Tato disertační práce se zabývá evolučním návrhem a optimalizací jak běžných, tak polymorfních digitálních obvodů. V práci jsou uvedena a vyhodnocena nová rozšíření kartézského genetického programování (Cartesian Genetic Programming, CGP), která umožňují zkrácení výpočetního času a získávání kompaktnějších obvodů. Další část práce se zaměřuje na nové metody syntézy polymorfních obvodů. Uvedené metody založené na polymorfních binárních rozhodovacích diagramech a polymorfním multiplexovaní rozšiřují běžné reprezentace digitálních obvodů, a to s ohledem na začlenění polymorfních hradel. Z důvodu snížení počtu hradel v obvodech syntetizovaných uvedenými metodami je provedena evoluční optimalizace založená na CGP. Implementované polymorfní obvody, které jsou optimalizovány s využitím CGP, reprezentují nejlepší známá řešení, jestliže je jako cílové kritérium brán počet hradel obvodu.

The importance of studying the physico-chemical properties of nano-dimensional materials has gained significant attention in the fields of semiconductors, pharmaceuticals, materials science, and atmospheric chemistry owing to the differences in physical properties between macro- and nano-dimensional solids. Nonetheless, direct studies of physical properties of materials at nanoscale is limited in part due to their inherent size constraints and experimental limitations. However, development of atomic force microscopy (AFM) led to the implementation of methods to characterize a wide range of physical properties, including – but not limited to – mechanical properties, electrical properties, viscoelastic properties, and surface tension. Herein, the dissertation focuses on AFM-based method development for characterization of atmospheric particles as well as understanding the relationship between structure and physical properties of organic solids at both macro- and nano-dimensions. In the atmospheric chemistry realm, the combined aerosol effect on the climate and environment has significant uncertainty in part due to lack of direct characterization of their physico-chemical properties. The difficulty in assessing the physical and chemical properties arises due to the presence of diversified aerosol sources, which in turn influences the size, morphology, phase states and chemical compositions. Sea spray aerosols (SSAs) are the second-largest source of aerosols in the atmosphere. Studying SSAs – especially in submicrometer-dimensions – requires high-resolution microscopy techniques such as AFM. AFM can be used for imaging of individual aerosols, quantifying organic volume fraction for core-shell morphologies, measuring water uptake, quantifying surface tension of individual droplets, and measuring mechanical and viscoelastic properties of materials. Herein, we employed AFM-based morphology and force spectroscopy studies to correlate the 3D morphology, phase state, and viscoelastic properties of selected single-component chemical systems found in sea spray aerosol (SSA). We established a quantitative framework toward differentiation of the solid, semisolid and liquid phase states of individual particles by utilizing both relative indentation depth (RID) and viscoelastic response distance (VRD) data obtained from the force−distance plots. Moreover, we established a semi-quantitative and quick phase assessment by measuring the aspect ratio (AR) that refers the extent of particle spreading as a result of impaction. Overall, the established AFM-based quantitative and semi-quantitative phase identification method can be utilized to assess the phases of aerosols irrespective of chemical identity. Next, we investigated the factors that may control the electrical and mechanical properties of pharmaceutical and organic semiconducting materials in nano- and macro-dimensions. Understanding the structure-property relationship of materials, especially in the nano-dimension, is necessary for proper drug design and development of organic semiconducting materials. In this context, cocrystals provide a means to modulate the physico-chemical properties of organic solids. For example, the modulation of the mechanical properties is important in the pharmaceutical industry for improving the tabletability. The mechanical properties may be affected by packing arrangement, interaction strength and type, and atomic and chemical composition. Herein, we report the influence of alkane and alkene functional groups on the mechanical properties of organic solids based on salicylic acid (SA). The approach affords both isostructural and polymorphic solids. The isostructural alkane functional solid exhibits a two-fold larger Young’s modulus (YM) compared to the cocrystal with the alkene, where the YM refers to the stiffness of the material. Here, the higher YM values are attributed to the presence of a bifurcated weak C-H···O interactions involving the alkane and neighboring SA molecules. On the other hand, in the case of alkene polymorphisms, molecular packing with column arrangement shows higher YM values compared to the herringbone arrangements. Thus, functional groups and crystal arrangements influence the stiffness of the solid organic cocrystals. Moreover, we report the modulation of mechanical properties of salicylic acid (SA) through cocrystallization by variation of propane and butane functionality with bipyridine coformers. We show that the variation of propane and butane functionality in bipyridine coformer with salicylic acid leads to synthesis of cocrystal and salt-cocrystal, respectively. The AFM nanoindentation study revealed that the Young’s modulus values follow the order salicylic acid < cocrystal << salt-cocrystal. The highest Young’s modulus values of the salt-cocrystal, among the studied systems, are attributed to the presence of strong N+–H···O– and O–H···O– interactions. On the other hand, higher Young’s modulus values of the propane-based cocrystal compared to the macro-dimensional salicylic acid are attributed to the stronger O–H ···N hydrogen bonding. Thus, homologous alkane functional groups can influence the mechanical properties of the organic solid crystals. Additionally, in situ solid-solid polymorphic phase transformation and nucleation of a metastable and elusive polymorph of SA cocrystals in combination with 4,4’-bipyridine were studied. Understanding the solid-solid phase transformations and nucleation mechanisms are important for proper control over the parameters associated with the synthesis of targeted crystalline solids with desired crystal structure. Using in situ powder X-ray diffraction (PXRD) and terahertz time domain spectroscopy (THz-TDS) data we showed that the Form II polymorph transforms to Form I over time. AFM imaging and nanoindentation techniques were utilized to follow and quantify in real-time the solid-solid polymorphic transformation of the metastable Form II to the thermodynamically stable Form I on a single crystal basis. AFM in situ single crystal data revealed that the metastable Form II has a rod-shaped morphology and relatively high elasticity (Young’s modulus), which transforms to prism-shaped nanocrystals of much smaller sizes with significantly reduced elasticity. The AFM imaging reveals that the single crystals on the order of 80-150 nm to undergo catastrophic changes in morphology that are consistent with cracking and popping owing to a release of mechanical stress during the transformation. The nucleation mechanism for the polymorphic transformation is not spatially localized and occurs over the entire crystal surface. The higher mechanical properties of the metastable Form II is due to the presence of the additional interlayer C-H···O interactions. Furthermore, we have studied the electrical properties of boron-based cocrystals. More specifically, cocrystallization of a nonconductive 2,4-difluorophenylboronic ester catechol adduct of a 4,4’-bipyridine (BEA) host with two aromatic semiconducting guests (pyrene and tetrathiafulvalene) generated conductive cocrystals with variable charge carrier mobilities. Charge carrier mobilities of the cocrystals with either pyrene or tetrathiafulvalene were measured using conducting probe AFM (CP-AFM). The incorporation of π-rich aromatic guests through face-to-face and edge-to-face π-contacts results in electrically conductive cocrystals. The cocrystal with tetrathiafulvalene as a guest shows approximately 7 times higher charge carrier mobility than the cocrystal with pyrene. Overall, the current dissertation demonstrates the AFM-based method development and applications towards materials characterization to measure the morphological, electrical, mechanical, and phase-states at both nano- and macro-dimensions. The high spatial precision of the methods developed enables us to better understand the controlling factors for materials design and processing across nano- and macro-dimensions.

This project investigated the genetic composition of natural populations of Anadara trapezia in Australia at three spatial scales : i) microgeographic (within an estuary, 50 metres to ~ 6 kilometres); ii) microgeographic (within populations, less than 50 metres); and, iii) macrogeographic (hundreds of kilometres along the coast of Australia). Allozyme polymorphism surveys using cellulose acetate strips have revealed, from 43 enzymes screened, 18 putative polymorphic loci. Comparisons of levels of heterozygosity among enzyme structural groups showed no significant differences, however, monomers were significantly more variable as a group than multimers. Significant differences in the level and distribution of polymorphism among functional groups of enzymes were observed. It appears that selection may be acting at the molecular level, not only on a particular locus, but on a group of functionally similar loci. At the macrogeographic scale, significant departures from random mating were observed in most populations. Significant differences in allele distribution among populations of A. trapezia along the east coast of Australia were found. At the macrogeographic scale, heterogeneity of allele frequencies may depend upon the distance separating the populations and surface water currents. Differentiation among population groups in this study is attributed to changes in the direction of the East Australian Current combined with onshore countercurrents. The systematic status of the disjunct western Australian population of A. trapezia was also evaluated as compared with the east coast populations. No evidence of genetic, hence evolutionary divergence was found. The results have serious implications in the management of fisheries as erroneous assumptions in fisheries management models may lead to depletion and near extinction of marine species. The research stresses the necessity of sampling at multiple scales and replication strategies. It also highlighted the complexities researchers are faced with in studies of marine bivalves, such as the presence of null alleles, deficiencies of heterozygotes, apparent inbreeding and the small geographic scales governing population structure. / Doctor of Philosophy (PhD)

[Truncated abstract] Traditional medicines are used by millions of people throughout the world as their primary source of medical care. A range of materials are in used traditional medicines including plant and animal parts. Even though the traditional medicine trade is estimated to be worth sixty billion dollars annually the trade remains largely unregulated. Unscrupulous practices by vendors to increase their profit margins such as substituting and adulterating expensive material with cheaper varieties go unchecked. This can be dangerous to consumers because some substitutions involve poisonous material. Also, animal parts from endangered species can find their way into traditional medicines, therefore there needs to be a way to identify them in traditional medicines to prosecute poachers. The traditional techniques used for the identification of material used in Traditional Chinese Medicine (TCM) include, morphological, histological, chemical and immunological analysis. However, these techniques have their limitations. This makes applying multiple techniques essential to provide thorough authentication of the material. DNA profiling provides a technique well suited to analysing material used in TCM. DNA profiling is advantageous over other techniques used to authenticate material used in TCM because it requires only a small sample amount, can determine the cultivator, be used on all forms of TCM and potentially distinguish the components of mixtures. ... Therefore, profiles of different species/individual are different and species? can be distinguished. Commercially sold traditional medicines are processed which is likely to degrade the DNA of the sample making extraction and amplification difficult. Here an organic Phenol:Chloroform extraction technique extracted DNA from commercial dried root samples. The extracted DNA was amplifiable using RAPD primers. The RAPD primers used here produced enough polymorphic bands to distinguish different plant species. They were used to distinguish commercial samples that were sold as three different species within the Panax genus, Panax ginseng, Panax quinquefolium and Panax notoginseng and genetically unrelated plant material; Potato and Eleutherococcus senticosus. Liquid samples and mixtures were also profiled with the RAPD primers to determine whether the RAPD primers provide enough distinguishing ability to analyse these forms of TCM. DNA was extracted from the liquid samples, one a ginseng drink and the other an ginseng extractum. However, there was no reliability in the production of PCR products. The analysis of the mixture samples found that not enough polymorphic bands were produced by the RAPD primers used here to identify Panax species within mixtures of two Panax species. While when P. ginseng was mixed with a genetically unrelated sample there was enough polymorphism to differentiate the two samples in the mixture. The results of this research show that RAPD analysis provides a simple and inexpensive technique to begin analysis of materials used in TCM. Using RAPD analysis it is possible to distinguish Panax plant species from each other. However, the RAPD primers used here did not provide enough reproducibility or polymorphism to analyse liquid and mixtures of Panax species plants.

The Cape parrot (Poicephalus robustus) is South Africa's only endemic parrot. It has become increasingly rare in recent years, with fewer than 500 birds left in the wild, and is now regarded as endangered. Possible factors contributing to this rapid decline in numbers include habitat loss, food shortage, disease and illegal trafficking and trading in the species. Habitat loss and food shortage have been brought about by the rapid destruction of the yellowwood trees in the afromontane forests in South Africa and have played a role in reducing the population numbers. The Psittacine beak and feather disease virus (PBFDV) has also contributed to the loss of some individuals, however it is the illegal trafficking of this rare and valuable species that has become of great concern. As the Cape parrot is becoming increasingly rare and therefore highly sought after, its commercial value has multiplied to the extent that illegal black market trapping is on the rise. The industry involved in breeding and conservation of endangered bird species, has a need for the proper establishment of studbooks, containing all available information on captive as well as tagged birds. Most of the information found in studbooks is based on morphological attributes of individual birds. Although this is useful, there is a need to add molecular information in order for complete identification of individuals, particularly in a species threatened by illegal trading and theft. A preliminary analysis of the amount of variation present in the population of interest is therefore required so that appropriate methods and techniques can be developed to identify individual birds. A RAPD analysis was conducted to assess the amount of variation in the Cape parrot and lay the foundations for the establishment of individual identification in the species. Blood samples from 30 parrots, consisting of both related and unrelated individuals, were obtained from three separate locations: Amazona in Assagay, Rehoboth Farm in Dargle, as well as from the Eastern Cape. 15 random primers were selected and used to conduct a randomly amplified polymorphic DNA (RAPD) analysis. RAPDs are extremely useful in situations where relatively inexpensive first approximations of the genetic variation are needed, such as in rare and endangered species. After successful optimisation of the technique in the species, the 15 primers were screened for all 30 individuals and the individual DNA fingerprints, analysed. Clear, distinctive and reliable DNA fingerprints were obtained for all individuals however, it was interesting to note despite the analysis of 85 loci using the 15 primers almost identical DNA fingerprints were produced between the individual birds. A population analysis into the amount of variation present between and within the three populations, as well as for the representative population as a whole, was conducted. Using various statistical programmes such as POPGENE and ARLEQUIN, heterozygosities, genetic distance measures, diversity indices, Wright's fixation index and AMOVAs were estimated. The amount of polymorphism detected in this investigation was 33 % and the heterozygosity, 0.37, which is a relatively high value for the uniformity displayed in the DNA profiles. The high GC content of the primers however, could be a possible explanation thereof. Relationship and kinship determination, sex determination as well as population assignment was possible despite not being able to identify each individual based on unique DNA fingerprints. The AMOVA analysis indicated significant variation on both the between (5.59 %) and within (94.41 %) levels of analysis. Little variation or differentiation was observed between the three subpopulations, which was confirmed with an FST value of 0.056. The variation experienced within each subpopulation was analysed using Shannon's index of phenotypic diversity. The Amazona population displayed the most variation with a value of 0.286 and the Rehoboth population, the least with 0.195. This was expected, with the individuals from the latter population comprising one extended family. Nei's measures of genetic identity revealed that the individuals from Amazona were more similar to the Eastern Cape population, which was again expected with regular exchanging of chicks between the two breeders. RAPD technology was successful in laying the foundations for individual identification in the Cape parrot. It was also successful in producing reproducible DNA fingerprints in the species that were able to determine relatedness to some extent, determine the sex of individuals and identify individuals from a particular subpopulation. Furthermore RAPD analysis gave a good indication of the variation found in the Cape parrot population, which is important for conservation purposes. In order to maximize conservation efforts and strategies in an endangered species, determining the level of genetic diversity and variation found in the remaining individuals of the population is of great importance. This information could provide powerful insight for conservation purposes and depending on the level of diversity detected, appropriate breeding programmes could be set up in order to increase the genetic variation and thereby reduce the chance of extinction of the species. The following important findings emerged from this investigation: • RAPD technology, once optimised for the species of interest, is successful in producing clear and reliable DNA fingerprints, provided the same protocol is followed carefully throughout the investigation. • An optimised protocol for fingerprinting the Cape parrot using RAPDs was established. • Possible sex identification, population assignment and a degree of kinship determination was determined using RAPDs. • Little variation was found within the representative Cape parrot population as a whole due to small population size and possible inbreeding. • As expected for an avian species, little genetic sub-division or differentiation was observed between the three populations analysed. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.

No abstract available. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.

A collection of 29 clinical streptococcal isolates obtained from the University of KwaZulu-Natal, Medical School, Durban Metro area (South Africa) were studied to establish their penicillin G susceptibility patterns often refered to as minimal inhibitory concentration (MIC) and to determine the genetic diversity among them using two genotyping methods, randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE) analysis. All isolates with MIC less than or equal to 0.12 µg/ml were considered susceptible, intermediate resistant if MIC was between 0.25 µg/ml and 4 µg/ml and resistant if greater than 4 µg/ml, The percentage of isolates with resistance was relatively high (75.9%), only 10.3% of isolates showed intermediate resistance and 13.8% of the isolates were completely susceptible to penicillin G. Some of the resistant isolates were highly resistant reaching penicillin G MIC levels of 5000 µ/ml. They were speculated to contain Path altered penicillin binding proteins and high level of crosslinking cell wall induced by the gene products of the MurMN operon. RAPD analysis was performed using three primers, MBPZ-1, MBPZ-2, and MBPZ-3, respectively. RAPD analysis allowed for the identification of 27 RAPD types with MBPZ-1 and MBPZ-3 and 26 RAPD types with MBPZ-2. Ninety-eight percent of these isolates were clustered into two groups, group I and group II, with 90% to 100% dissimilarity among them. Fifty two percent of the isolates of MBPZ-1 group I were in MBPZ-2 group I, 72% isolates of MBPZ-1 group I were in MBPZ-3 group I, and 72% of the isolates of MBPZ-2 group I were in MBPZ-3 group 1. This shows the discriminatory ability of the primers used in this study. Despite clustering of isolates, relatively high diversity was seen. PFGE analysis of macrorestriction fragments obtained after digestion of chromosomal DNA by restriction enzyme, SmaI showed 24 PFGE patterns. The 24 PFGE patterns were divided into three groups (I, II and III) of isolates, with an average of 85% dissimilarity (15% homology) among them. At 25% homology, four clusters, A (13 isolates), B (9 isolates), C (4 isolates), and D (4 isolates) were observed. Two pairs of isolates in group I, cluster A, showed 100% homology. This suggested that each represent the same strain. Four isolates of group I, cluster B, also exhibited 100% homology. This study showed that most of streptococcal isolates with the same penicillin G susceptibility patterns grouped together in a phylogenetic tree by both RAPD and PFGE analysis. There was also some similarity between the results obtained by RAPD analysis and PFGE analysis. Seventeen and nine of the 29 isolates grouped into group I and group II, respectively, two pairs of isolates were indistinguishable, and two pairs of islates were closely related by both RAPD (using MBPZ-3) and PFGE analysis. Although, RAPD analysis is sensitive, specific, faster and cost effective, the ease with which PFGE analysis can be performed, high discriminatory power, reproducibility of the results, and the polymorphism seen in the patterns, suggests that PFGE method has the potential to be very useful for epidemiological evaluations of nosocomial streptococcal infections in KwaZulu-Natal. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.

Theileria parva is a complex protozoan parasite causing East Coast fever in Eastern and Central Africa. Vaccination using live parasites is an effective control measure and has been used in Zambia based on locally isolated and introduced T. parva stocks. Diversity among T. parva populations was investigated in parasites from two Zambian provinces with different disease epidemiologies and control histories. Isolates from the pre-vaccination era, local and exotic stocks used for vaccination, and one recent field isolate were cloned and passaged in vitro to study genomic stability over time. The results of the data from three genome-wide probes indicate a marked homogeneity and stability among the Zambian isolates in contrast to East African isolates. Results from Southern blot profiles and the polymorphic immunodominant molecule (PIM) sequence analysis suggest a common origin for the Zambian isolates from the pre-vaccination era, except for one isolate (Zam5) from Southern Province. This isolate showed characteristics suggesting a buffalo origin. Assays for genotype characterisation were developed using five allelic markers. Multilocus characterisation revealed identical profiles in a recent Zambian isolate from Southern Province and two components of an exotic cocktail vaccine, indicating the escape of one of the vaccine stocks in the field. Characterisation of T. parva field populations by RFLP-PCR assays after immunisation revealed the presence of dominant genotypes from those that had been used for vaccination. Circumstantial evidence for the involvement of one of the exotic vaccine parasites in epidemics in Southern Province is presented and a hypothesis formulated for the rapid spread of this genotype. Analysis of the characterisation data suggested the existence of two groups of T. parva parasites of different origin. The classic T. parva group, characterised by a dimorphism of the p150, p104 and p32 loci and the absence of a p67 insert and a buffalo-derived group which showed a polymorphism of p150, p104 and p32 and the presence of a p67 insert. There is evidence that recombination occurs, resulting in parasites that have characteristics of both groups. The relevance of these recombinant parasites in the epidemiology of the disease seems low. Characterisation of larger samples from areas of regular buffalo-cattle contact is necessary to clarify this. Sequence analysis of the most discriminative locus (PIM) was undertaken and gene conversion could be the main mechanism generating diversity. A more appropriate nomenclature for T. parva is proposed based on the growing evidence of molecular differences among isolates and stocks.

[Truncated abstract] For the convenience of the reader, detailed figures and tables have been enlarged and compiled in Appendix 2, at the end of this thesis. This thesis is presented as an approach to identify, annotate and detect genomic duplication and polymorphism within large genomic regions. To demonstrate this, I have used as a model, the genomic region known as the Regulators of Complement Activation (RCA). The RCA complex is located on the long arm of chromosome 1 at position 1q32 and is a reservoir of complement regulatory proteins. The genes of the RCA share many similarities implying that all have arisen through multiple complex duplication events. My analysis of this region in the following chapters demonstrates the complexity of this duplication and identifies the many functional units within the RCA. It was my aim at the beginning of these studies to demonstrate an approach that could define the Ancestral Haplotypes (AHs) of the RCA gene cluster. To do this, extensive genomic analysis was required and the ever-increasing availability of genomic sequence has made this thesis possible. Each of the chapters serves to address the following aims set out at the beginning of this thesis: 1. Further characterise the relationship between the genes (Complement Control proteins-CCPs) and domains of the Regulators of Complement Activation (RCA). 2. Identify and examine the duplicated elements within the RCA. - 6 - 3. Examine the effects of retroviruses and other insertions and deletions (indels) in generating the divergence of duplicated genes. 4. Investigate the applicability of the Genomic Matching Technique (GMT) to define AH within the region. 5. Examine association of AHs with CCP implicated diseases. 6. Determine the GMT applicability in non-human species

Dentro do gênero Adesmia, as técnicas moleculares ainda não foram empregadas na caracterização de germoplasma e na análise da diversidade genética das espécies brasileiras que compôem o gênero. Portanto os objetivos deste trabalho foram: caracterizar, com a utilização de marcador molecular do tipo RAPD, as espécies brasileiras do gênero Adesmia DC; com base nestas informações estabelecer relações de diversidade genética entre as espécies e os acessos analisados; relacionar dados de diversidade com dados morfológicos e de reprodução.