The first chapter gives an overall introduction to Arachis hypogaea L., the crop that was studied in this thesis. The literature reviewed touched on various aspects of the crop in general such as the importance of peanut as a crop, its botanical description, origin and distribution world-wide. Different marker studies done on A. hypogaea L. and their extents have also been reviewed. The chapter also looked at the oryzacystatin-1 (OC-1) gene as a possible candidate gene for peanut improvement in South Africa (Chapter I). Agronomic data was collected in an attempt to observe and evaluate variability amongst the 18 commercially cultivated peanut lines from South Africa. The growth habits, growth seasons, testa colours, total oil percentages and oleic and linoleic acid ratios were the agronomic characters used to study variation (Chapter II). One South African commercially cultivated peanut line, JL 24, was transformed with the oryzacystatin-1 (OC-1) gene, which was isolated from rice. Three to four week old plantlets were vacuum infiltrated with Agrobacterium during the transformation process and various methods were used to analyze the putative transformants (Chapter III). Two types of DNA markers along with phynotypic data were used to examine polymorphism among 18 South African peanut lines and a transformed line. The amplified fragment length polymorphisms (AFLPs) and simple sequences repeats (SSRs) were the two DNA markers used in this study to determine the level of variation amongst these lines (Chapter IV). / Dissertation (MSc (Genetics))--University of Pretoria, 2008. / Genetics / unrestricted

DNA micro-array technology is a new and powerful technology that could substantially increase the speed of forest tree breeding programmes. This thesis represents a compilation of investigations that focus on the exploitation of DNA micro-array technology for genetic marker analysis of Eucalyptus trees. The major focus of the studies presented in this thesis was on the assessment and development of micro-array-based DNA fingerprinting in Eucalyptus. A DNA chip for Eucalyptus was not available at start of the study. As a result of this study a 384-prototype chip was developed to evaluate the potential of micro-arrays for fingerprinting closely related Eucalyptus clones, species and hybrids. These studies show that micro-arrays are an efficient DNA marker technology for genome-wide fingerprinting of complex organisms for which no sequence data exist. However, cross-hybridisation and the lack of dedicated software products remain a challenge. The 384-probe array developed in this study was subsequently employed for the detection of putative markers associated with tolerance to Chrysoporthe austroafricana in Eucalyptus grandis. Putative tolerance-associated markers were identified by bulk segregant analysis (BSA) and converted to cleaved amplified polymorphic sequence markers for further characterization in segregating Eucalyptus populations. BSA revealed a total of 109 scorable, polymorphic loci, of which nine appeared to be associated with tolerance or susceptibility. Two DArT markers were converted to cleaved amplified polymorphic sequence (CAPS) markers, which discriminate susceptible and tolerant individuals. These PCR markers can be used for the rapid screening for disease tolerance in Eucalyptus planting and breeding stock. The collection of studies included in this thesis demonstrated that DArT is an efficient DNA marker technology for genome-wide genotyping, particularly for application in less-studies plant genomes. Whole-genome profiling using DArT raises significant opportunities for tree breeding programmes and for future genome analysis of Eucalyptus. / Thesis (PhD)--University of Pretoria, 2011. / Genetics / Unrestricted

Thesis (Master, Microbiology & Immunology) -- Queen's University, 2013-09-30 22:46:35.072

The aim of my thesis is to describe and to compare individual aspects of invalid and putative marriage in the Czech Republic and the Netherlands and to analyse the reasons which lead to existence of these marriages. The reason for which I have chosen this topic is the fact that marriage is one of the most important legal institutions of family law and since the commencement of the new statute 89/2012 Sb., some requirements for marriage have been changed. The Dutch law is based on very similar principles as the Czech law, but the application of these principles is different in many ways. The thesis is composed of six chapters, the introduction and the conclusion. The first chapter of my thesis characterizes marriage as a legal institution in terms of its concept, purpose and function. It also compares marriage to other forms of living arrangements that are possible in the Czech Republic. The second chapter focuses on formation of marriage, the possible forms of entering into marriage, and analyses the conditions that must be met in the marriage to be valid, including special requirements for entering into marriage in unusual situations. The next chapter characterizes the legal consequences of marriage defects depending on their severity. The fourth chapter deals with invalid marriage and divides...

The phospholipid phosphatidylinositol and its phosphorylated derivatives, collectively referred to as phosphoinositides, form the basis for a multifaceted signaling pathway regulating many different cellular processes in eukaryotic cells. Phosphatidylinositol 4,5-bisphosphate, PI(4,5)P2, assumes a central position in this complex pathway. It can serve as a precursor for the generation of second messengers but can also act as a ligand to partner proteins. In order to mediate their physiological effects properly, the location and quantity of PI(4,5)P2 and other phosphoinositides have to be tightly controlled by enzymes. In general, phospholipid kinases lead to the activation of the pathway, whereas phospholipid phosphatases attenuate or terminate the signaling cascade. The SAC domain-containing protein 9 from Arabidopsis thaliana has been identified as a putative phosphoinositide phosphatase, but very little has been published on this particular protein. In my dissertation research, I broadened our knowledge of this protein and the effects seen in Arabidopsis plants carrying the mutant allele. I used molecular, genetic, and biochemical approaches to analyze the function of the putative phosphoinositide phosphatase, SAC9. To understand its physiological role, I investigated the cellular effects of a mutation in the SAC9 gene at the light microscopy, confocal microscopy, and transmission electron microscopy levels. My studies show that AtSAC9 is a soluble protein with an apparent molecular mass of 180 kDa and that it most likely is a phosphoinositide phosphatase. Furthermore, I show that the mutation of SAC9 induced unique cell wall defects that most likely have contributed to the stuntedness of the root. However, the cortical microtubule cytoskeleton was not disturbed in elongating root cells. These data are augmented by applying a novel approach for the mathematical analysis of cortical microtubule orientation.

O presente trabalho refere-se ao estudo do título putativo no âmbito da usucapio romana. A iusta causa é um dos requisitos fundamentais da usucapio, diante disso, a jurisprudência romana discutia se o usucapião deveria se proceder apenas diante a existência do título verdadeiro, ou se bastava a crença por parte do possuidor (título putativo) para que a usucapio acontecesse. A doutrina romanística, ao analisar os textos romanos, sugere que alguns textos referem-se a fortes indícios da admissão desse título, a exemplo do fragmento D, 41, 10, 5 de Nerácio, considerado, para muitos, o principal texto referente ao título putativo, e o fragmento D. 41, 10, 4, 2, de Pompônio. Entretanto, esse entendimento não é pacífico, na medida em que há quem considere que, na verdade, os referidos fragmentos do Digesto não guardavam relação com a admissão do título putativo, mas sim, que os referidos casos tratavam da verdadeira dispensa da causa para a admissão de uma usucapio que se procederia exclusivamente com base na boa-fé. O trabalho aborda a questão da causa na usucapio em suas diferentes modalidades: pro derelicto, pro emptore, pro legato, pro donato, pro dote, pro solutio, pro herede, também em relação aos chamados títulos inominados e na modalidade que, para muitos, é o principal ponto da controvérsia sobre o título putativo, usucapio pro suo. O estudo das diferentes espécies da usucapio é acompanhado pelos respectivos textos romanos. / This paper refers to the study of the putative causa within the Roman usucapio. The iusta causa is one of the fundamental requirements of usucapio, thereby, the Roman jurisprudence used to discuss whether the prescription should be undertaken only on the existence of the true causa, or if the belief of the holder was enough (putative causa) for the usucapio to happen. The romanistics studies, analyzing the Roman texts, suggests that some texts refer to strong admission of the evidence of that causa, like Neratius fragment D, 41, 10, 5, considered by many, the main text of the putative causa, and Pomponius fragment D. 41, 10, 4, 2. However, this is not a peaceful understanding, in the way that, there are some that consider that, in fact, those Digest fragments did not keep relation with the admission of the putative causa, but that those cases dealt with the real cause for the dismissal of acceptance of a usucapio that proceed exclusively based on good faith. This paper addresses the question of the causa in usucapio in its different kinds: pro derelicto, pro emptore, pro legato, pro donato, pro dote, pro solutio, pro herede, also in relation to others causae, and in the form that, for many, is the main point of controversy over the putative causa, the usucapio pro suo. The study of different kinds of usucapio is accompanied by their texts of Roman law.

O presente trabalho refere-se ao estudo do título putativo no âmbito da usucapio romana. A iusta causa é um dos requisitos fundamentais da usucapio, diante disso, a jurisprudência romana discutia se o usucapião deveria se proceder apenas diante a existência do título verdadeiro, ou se bastava a crença por parte do possuidor (título putativo) para que a usucapio acontecesse. A doutrina romanística, ao analisar os textos romanos, sugere que alguns textos referem-se a fortes indícios da admissão desse título, a exemplo do fragmento D, 41, 10, 5 de Nerácio, considerado, para muitos, o principal texto referente ao título putativo, e o fragmento D. 41, 10, 4, 2, de Pompônio. Entretanto, esse entendimento não é pacífico, na medida em que há quem considere que, na verdade, os referidos fragmentos do Digesto não guardavam relação com a admissão do título putativo, mas sim, que os referidos casos tratavam da verdadeira dispensa da causa para a admissão de uma usucapio que se procederia exclusivamente com base na boa-fé. O trabalho aborda a questão da causa na usucapio em suas diferentes modalidades: pro derelicto, pro emptore, pro legato, pro donato, pro dote, pro solutio, pro herede, também em relação aos chamados títulos inominados e na modalidade que, para muitos, é o principal ponto da controvérsia sobre o título putativo, usucapio pro suo. O estudo das diferentes espécies da usucapio é acompanhado pelos respectivos textos romanos. / This paper refers to the study of the putative causa within the Roman usucapio. The iusta causa is one of the fundamental requirements of usucapio, thereby, the Roman jurisprudence used to discuss whether the prescription should be undertaken only on the existence of the true causa, or if the belief of the holder was enough (putative causa) for the usucapio to happen. The romanistics studies, analyzing the Roman texts, suggests that some texts refer to strong admission of the evidence of that causa, like Neratius fragment D, 41, 10, 5, considered by many, the main text of the putative causa, and Pomponius fragment D. 41, 10, 4, 2. However, this is not a peaceful understanding, in the way that, there are some that consider that, in fact, those Digest fragments did not keep relation with the admission of the putative causa, but that those cases dealt with the real cause for the dismissal of acceptance of a usucapio that proceed exclusively based on good faith. This paper addresses the question of the causa in usucapio in its different kinds: pro derelicto, pro emptore, pro legato, pro donato, pro dote, pro solutio, pro herede, also in relation to others causae, and in the form that, for many, is the main point of controversy over the putative causa, the usucapio pro suo. The study of different kinds of usucapio is accompanied by their texts of Roman law.

<P> Type II Secretion (T2S) and type IV pilus (T4P) systems in Gram-negative bacteria share many features that suggest a common ancestral origin. This study examined the role of the minor pilins FimU, PilV, PilW, PilX and PilE, as well as the putative adhesin PilYl in both the T4P and T2S systems, and elucidated the role of these proteins in pilus assembly. Genetic analysis of the major pilin cluster and the minor pilin operon revealed that the major pilin alleles are associated with a specific set of minor pilins, and that unrelated strains of the same major pilin type have identical minor pilin genes, suggesting that the two gene clusters were horizontally acquired as a 'pilin island'. We observed that the minor pilins required a specific stoichiometric ratio for proper assembly, as overexpression either completely abolished, or significantly reduced twitching motility in mutant backgrounds. We demonstrated that the minor pilins were incorporated into the pilus fibre, and that they were dependent on PilA for surface localization. The T4P minor pilins were also shown to play a role in the secretion of effectors through the T2S system, as elastase and haemolytic phospholipase C secretion was reduced in minor pilin mutants, while overexpression of FimU or PilX significantly increased secretion of T2S exoproteins. Therefore, the minor pilins may participate in T2S substrate recognition. We found that PilYl was not essential for assembly in the absence of retraction, but that its absence caused changes in the levels of other T4P biogenesis proteins, namely FimU, PilW, PilF and PilQ secretin multimers. Finally we show that the minor pilin, PilX functions as a strain-specific factor, potentially through specific interactions with non-conserved residues of PilQ that are necessary to induce opening of the secretin. </p> / Thesis / Doctor of Philosophy (PhD)

<p> FKBP65 is a 65 kDa FK-506 binding protein containing 4 putative peptidyl prolyl isomerase (PPiase) domains, whose expression level parallels that oftropoelastin, the soluble precursor ofelastin. Studies from other laboratories have established that FKBP65 associates with tropoelastin (TE) in the endoplasmic reticulum (ER) and dissociates from TE before reaching the Golgi apparatus (Patterson et al., 2000). TE contains 12% proline residues, which are often found in VPGVG repeats, and it has been suggested that these repeats formp-turns and subsequently P-spirals (Urry et al., 1992). The formation ofthe P-spiral is thought to be essential to endow the elastic properties of the elastin fibers. In order to form a P-turn, the proline residue at position 2 ofthe VPGVG sequence must be in trans conformation (Urry et al., 1995). Therefore, it was hypothesized by Davis and coworkers (Davis et al., 1998) that FKBP65, as a PPiase, may play an important role in the folding oftropoelastin by enhancing the formation ofP-turns in the ER, and thus elastic fiber formation. In the present study we have studied the coacervation (a reversible, temperature-dependent, self association process) ofTE and recombinant elastin model polypeptide, EP4, in the absence or presence ofrecombinant FKBP65 (rFKBP65). rFKBP65 was shown to enhance the coacervation process of TE, by lowering the coacervation temperature (T c) and increasing the overall extent of coacervation. In the kinetic study ofcoacervation ofTE at a constant temperature, rFKBP65 increased both the initial rate ofthe coacervation process and the overall extent ofcoacervation. These effects are specific to rFKBP65, as FKBP12 has no effect on the coacervation process. Rapamycin, an inhibitor ofthe PPiase activity ofFK-506 binding proteins, did not alter rFKBP65's effect on TE coacervation. </p> <p>In contrast to TE, rFKBP65 affected the coacervation process ofEP4 by increasing the T c, and by enhancing the dissociation of coacervates when temperature is decreased. Once again, these effects are specific to rFKBP65, as FKBP12 and BSA were shown to have no effect on the coacervation ofEP4. The effect of small pH changes on rFKBP65 was also investigated, and it was found that lowering the pH from 7.5 to 6.0 had no effect on rFKBP65's secondary structure or coacervation-altering activity. </p> <p> In summary, this study, along with an earlier study from this laboratory, has shown that FKBP65 affects the coacervation process ofTE. In addition, the coacervation pro·cess of an elastin model polypeptide, EP4, is also modulated by FKBP65. However, the mechanism ofthese effects remains unclear. Nevertheless, along with the data established by other laboratories, FKBP65 does appear to be a strong candidate as a molecular chaperone for tropoelastin, and may play an important role in the elastogenesis process. </p> / Thesis / Master of Science (MSc)

<p> An area of current research interest involves the diacylglycerol kinase (DGK) family. Diacylglycerol kinases (DGKs) are a group of enzymes that phosphorylate diacylglycerol (DAG), a second messenger involved in cell signaling. The product of this reaction, phosphatidic acid (PA), also has signaling roles. An interesting isoform is DGKε, that although it has no identifiable regulatory domains other than the C1 domains. In addition, the catalytic domain is homologous to that of other DGK isoforms; however, DGKε exhibits an unusual specificity toward acyl chains of DAG, selectively phosphorylating an arachidonoyl-DAG substituted at the sn-2 position. Recently, researchers have identified an N-terminal hydrophobic domain of about 19 amino-acids in human DGKε. The present study attempted to identify the function of the N-terminal putative transmembrane domain of human DGKε and its relationship to the activity and substrate specificity of this enzyme by designing a truncated form of DGKε lacking the putative transmembrane domain.</p> <p> We have shown that the putative transmembrane domain of DGKε is not required for enzyme activity or for substrate specificity. In a mixed micellar assay the enzyme-catalyzed reaction followed surface dilution kinetics with respect to diacylglycerol and followed Michaelis-Menten kinetics with respect to ATP. The results show that the truncated form of the enzyme maintains substrate specificity for lipids with an arachidonoyl moiety present at the sn-2 position. The truncation increased the catalytic rate constant for all three substrates used in this study. It appears unlikely that the putative transmembrane domain, a segment unique to DGKε, has no functional role. It is possible that the hydrophobic segment may have a role in enzyme regulation by associating the enzyme in oligomers that are inactive in quiescent cells and get activated upon dissociation into monomers by increased levels of DAG in the membrane. We have shown that the presence of higher molecular species in the gel is not dependent on the presence or absence of the putative transmembrane domain. The only difference between the full-length and truncated enzyme is the monomer to dimer ratio. It appears likely that another segment of DGKε besides the putative transmembrane domain may be involved in oligomerization and that oligomerization is either transient or very weak. The absence of the hydrophobic domain of DGKε seems to cause no drastic changes either in the activity, the substrate specificity, or the state of oligomerization of the enzyme.</p> <p> Therefore, the next question is whether the hydrophobic domain of DGKε inserts itself in the membrane as a transmembrane helix or it only helps associate the enzyme to the surface of the membrane. We studied the topology of theN-terminal domain of DGKε in intact and permeabilized cells by indirect immunofluorescent microscopy. The results show that the N-terminal domain of the protein is present in the cytosol. The data supports a model in which the hydrophobic domain of DGKε forms a hydrophobic loop that attaches to the inner layer of the plasma membrane or that the hydrophobic domain attaches to the inner leaflet through its nonpolar surface of a horizontal helix. The first hypothesis is supported by the presence of a Pro residue in the middle of the hydrophobic domain. This Pro would introduce a kink in the helix creating a loop, but the absence of one or more glycine residues proximal to proline may hinder the formation of the loop. The second hypothesis is sustained by the presence of a polar surface on one side of the helical wheel. This orientation indicates the presence of a slightly horizontal helix attached to the surface of the inner layer of the plasma membrane.</p> <p> Regardless of the orientation of the helix, the weak association of the enzyme with the membrane is supported by previous data on the ease of extractability of the enzyme with high salts and on the Triton X-114 phase partitioning.</p> / Thesis / Master of Science (MSc)