Drought is one of the most important stress factors affecting plants. Increasing drought resistance via plant breeding is currently the main approach for improvement of plant productivity and reduction of water usage. Plant breeders and scientists search for reliable and easy methods of the determination of drought sensitivity in different genotypes. A precise knowledge about processes underlying plant stress response and defence against limited water availability is necessary for this. The aim of this study was a detailed analysis of photosynthetic processes and changes in leaf proteome (again aimed especially at proteins involved in photosynthesis) during drought stress. This analysis was a part of a complex project focused on the possible causes of genetic variability and drought resistance in agronomically important crops. Another goal was to analyse whether the measurement of selected photosynthetic parameters can be used for the determination of drought sensitivity per se and/or for the prediction of the response of hybrids based on known behaviour of their parents. Young plants of maize inbred lines and their hybrids were used as a model for this analysis, which was made under conditions of moderate and more severe drought. Two inbred lines which were (together with their F1 hybrids) used...

Pseudoroegneria spicata and Elymus wawawaiensis are two native perennial bunchgrasses of North America's Intermountain West. Frequent drought, past overgrazing practices, subsequent weed invasions, and increased wildfire frequency have combined to severely degrade natural landscapes in the region, leading to a decline in the abundance of native vegetation. Being formerly widespread throughout the region, P. spicata is a favorite for restoration purposes in the Intermountain West. Elymus wawawaiensis, which occupies a more restricted distribution in the Intermountain West, is often used as a restoration surrogate for P. spicata. However, since most restoration sites are outside the native range of E. wawawaiensis and as the use of native plant material may be more desirable than a surrogate, the use of E. wawawaiensis as a restoration plant material has been somewhat controversial. The main goal of my research was to identify plant materials of these species with superior seedling growth, drought tolerance, and defoliation tolerance, traits that may contribute to enhanced ecological function in restored rangeland plant communities. I conducted a growth-chamber study to evaluate morphological and growth-related traits of germinating seedlings of these two species. My study suggested that, while the two bunchgrasses are similar in many ways, they display fundamentally different strategies at the very-young seedling stage. While P. spicata exhibited greater shoot and root biomass to enhance establishment, E. wawawaiensis displayed high specific leaf area (SLA) and specific root length (SRL), two traits commonly associated with faster growth. According to the eco-physiology literature, plants with greater stress tolerance display lesser growth potential. However, my greenhouse study showed that E. wawawaiensis was relatively more drought tolerant than P. spicata, despite higher expression of growth-related traits, e.g., SLA and SRL. While the two species displayed similar water use efficiency when water was abundant, E. wawawaiensis was also more efficient in its water use when drought stress was imposed. In a field study, I found E. wawawaiensis to be twice as defoliation tolerant as P. spicata. This study showed that P. spicata is typically more productive in the absence of defoliation, but E. wawawaiensis was more productive after defoliation due to its superior ability to recover and hence is a better candidate for rangelands that will be grazed. Hence, my study showed that E. wawawaiensis, despite being regarded as a surrogate for P. spicata, exhibits superior seedling establishment, drought tolerance, and defoliation tolerance. Therefore, E. wawawaiensis has advantages as a restoration species for the Intermountain West.

L’amélioration génétique de la luzerne (Medicago sativa), une légumineuse fourragère autotétraploïde, pourrait bénéficier de l’allele mining. Cette méthode consiste à rechercher, dans la diversité naturelle, des allèles ayant potentiellement un effet sur le phénotype. Pour évaluer l’intérêt de cette stratégie qui exploite la diversité naturelle, cinq gènes impliqués dans des caractères sélectionnés ont été retenus : CAD1 et CCoaOMT (digestibilité), CONSTANS-like (rendement fourrager), NHX1 (tolérance à la salinité) et WXP1 (tolérance à la sécheresse). La diversité de ces gènes a été étudiée en séquençant 387 génotypes cultivés et 20 génotypes sauvages. L’analyse des données confirme la présence d’un goulot d’étranglement durant la domestication et la sélection de la luzerne. CONSTANS-like et WXP1 révèlent de nombreux variants alors que CAD1, CCoaOMT et NHX1 sont très peu variables. Des variants ayant un effet potentiel sur le phénotype ont été identifiés dans les zones de la séquence protéique qui sont conservées au sein des Faboideae. L’impact sur le phénotype de deux variants du gène CONSTANS-like a été étudié : Constans-634, à l’origine d’un codon stop prématuré et Constans-4111, situé dans une région conservée du gène. Pour cela des croisements ont été réalisés afin d’obtenir une descendance avec toutes les doses possibles (AAAA, AAAB, AABB, ABBB and BBBB) pour chaque mutation étudiée. Des marqueurs KASPar ont été développés afin de déterminer les doses de mutations chez les descendants. Aucun génotype homozygote muté pour Constans-634 n’a été détecté parmi les 1505 descendants. Ce variant a induit une floraison plus précoce de trois jours pour les génotypes portant trois doses d’allèle muté. Le variant Constans-4111 a induit un effet additif sur la hauteur de tige. Les génotypes homozygotes de type sauvage étaient en moyenne 11,8 cm plus petits que les génotypes homozygotes portant trois ou quatre doses du variant. L’intégration de la stratégie allele mining dans les schémas de sélection des espèces végétales autotétraploïdes hétérozygotes a été discutée. / Lucerne (Medicago sativa) is an autotetraploid forage legume, whose breeding could beneficiate from allele mining. This strategy is based on the natural diversity and consists in seeking alleles with a potential effect on the phenotype. The interest of this approach was evaluated by studying five genes of agronomic interest: CAD1 and CCoaOMT (digestibility), CONSTANS-like (forage yield), NHX1 (salt tolerance) and WXP1 (drought tolerance). The diversity of these five genes was evaluated by sequencing 387 genotypes of cultivated accessions and 20 genotypes of wild accessions. The results confirmed a bottleneck during lucerne domestication and selection. CONSTANS-like and WXP1 were very variable whereas CAD1, CCoaOMT and NHX1 contained very few variants. Variants with a potential strong impact on the phenotype were identified in conserved parts of protein sequence within the Faboideae. The impact on phenotype was studied for two mutations of the CONSTANS-like gene: constans-634, causing a premature stop codon and constans-4111, located in a conserved region of the gene. Genotypes carrying one to three doses of the mutations (AAAB, AABB and ABBB) were polycrossed in order to obtain offsprings with every allele combination (AAAA, AAAB, AABB, ABBB and BBBB). KASPar markers were developed to determine the mutation doses in offspring progeny. No homozygous genotype was found for constans-634 in the 1505 offspring progeny. This mutation induced a premature flowering of three days for the genotypes carrying three doses of the mutation. The mutation constans-4111 induced an additive effect on stem height and the homozygous genotypes without the variant where on average 11.8 cm shorter than homozygous genotypes carrying three or four doses of the variant. The application of allele mining strategy in plant schemes of heterozygous autotetraploid species was discussed.

Цель работы – отобрать генотипы растений рода Alissum устойчивые к повышенным концентрациям ионов никеля, меди и цинка. Были выбраны 3 вида растений из рода Alissum sp. семейства Brassicaceae, а именно: A. litvinovii, A. tortuosum, A. оbovatum. В ходе эксперимента из семян были получены растения, которых подвергли микроклональному размножению. Растения культивированы на средах, содержащих Cu2+, Ni2+ или Zn2+ в концентрации 50 – 100 – 200 µМ/л. В течение 6 недель проводили морфологические измерения и описания. После эксперимента было определено содержание ионов Ni2+ в сырой биомассе с использованием реактива Чугаева. Отобраны генотипы растений, устойчивые к избытку тяжелых металлов в среде и способные к активному росту в условиях стресса, генотип A. tortuosum № 6 превысил контроль в 2,6 раза, достигнув размера 46,7 мм (200 µМ Zn2+). Растения показали аккумулирующие способности в отношении ионов никеля в условиях in vitro. Генотип № 5 A. оbovatum аккумулирует 1,59 мг/г Ni2+ сырой массы, что в 8 раз превысило контроль (200 µМ Ni2+). Данное исследование подтверждает перспективность использования растений из рода Alyssum для фиторемедиации. Выпускная квалификационная работа магистра – 61 стр., 21 рис., 5табл., 81 литературных источников, из которых русскоязычных 50, англоязычных 31. / The purpose of the work is to select plant genotypes of the genus Alissum that are resistant to increased concentrations of nickel, copper and zinc ions. Three species of plants from the genus Alissum sp. Brassicaceae family, namely: A. litvinovii, A. tortuosum, A. obovatum. During the experiment, plants were obtained from seeds, which were subjected to microclonal propagation. Plants were cultivated on media containing Cu2+, Ni2+ or Zn2 + at a concentration of 50 – 100 – 200 µM/L. Morphological measurements and descriptions were performed over 6 weeks. After the experiment, the content of Ni2+ ions in the raw biomass was determined using the Chugaev reagent. Plant genotypes that are resistant to an excess of heavy metals in the medium and capable of active growth under stress were selected; A. tortuosum genotype № 6 exceeded the control by 2.6 times and reached a size of 46.7 mm (200 µM Zn2+). Plants showed an accumulating ability with respect to nickel ions in vitro. Genotype № 5 A. obovatum accumulates 1.59 mg/g Ni2+ wet weight, which is 8 times higher than the control (200 µM Ni2+). This study confirms the promise of using plants from the genus Alyssum for phytoremediation. The final qualifying work of the master is 61 pages, 21 figures, 5 tables, 81 literary sources, of which 50 are Russian-speaking and 31 are English-speaking.

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Soybean (Glycine max L.) is an important legume crop often exposed to heat and drought stresses during reproductive and early-seed setting stages, resulting in lower yields and seed quality. Ten soybean cultivars were phenotyped for individual (drought or heat) and combined stress tolerance. Under drought, reduced stomatal conductance and increased canopy temperature significantly reduced seed number (46%) and weight (35%). Heat stress alone reduced seed number (19%) and weight (23%) compared to control. Moreover, a degree increase in daytime temperature above 32 °C during the reproductive stage reduced seed weight by 4% and 7% under well-watered and drought conditions, respectively. Seed protein was increased under drought, while it declined under heat and combined stress compared to control. In contrast, oil content showed the opposite trend. Weak correlations between phenotypic traits under individual and combined stress suggest that selecting cultivars for individual stress tolerance may not work under combined stress conditions.

The genus Salmonella consists of pathogenic gram negative organisms which infect intestines of birds, animals and humans. They are the causative agents of salmonellosis which is characterised by diarrhoea, nausea, fever and abdominal cramps. If not treated in time, salmonellosis can also be fatal. Salmonella genus is divided into two species Salmonella bongori and Salmonella enterica. Salmonella enterica is further divided into six subspecies out of which the subspecies enterica has many of the pathogenic serovars of this species. Salmonella typhimurium is a server in the subspecies enterica of Salmonella enterica species. Transmission of salmonellosis takes place through contaminated food and water. When the organism enters a host, it encounters a range of hostile environments such as acidic pH, lack of oxygen as well as immune response of the host. In order to establish infection, the bacterium needs to survive under stressful conditions and propagate itself. Various proteins are induced in cells under unfavourable conditions that protect them in such situations. One such group of proteins belongs to the Universal Stress Protein (USP) family. Universal Stress Proteins are a set of proteins induced in organisms when it is exposed to a variety of environmental insults including heat shock, nutrient starvation, presence of toxic compounds, etc. Although survival in adverse conditions is mediated by induction of this group of proteins, the precise mechanism of cellular protection has not been elucidated yet. The functional role of a protein is directly related to its three-dimensional structure and hence important insights can be gained regarding the role of these proteins by determining their structures. The structures of two Universal Stress Proteins from S. typhimurium; a single domain protein, YnaF and another tandem USP domain protein, YdaA were determined by X-ray crystallography and biochemical analysis was carried out on them. Guided by structure, plausible roles for both the proteins in stress tolerance of S. typhimurium have been proposed. Additionally, work was also carried out on phosphomannose isomerise from S. typhimurium. Phosphomannose isomerase is a housekeeping enzyme which catalyses the interconversion of mannose-6-phosphate and fructose-6-phosphate. Mannose is important for mannosylation of various lipids and proteins which form an important component of bacterial and fungal cell walls. Presence of a functional phosphomannose isomerise enzyme is important as it helps the organism survive adverse conditions by forming a strong cell wall which shields it from harmful environments. Moreover, phosphomannose isomerase was also found to be essential for virulence of Leishmania mexicana and Cryptococcus neoformans. The structure of phosphomannose isomerase from S. typhimurium was determined in our laboratory in the year 2009. However, in the earlier studies, the catalytically important residues had not been identified and mechanism of isomerisation was not established. Structural analysis, site directed mutagenesis and biochemical assays were used to identify key residues in the active site of StPMI. Identification of these residues might help in deciphering the catalytic mechanism which will eventually be useful to develop inhibitors that arrest the growth of Salmonella as well as other microorganisms. The work reported in this thesis describes the efforts made to enhance our understanding of functional aspects of the two Universal Stress Proteins, YnaF and YdaA and phosphomannose isomerase from S. typhimurium. Chapter 1 begins with a brief introduction to the kinds of unfavourable environments encountered by microorganisms and their strategies of adaptation. This is followed by a review of the literature on Universal Stress Proteins, which are induced in many organisms in response to arrest of or perturbations in the growth rate. Structural, biochemical and evolutionary aspects of members of the family have also been discussed. Subsequently, a brief description of the earlier work carried out on another enzyme important in stress tolerance, phosphomannose isomerase, has been documented. A detailed account of mechanisms of isomerisation carried out by aldose ketose isomerases and identification of important strategies for determination of mechanism of phosphomannose isomerase catalysed reaction have then been provided. The chapter ends with a summary of aims and objectives of the present work. Chapter 2 describes the various experimental techniques and computational methods used during the course of this thesis work. Isolation of plasmids, overexpression and purification of protein, site directed mutagenesis, biochemical assays, crystallisation of proteins, X ray diffraction data collection form a part of the experimental aspect and have been described in detail. Brief descriptions of the programs used and principles behind computational methods used for structure determination (including data processing, phasing, model building and refinement), validation and analysis have also been provided. Chapter 3 includes the structural and functional studies carried out on YdaA, a tandem USP domain protein from S. typhimurium. Expression, purification, crystallisation and structure determination of YdaA in its native and ADP bound forms are described in the chapter. Biochemical assays with radiolabelled ATP showed that YdaA was an ATPase. The crystal structure of YdaA complexed with ATP revealed the presence of ADP (hydrolysis product of ATP) only in the C-terminal domain of the protein. Based on structural analysis and presence of ATP binding motif in the C-terminal domain, it could be hypothesized that ATP hydrolysis activity of the protein is confined to the C-terminal domain of the protein. The N-terminal domain of the protein was found to play another interesting role. A zinc binding site could be identified in the N terminal domain based on structural analysis and elemental X-ray absorption studies done at the synchrotron. Site directed mutagenesis and biochemical experiments suggested that zinc binding in the N-terminal domain was not related to ATPase activity of the C-terminal domain. Additionally, an intermediate of lipid A biosynthesis pathway UDP-(3-O-(R-3-hydroxymyristoyl))-N-acetyl glucosamine was found bound to the N-terminal domain of YdaA. Lipid A is the membrane anchor of polysaccharides in the outer membrane of gram negative organisms and the intermediate occurs at the committed step of the pathway. However, no similarities could be identified between YdaA and members of the relevant biosynthetic pathway. Therefore, YdaA is unlikely to play a catalytic role in the same pathway but can function as a carrier molecule. A plausible link between the N- and C-terminal domains of YdaA could be identified by structural analysis. Many catalytically suitable residues from the N-terminal domain were found to be close to the β-phosphate of ADP bound to the C-terminal domain. Hence YdaA was identified to be a zinc binding ATPase which might play some yet unidentified role in lipid A biosynthesis pathway. Chapter 4 describes the attempts made towards understanding the functional role of YnaF, a single domain USP from S. typhimurium. A description of the expression, purification, crystallisation and X ray diffraction techniques used for structure determination of YnaF and its single site mutant have been provided in detail. Gel filtration, dynamic light scattering studies and the crystal structure determination of YnaF showed a tetrameric organisation of four USP protomers stabilised in the centre by chloride ions. Additionally, YnaF crystallised with a bound ATP even though ATP was not included in the crystallisation cocktail. Biochemical assays on YnaF with radiolabelled ATP showed that it was inactive with respect to ATP hydrolysis. When selected mutations that disrupt chloride binding were made, YnaF was converted to an active ATPase. The crystal structure of the mutant complexed with an ATP analogue revealed key differences at the active site in comparison with that of the wild type and allowed identification of residues that might be important for ATP hydrolysis in this group of proteins. Hence YnaF might play the role of a sensor protein in some signal transduction pathway involving chloride ions in bacteria. A structure based analysis and comparison of USPs from the Protein Data Bank with the structures of YnaF and YdaA is summarised at the end of this chapter. Chapter 5 describes the efforts carried out towards determination of mechanism of isomerisation catalysed by phosphomannose isomerise (PMI). Earlier reports suggest that the enzyme catalyses the reversible isomerisation of mannose-6-phosphate and fructose-6-phosphate via formation of a cis-enediol intermediate. The structure of phosphomannose isomerase from S. typhimurium has been reported by our laboratory. The enzyme is a monomer with three domains; a catalytic domain, a carboxy terminal domain and an α-helical domain. Residues from the catalytic domain were found to coordinate a zinc ion. Overexpression, purification, co crystallisation experiments and soaking studies carried out on crystals of PMI and its single site mutants are outlined in this chapter. The structure of a complex of PMI with mannose-6-phosphate at pH 7.0 revealed the presence of a blob of density close to the zinc binding site which was confirmed to be the active site by analysis of conservation of residues in the site. Based on site directed mutagenesis, activity studies and analysis of structure of PMI, zinc was identified to play an important role in maintaining the structural integrity of the active site. Electrostatic surface analysis of the structure of PMI revealed that the zinc ion might also play the role of anchoring phosphate moiety of the substrate in a highly negatively charged active site pocket. Activity assays following site directed mutagenesis studies eliminated the role of Glu264 in catalysis and implicated two lysines, Lys86 and Lys132 as the possible base in the reaction. The plausible role of a highly conserved residue Arg274 was also proposed based on comparison of structures of wild type and mutant PMIs. The future prospects of the work are briefly discussed towards the end of the thesis. Further experiments and analysis required to obtain better understanding of the functions of these proteins have been discussed. The Appendix section describes extensive crystallisation attempts that were carried out on the enzyme sorbitol-6-phosphate-dehydrogenase from S. typhimurium which catalyses the isomerisation reaction between sorbitol-6-phosphate and glucose-6-phosphate using NADPH as the cofactor. Needle shaped crystals were obtained which diffracted to a poor resolution of 7-8 Å at our in house X ray facility. Attempts to improve the quality of the crystals like co crystallisation with substrate and its analogues, soaking in various compounds and seeding are briefly described. The following manuscripts based on work described in this thesis have been published or will be communicated for publication. 1. Structural and functional analysis of two universal stress proteins YdaA and YnaF from Salmonella typhimurium: possible roles in microbial stress tolerance. Bangera M., Panigrahi R., Sagurthi S.R., Savithri H.S., Murthy M.R.N. Journal of Structural Biology, 2015 Mar; 189 (3): 238-50. 2. Structural and functional insights into phosphomannose isomerise: role of zinc and catalytic residues. Bangera M., Savithri H.S., Murthy M.R.N. Manuscript under preparation

Une question centrale de la génétique moderne est de mieux comprendre comment la variation génétique présente au sein d’individus d’une même espèce influence la diversité phénotypique et l’évolution. La levure modèle Saccharomyces cerevisiae offre une occasion unique d’apporter des éléments de réponse à cette question à travers la dissection de l’architecture génétique de la variation de tolérance à des stress environnementaux à l’échelle d’une population. Mon étude révèle un niveau supplémentaire de complexité de la relation génotype-phénotype où finalement les caractères supposés les plus simples, dits Mendéliens (déterminisme strictement monogénique) peuvent se révéler être complexes (déterminisme multigénique) selon le fond génétique en raison de l’action de gènes modificateurs, d’interactions épistatiques et/ou de suppresseurs. Toutefois, les processus évolutifs peuvent être bien différents en fonction des espèces. Afin de mieux les décrypter, je me suis également intéressée à Lachancea kluyveri, une levure phylogénétiquement distante de S. cerevisiae. Cette espèce présente une diversité génétique plus élevée et constitue une ressource encore peu exploitée. L’exploration de la diversité phénotypique et la détermination de leurs origines génétiques initiées dans cette étude sont extrêmement prometteuses et apportent de solides fondations pour l’étude à la fois de l’architecture génétique des caractères et de l’évolution de la relation génotype-phénotype au sein de diverses espèces de levures. / A central issue of modern genetics is to better understand how genetic variations between individuals within a species influence the phenotypic diversity and the evolution. The budding yeast Saccharomyces cerevisiae as a model organism offers a unique opportunity to address this issue through the dissection of the genetic architecture of stress tolerance across a population. My study reveals an additional level of complexity of the genotype-phenotype relationship. Indeed, simple Mendelian traits (monogenic determinism) may become more complex (multigenic determinism) depending on genetic background due to the action of modifier genes, epistatic interactions and / or suppressors. However, evolutionary processes can be very different depending on the species. That is why a non-conventional yeast species namely Lachancea kluyveri (formerly S. kluyveri) was also studied. This species distantly related to S. cerevisiae has a higher genetic diversity and remains a relatively unexplored resource. The exploration of the phenotypic diversity and the determination of the genetic origins initiated in this study lay foundations for the analysis of the genetic architecture of traits and the evolution of the genotype-phenotype relationship within diverse yeast species.

Freshwater green algae within species complex Haematococcus pluvialis are best natural producers of secondary carotenoid astaxanthin famous for its strong antioxidant activity. Despite worldwide distribution, great biotechnological potential and extensive literature dealing with different approaches to the cultivation of these organisms, there still remains surprising gaps in knowledge of their complex life cycle and behaviour. First uncertainty concerns asexual cell division of zoospores, hypothetically limited to five cell doublings (Lee & Ding 1994). No such genetic limitation was observed in this thesis, number of cell divisions is thus influenced mainly by cultivation conditions. There is still considerable uncertainty regarding to the asexual reproduction, as well. Ambiguity might be caused by scarce observation of the whole process of gametogenesis and conjugation of gametes. Published studies differed in some particular points. Unfortunately, we did not succeed in induction of gametogenesis despite broad scale of tested cultivation conditions applied to two different species within the H. pluvialis complex, namely H. pluvialis and H. rubicundus. This failure might be caused by heterothalism of the studied strains. Haematococcus pluvialis inhabits mainly ephemeral pools. Its ability to...

The current study describes the impact of trauma on the perceptual thinking processes of participants who have sustained repeated criminal victimisation in South Africa. Fifteen adult males, (mean age 40.73 years) participated voluntarily in this descriptive study. The Rorschach Inkblot Method (RIM) is the psychometric instrument used to describe the participants and Exner’s Comprehensive System (CS) is the method of interpretation employed. The Perceptual Thinking Index (PTI) is the cluster of variables selected to describe each participant’s level of reality testing and perceptual functioning. Together with this, the D and Adjusted D Score variables are discussed to describe the participants’ overall and current coping capacities. From the research findings yielded in this study, it appears that the majority of these participants (73%) can be described as experiencing problems in terms of their perceptual thinking processes. They are also unable to appropriately engage in accurate reality testing. The results of the research study may serve as an incentive for further studies of this nature as according to the RIM, participants with this level of impaired reality testing generally present with psychotic features. However, these research results were found in participants who present with an absence of a psychotic or any schizophrenic type disorder diagnoses. Other researchers who used the RIM to assess trauma victims have found results similar to this. Furthermore, participants who present with this level of impaired reality testing, generally experience severe problems in coping with basic psychological aspects of daily functioning. This however, does not appear to be the case with this sample group, which was one of the motivating factors for conducting this research. A concluding suggestion is made for possible research into investigating how, and at what expense, other psychological features of functioning are employed, in order to manage in a seemingly well-adaptive manner. / Dissertation (MA (Clinical Psychology))--University of Pretoria, 2006. / Psychology / unrestricted