pinnatifolius than its alien congener. The most commonly recorded herbivores were sap-sucking species such as Myridae. The magpie moth, Nyctemera amica was the most common folivore on both Senecio species and laboratory studies demonstrated a clear preference by ovipositing females and feeding larvae of this species for the native Senecio species, over the alien. Field surveys supported these findings, recording greater leaf damage on the native species than the invader. Herbivory levels were lower, rather than higher, in mixed populations than in pure populations, thus there was no evidence that the presence of one species enhanced herbivory in the other. Field pollination trials were conducted to determine whether competition for pollinators or facilitation of pollination occurred in mixed Senecio populations. The presence of the native S. pinnatifolius affected pollinator visitation rates to the alien Senecio; bee visits to S. madagascariensis were significantly reduced by the presence of S. pinnatifolius, whilst syrphid visits increased. However, altered visitation rates were not reflected in seed set. The presence of the alien species had no impact on pollinator visits to the native. Surprisingly, S. pinnatifolius seed set was higher in mixed populations than in pure populations. This might be due to abiotic factors, lower rates of herbivory at these sites or transfer of pollen between species resulting in the production of hybrid seed (if S. madagascariensis has greater male fitness). Hybridisation in the field was investigated using AFLP techniques. No mature hybrid plants were recorded in mixed populations, but hybrid seeds were produced by both species. Senecio pinnatifolius maternal parents produced higher numbers of hybrid seed than expected based on the relative frequencies of the two species, whilst hybridisation in S. madagascariensis was lower than expected. This may indicate greater male fitness of the invader. A range of complex indirect interactions can occur between invasive and native species, with these interactions having the potential to influence the success or failure of the invader and its impacts on co-occurring natives. The Discussion addresses the findings of the studies described here in the context of invasion biology theory.

This thesis outlines a study of the physical changes that hybridisation imposes on single-stranded DNA (ssDNA) immobilised by one end to a substrate, and of how such physical changes can be exploited to detect specific sequences of DNA in a target solution. The system studied was composed of a mixed monolayer of 20mer ssDNA with C6 alkanethiolate modifications on their 3??? ends and short-chain hydroxyterminated alkanethiolates, on a gold substrate. It was prepared using the self-assembly properties of alkanethiols on gold. Atomic force microscopy images showed that the end-immobilised ssDNA is flexible enough to lie over the diluent hydroxy-terminated self-assembled monolayer (SAM). Hybridisation was shown to cause the DNA to become more rigid and stand up off the substrate due to an increase in persistence length. Such physical changes of the DNA upon hybridisation were significant enough to be exploited in the development of a DNA recognition interface. The recognition interface was designed with the view of keeping it both simple to make and simple to use, and was coupled with electrochemical transduction. A label-free recognition interface was developed that relied on the oxidation of the sulfur head group of the alkanethiolate SAM to detect hybridisation (firstly air oxidation and then electrochemical oxidation). It produced a positive signal upon hybridisation with complementary target DNA. Improvements in the reliability and robustness of the recognition interface were made using a labelled approach. The labelled version employed electroactive molecules as labels on the 5??? ends of the probe DNA strands. Two labels were investigated ??? anthraquinone and ferrocene. The flexibility of the ssDNA ensured that the redox labels were able to directly access the underlying gold electrode. Hybridisation was expected to remove the labels from the electrode due to an increase in the DNA???s persistence length, and thus perturb the electrochemical signal. The use of ferrocene as a label provided a ???proof-of-concept??? for the system. The labelled recognition interface provides a foundation for the future development of a simple, reliable, and selective DNA hybridisation biosensor.

Dysfunctional or over and under expressed enzymes play a crucial role in a variety of diseases. A tool that can identify dis-regulated enzymes in individual patients would be beneficial and would allow personalised treatment. For this purpose, a 10,000 membered ‘spit-and-mix’ PNA-encoded peptide library with a cell penetrating peptide was synthesised and interrogated with K562 cell lysate and intact K562 cells. This allowed the specific enzyme activity pattern for ABL tyrosine kinase from both inside a cell and a lysate to be obtained. Hybridisation of this library with a DNA-microarray resulted in bio-fouling by the cell lysate, thereby preventing analysis of the phosphorylation pattern. To allow extraction and purification of the peptide library from the cell lysates, a His-tag was incorporated into the library, and enabled successful library analysis. In addition to this 10,000 member library, a focused 100 PNA-encoded peptide library was synthesised. The library included peptide sequences known to be phosphorylated by specific tyrosine kinases deregulated in acute lymphoblastic leukaemia (ALL) with a PNA-tag complementary to a DNA microarray. Different ligation methods to conjugate the peptides to PNA-tags were screened – this included amide coupling, copper catalysed azide–alkyne cycloaddition, strain promoted azide–alkyne cycloaddition and Diels–Alder cycloaddition. The inverse electron demand Diels–Alder cycloaddition between a tetrazine and norbornene was chosen as the preferred ligation method, and the reaction conditions optimised. To purify the library from cell lysate, a His-tag was again coupled to each member using the strain promoted azide–alkyne cycloaddition. To test the tetrazine ligation, fluorescence in situ hybridisation (FISH) was used in cells, whereby a fluorophore was ligated onto a tetrazine–conjugated PNA probe. This was hybridised onto an mRNA in fixed cells. Results indicated that the ligation needed further optimisation.

The C4 cycle represents a series of biochemical and anatomical modifications that are targeted to overcome the effects of photorespiration caused by the oxygenase capability of Ribulose Bisphosphate Carboxylase/Oxygenase (RuBisCO). The cycle has evolved independently in over 60 lineages, which suggests that recruitment of genes into the C4 cycle is a relatively easy process. However, the mechanisms by which the anatomy and cell-specificity of the components of the C4 cycle is achieved is poorly understood. Preliminary work in maize indicated several components of the C4 cycle may be targeted by microRNAs (miRNAs). To explore this, a library of sRNA sequences from mature leaf tissue of the model C4 species Cleome gynandra L. was generated and then searched against a list of expressed sequence tag sequences for candidate genes of the C4 cycle. To complement this, transgenic C. gynandra containing the viral p19 protein, which is capable of suppressing miRNA activity, were produced. A limited subset of the candidate C4 genes showed a high level of sRNA read alignment. In C. gynandra plants expressing p19 photosynthesis was compromised and transcripts of several genes (most notably RbcS and RCA) were upregulated. These data were complemented by examining the effect of illumination on developing C. gynandra cotyledons, and attempts to generate a hybrid between C. gynandra and the C3 C. hassleriana Chodat. RbcS also showed elevated abundance in etiolated cotyledons, although this rapidly declined after illumination. The remainder of the C4 genes profiled accumulated in etiolated tissue, but were upregulated within 6 hours of illumination. Therefore, this study has illustrated that miRNA activity may play a role in maintaining the C4 photosynthetic cycle at optimum efficiency, although it has not been possible to identify at which point(s) this regulation is applied. Secondly, RbcS appears to be subject to multiple regulatory mechanisms in C. gynandra, and it is possible that miRNAs have a role in negatively regulating expression of RbcS.