The aim of this study was to assess the feasibility of using the budding yeast Saccharomyces cerevisiae as a system in which to analyse plant promoters. The promoters chosen for study were the 19S and 35S promoters of cauliflower mosaic virus (CaMV) which, like cellular plant promoters, are transcribed in the plant nucleus by host cell RNA polymerase II. A complete CaMV genome was introduced into yeast on a 2 micron plasmid-based vector and using Northern blot analysis, several CaMV-hybridising transcripts were detected. More precise information on the activity of the promoters was obtained by constructing gene fusions in which the 19S and 35S promoters were linked to the bacterial lacZ gene. Biochemical assays for β-galactosidase showed that cells harbouring the 19S-lacZ gene expressed β-galactosidase but those harbouring the 35S-lacZ gene did not. The insertion of a yeast transcription termination signal upstream of the 19S promoter did not abolish or diminish expression of the 19S-lacZ gene. β-galactosidase was present at low levels in cells expressing 19S-lacZ, constituting less than 0.01% of total cell protein. The 5'ends of 19S-lacZ transcripts present in yeast were mapped by primer extension. The major RNA species initiated approximately 250bp upstream of the 19S-lacZ coding region, indicating the existence of a fortuitous promoter in this region of the CaMV DNA. Two less abundant RNA species initiated within the 19S-lacZ open reading frame at positions +9 and +25bp and may be produced from the genuine 19S promoter. There is evidence to suggest that one or both of these shorter transcripts is the functional mRNA for β-galactosidase. All three classes of RNA were polyadenylated. Coupling of the 19S-lacZ gene to a yeast enhancer (the GAL UAS) produced a 5-fold increase in β-galactosidase activity. At the transcriptional level, activation of the enhancer resulted in a massive increase in the level of the RNA initiating at -250bp but had a minor influence of the levels of the two RNA species initiating at +9 and +25. A series of deletion mutations within the 19S promoter was constructed using Ba131 nuclease. Analysis of these mutations in yeast revealed that sequences from -500 to -193bp and from -137 to -62bp were not required for 19S promoter function, but a deletion from -62 to -21bp (which removes the putative TATA box) severely reduced 19S-1acZ gene expression. Transgenic tobacco plants containing the 19S promoter deletions fused to a CAT gene were produced by A.tumefaciens-mediated gene transfer but the analysis of these plants was not completed.

No description available.

A series of studies was conducted to examine the feasiblity of a bottom-up approach to the study of judgment of affective feeling states from facial behavior. Previous work on the judgment of emotion from facial expressions has taken a more top-down approach; observers judged the emotional meaning of a wholistic facial expression. Individual facial movements have sometimes then been identified within that complex expression, but the meaning of those individual movements has not been studied. A bottom-up approach begins by exploring the meaning of individual facial movements instead of complex facial expressions. In this approach the relationship between the emotional meaning of individual facial movements and complex facial expressions can be explored. It is argued that such an approach has the potential to explain judgment of not only a limited set of basic emotional expressions, but the full range of emotionally tinged feelings that individuals both experience in themselves and judge in others. Individual action units, as scored by Ekman and Friesen's (1978) Facial Action Coding System (FACS), and selected combinations of action units were presented to observers in three pairs of studies. Filmstrip sequences were used in the first pair of studies, and still photographs in the other two pairs. In the first study of each pair, observers judged the degree of pleasure and arousal expressed by the face. In the second study of each pair, observers rated how well each of a set of emotion terms described the feeling expressed by the face. Observers were found to reliably attribute meaning to individual action units on both scales. Additionally, pleasure and arousal judgments predicted emotion term ratings. The meaning attributed to combinations of action units was found to be related to the meanings of the individual action units occurring alone. Resultant ratings were shown to be meaningful within a dimensional model of emotion space. / Arts, Faculty of / Psychology, Department of / Graduate

The process by which information encoded m an organism's DNA is used in the synthesis of functional cell products is known as gene expression. In recent years, sequencing of RNA (RNA-seq) has emerged as the preferred technology for the simultaneous measurement of transcript sequences and their abundance. The analysis of RNA-seq data presents novel challenges and many methods have been developed for the purpose of mapping reads to genomic features and expression quantification. In the first part of my thesis I developed an R based pipeline for pre-processing, expression estimation and data quality assessment of RNA-seq datasets, which formed the basis for my subsequent work on the evolution of gene expression regulation in mammals. Since changes in gene expression levels are thought to underlie many of the phenotypic differences between species, identifying and characterising the regulatory mechanisms responsible for these changes is an important goal of molecular biology. For this, I studied the regulatory divergence of liver gene expression and of isoform usage between mouse strains. I demonstrate that gene expression diverges extensively between the strains and propose that the regulatory mechanism underlying divergent expression between two closely related mammalian species is a combination of variants that arise in cis and in trans. Isoform usage diverges to a lesser extent and appears to display a larger contribution of trans acting regulatory elements to its regulation, suggesting that isoform usage may be under different evolutionary constraints. These observations have important implications for understanding mammalian gene expression divergence and for understanding how speciation occurs.

No description available.

Nonverbal indices of pain are some of the least researched sources of data for assessing pain. The extensive literature on the communicative functions of nonverbal facial expressions suggests that there is potentially much information to be gained in studying facial expressions associated with pain. Results from two studies support the position that facial expressions related to pain may indeed be a source of information for pain assessment. A review of the literature found several studies indicating that judges could make discriminations amongst levels of discomfort from viewing a person's facial expressions. Other studies found that the occurrence of a small set of facial movements could be used to discriminate amongst several levels of self-reported discomfort. However, there was no research directly addressing the question of whether judges ratings would vary in response to different patterns of the identified facial movements. Issues regarding the facial cues used by naive judges in making ratings of another person's discomfort were investigated. Four hypotheses were developed. From prior research using the Facial Action Coding System (FACS) (Ekman S. Friesen, 1978) a small set of facial muscle movements, termed Action Units (AUs), were found to be the best facial movements for discriminating amongst different levels of pain. The first hypothesis was that increasing the number of AUs per expression would lead to increased ratings of discomfort. The second hypothesis was that video segments with the AUs portrayed simultaneously would be rated higher than segments with the same AUs portrayed in a sequential configuration. Four encoders portrayed all configurations. The configurations were randomly editted onto video tape and presented to the judges. The judges used the scale of affective discomfort developed by Gracely, McGrath, and Dubner (1978). Twenty-five male and 25 female university students volunteered as judges. The results supported both hypotheses. Increasing the number of AUs per expression led to a sharp rise in judges' ratings. Video segments of overlapping AU configurations were rated higher than segments with non-averlapping configurations. Female judges always rated higher than male judges. The second study was methodologically similar to the first study. The major hypothesis was that expressions with only upper face AUs would be rated as more often indicating attempts to hide an expression than lower face expressions. This study contained a subset of expressions that were identical to ones used in the first study. This allowed for testing of the fourth hypothesis which stated that the ratings of this subset of expressions would differ between the studies due to the differences in the judgment conditions. Both hypotheses were again supported. Upper face expressions were more often judged as portraying attempts by the encoders to hide their expressions. Analysis of the fourth hypothesis revealed that the expressions were rated higher in study 2 than study 1. A sex of judge X judgment condition interaction indicated that females rated higher in study 1 but males rated higher in study 2. The results from these studies indicated that the nonverbal communication of facial expressions of pain was defined by a number of parameters which led judges to alter their ratings depending on the parameters of the facial expressions being viewed. While studies of the micro-behavioral aspects of facial expressions are new, the present studies suggest that such research is integral to understanding the complex communication functions of nonverbal facial expressions. / Arts, Faculty of / Psychology, Department of / Graduate

The building for the centre for youth sub cultural expression focuses on the idea of resistance as form giver in architecture. Resistance [a notion to dislike of or opposition to a plan, an idea] refusal to obey. The aim of the discourse is to express how an abject psychological perception of a group can be transformed positively through architecture. Architecture should not just become a sculptural manifestation but should communicate and respond to its context. The goal is to create an environment that acts as a platform for sub cultural expression but also becomes a domain for youth identity formation. The emphasis is on the exposure of sub cultural arts and artists, thereby potentially establishing a foundation where the South African visual culture can be booted by a fresh and youthful injection of ideas, medium and content. / Dissertation (MArch (Prof))--University of Pretoria, 2007. / Architecture / unrestricted

archives@tulane.edu / G protein-coupled receptors (GPCRs) constitute the largest family of human proteins with approximately around 800 genes and, are therapeutic targets for more than 35% of the FDA-approved drugs with around 180 billion dollars in sales. The therapeutic potential of GPCRs is still not completely utilized. Structure-guided drug discovery will definitely aid in identifying novel compounds with therapeutic responses while minimizing adverse effects in patients. Multiple protein engineering strategies including deletions, truncations, insertion of stable soluble protein or thermostabilizing mutations have been utilized to obtain the structural information of these membrane proteins. Yet to date, the high-resolution structure of only 63 GPCRs have been determined, with only a few of these structures being of active protein. Heterologous expression of most GPCRs results in low yields of active receptors and additional strategies are required to improve active receptor yields for these “hard-to-express” GPCRs. Yeast provides a unique heterologous expression platform to produce mammalian GPCRs, as it also allows us to measure downstream signaling activity with an easy and inexpensive high-throughput assay via its MAPK response pathway. The adenosine A2A receptor (A2AR) shows exceptional expression and trafficking to the plasma membrane in yeast; however, this is not the case for other adenosine receptors. A2AR has a longer C-terminus than the other adenosine receptor subtypes, which may contribute to its exceptional trafficking to the plasma membrane. To test the possibility to improve trafficking of the adenosine A1 receptor (A1R), chimeric receptors containing the seven transmembrane domains of A1R and the full-length or truncated A2AR C-terminus were constructed. The chimeric receptor showed improved localization to the plasma membrane and was capable of binding radioligand with native-like A1R affinity. Functionally active A1R receptor variants were produced at a theoretical yield of 95 pmol/mg total membrane protein, estimated using radioligand binding data, which is greater than three-fold higher than previously reported yields from other heterologous expression systems, and should facilitate biophysical characterization and drug discovery efforts. Recent efforts to determine the high-resolution crystal structures for the adenosine A1 and A2A receptors have utilized modifications to the native receptors in order to facilitate receptor crystallization and structure determination. One common modification is a truncation of the unstructured C-terminus, which has been employed for all the adenosine crystal structures obtained to date. Here, the presence of the full-length C-terminus is shown to affect downstream signaling using a yeast MAPK response-based fluorescence assay. Upon ligand binding, the A1d291R or A2Ad316R variants were unable to couple to human-yeast chimeric G-protein chimeras to generate a downstream signal in yeast, though full-length receptors showed native-like G-protein coupling. Further, constructs transfected into HEK-293 cells showed similar behavior – i.e. the variants with C-terminal truncations lacked cAMP-linked signaling compared to the full-length receptors. Although the C-terminus was essential for Ga protein- associated signaling, chimeras of A1R with a C-terminus of A2AR coupled to the A1R-specific Ga (i.e. Gai1 versus Gas). This surprising result suggests that the C-terminus is important in signaling, but not specificity, for the interaction with Ga protein. Based on these results for chimeric A1R variants, chimeric variants for adenosine A3R and tachykinin 2 receptor (NK2R) were constructed. The A3/A2AR variant was constructed using an approach similar to that for A1/A2AR; the resulting construct showed a two-fold increased expression as compared to the wild-type A3R. This chimeric variant showed native-like signaling activity in engineered yeast strains with a modified MAPK pathway, whereas the wild-type receptor showed no activity. In order to improve the functional expression of human NK2R required for high-resolution structural studies, NK2R chimeras utilizing sequences from rat NK2R previously shown to have a higher functional expression in yeast than human NK2R were utilized. Herein, we show that NK2R chimeras incorporating the rat NK2R C-terminus demonstrated improved expression, ligand binding, and downstream signaling in engineered yeast strains. Taken together, these results highlight the utility of the chimeric receptor engineering strategy to facilitate the expression of active receptors. / 0 / Abhinav Rabindra Jain

Newspapers often report on the violation of learners’ rights. Many of these violations and critical incidents are related to the right to freedom of expression, which is internationally viewed as a core right in a democracy. My inquiry focused on grade 11 learners’ understanding of the right to freedom of expression. It is important to understand learners’ understanding of the right to freedom of expression, as they are the leaders of tomorrow and the right to freedom of expression is central to the survival of democracy. This inquiry was informed by an interpretivist paradigm. Atlas.ti™ was used to systematically analyse the data and categorise it into three hermeneutic units. This computer-aided qualitative data analysis software (CAQDAS) tool facilitated the process of analysis and enhanced the validity of the research. During the inductive process of analysis two patterns crystallised, viz. the absolutising of the right to freedom of expression by some learners and the lack of skills to implement the limitation to the right to freedom of expression in schools. The main findings indicated that some learners did not have knowledge regarding the right to freedom of expression, although most learners were aware that they could speak their minds under this right. Furthermore, learners didn’t seem to know how to exercise the right to freedom of expression. One of the factors disrupting the implementation of the right to freedom of expression in schools is the perpetuation of an authoritarian culture in schools. Theoretical and practical recommendations are suggested and avenues for future research are identified. / Thesis (PhD (Education Management))--University of Pretoria, 2005. / Education Management and Policy Studies / unrestricted

Plant-parasitic nematodes are the cause of devastating yield loss in vital agricultural crops around the world. Heterodera glycines, also referred to as soybean cyst nematode, is the main pathogen of Glycine max (soybean) causing more loss than all other pathogens of G. max combined. The resultant economic impact due to H. glycines in United States soybean production alone is estimated to account for an annual one-billion-dollar loss. Natural resistant genotypes have been found in trials to combat this pathogen. Of the resistant varieties identified, G. max[Peking/PI 548402] and G. max[PI 88788] are the major sources of resistance. Identification of genes expressed in the cells of which the nematode parasitizes, the syncytia, exclusively undergoing the resistant/incompatible reaction from the two major sources of resistance mentioned previously have identified a number of candidate genes presumed to function in defense to H. glycines parasitism. Prior to this work, success has been obtained by selection of a number of these candidate genes in functional analysis to show involvement in defense. This work is aimed at functionally identifying signaling components involved in the defense reaction. Reverse genetic studies of NON-RACE SPECIFIC DISEASE RESISTANCE 1 Glycine max homolog, Gm-NDR1-1, has confirmed a functional role in the defense to H. glycines to G. max. Gene expression studies revealed both effector-triggered immunity (ETI) and pattern-triggered immunity (PTI) components to be regulated by Gm-NDR1-1. Furthermore, induction in the heterologous expression of Gm-NDR1-1 in Gossypium hirsutum (cotton) suppressed Meloidogyne incognita parasitism. Harpin treatment has been evaluated due to the knowledge of NDR1’s capability of being harpin-induced (HIN1). Expression studies of the harpin treatment did in fact induce Gm-NDR1-1. The analysis further provides evidence of NDR1 role in defense by displaying the harpin-induced response of NDR1 in resistance to infection of Rotylenchulus reniformis. Receptors are known to function through signaling components in plant defense. Therefore, the conserved downstream signaling component of multiple diverse stimuli, mitogen-activated protein kinases (MAPKs) were functionally characterized in G. max for their role in resistance to H. glycines via the reverse genetic parasitism assays and evaluated to observe the effect on defense gene expression.