Includes bibliographical references. / Plants rely on an elaborate multi-layered defence system to perceive and effectively respond to disease causing pathogens. The defence-related cir1 (constitutively induced resistance 1) mutant was first isolated in an effort to identify components of the Arabidopsis thaliana defence system essential for resistance against pathogens. The cir1 mutant has previously been described as having increased resistance to the virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000 and oomycete pathogen Hyaloperonospora parasitica Noco2 and was shown to constitutively express salicyclic acid-, jasmonic acid/ethylene- and reactive oxygen intermediate-responsive genes. Genetic analysis and mapping studies of the mutation revealed that it is recessive and may be encoded by one of eight genes located within a 309.10 kb region on the lower arm of chromosome four.

Includes bibliographical references. / Polyphenols in plants are known to act as antioxidants, antimicrobials, antifungal, photoreceptors, visual attractors and as light screens. In this study polyphenols in angiosperms found in southern Africa and called resurrection (desiccation tolerant) plants were studied. These plants are Myrothamnus flabellifolius, Xerophyta viscosa, Xerophyta humilis, Xerophyta schlecterii, Xerophyta villosa. Craterostigma wilmsii, Craterostigma plantagineum, Craterostigma pumilum and Eragrostis nindensis. These plants are able to tolerate water stress without undergoing permanent damage. During drying these plants are subjected to different stresses and one such stress is oxidative stress. It has been suggested that polyphenols function as stress protectants in plant cells by scavenging reactive oxygen species (ROS) produced during a period of oxidative stress. In this study the total phenolic content and the related antioxidant capacity of the plants leaf extracts were analysed.

Bibliography: leaves 147-152. / Brain Factor 1 (BF-1), a winged helix transcription factor, displays a restricted pattern of expression within the developing forebrain, playing a critical function in the regulation of neuronal progenitor cell proliferation and differentiation within the developing forebrain. The molecular mechanisms by which BF-1 carries out its function remain to be elucidated. Hence, this study aimed to investigate and characterise the molecular mechanisms by which BF-1 function is regulated during neuronal development.

Plants have evolved a complex system of defence to prevent pathogen establishment. The Arabidopsis thaliana cir1 (constitutively induced resistance 1) mutant displays enhanced resistance to infection by the virulent bacterial pathogen Pseudomonas syringae and constitutively expresses a number of defence genes. Evidence suggests that CIR1 is a negative regulator of plant immunity important in the absence of pathogen attack. Genetic mapping experiments indicate that cir1 is located on the lower arm of chromosome 4 of A. thaliana and may be one of 8 known genes in the region. Analysis of T-DNA knockouts of these 8 genes suggests that AT4G11100 is the mostly likely candidate for CIR1. This project established that the disease resistance phenotype of cir1 is temperature dependent and linked to reduced plant growth. Genetic crosses between cir1 and at4g11100 T-DNA knockout mutants revealed that the mutants complement and therefore AT4G11100 is not CIR1. However, like cir1, the at4g11100 T-DNA knockout mutants display enhanced disease resistance. Over expression of AT4G11100 leads to increased susceptibility to infection by Pseudomonas syringae (Pst) and reduced induction of the salicylic acid defence gene PR2 following Pst infection, suggesting that AT4G11100 may too be a negative regulator of immunity. Additionally, a plant line with exceptionally high AT4G11100 expression levels displayed distinct leaf morphology, possibly implicating AT4G11100 in leaf development.

Centromeres are the site on a chromosome that allows for proper chromosome segregation during cell division. The kinetochore, a DNA-protein complex that forms at the centromere, attaches the chromosome to the spindle fiber and therefore ensures faithful separation of the chromosomes. Cse4p was identified in the budding yeast S. cerevisiae and is protein that resembles histone H3. Due to its centromere specific localization led to the proposal of the centromere specific nucleosome(s). Further identification of Cse4p homologues in many diverse organisms, including mammalian systems, supported the idea that the centromere specific nucleosome is an evolutionarily conserved structure. The localization of the centromere specific histone H3-like proteins (CenH3) to the centromere is one of the remaining questions in centromere biology. It is known that the CenH3 proteins have two domains, the conserved Histone Fold Domain (HFD), and a unique N-terminus, and that each domain has a distinct function. I analyzed the function of the Cse4 N-terminus and HFD in centromere localization and showed that the N-terminus has no function in centromere localization, but rather is involved in regulating Cse4 protein turnover. The HFD, therefore, contains all the centromere targeting information and is necessary and sufficient for Cse4 function. Further analysis revealed that the specific DNA contacts of the Cse4 HFD are important for localization to the centromere. In the budding yeast, S. cerevisiae, the centromere DNA is defined by a specific DNA sequence that is divided into three distinct regions, CDEI, II, and III. Mutations of these regions affect centromere function to varying degrees. Analysis of Cse4p localization to mutant centromere constructs revealed that CDEII is necessary for Cse4 centromere localization, but that the intrinsic bend of this element is not involved in Cse4p centromere localization. Cse4p localization to two other centromere mutants, cen3-X35 and cen3-X50, was analyzed. Cse4p could recognize cen3-X35, a construct with a CDEII deletion, but failed to recognize cen3-X50, a CDEII and CDEIII mutant centromere construct. Furthermore, the CBF3 complex was present at both of these centromere constructs, indicating that the CDEIII by deleted in cen3-X50 directly affects Cse4p centromere localization.

The earliest steps of nascent protein folding are critical to the overall folding efficiency. Folding events start as soon as the protein is translocated into the endoplasmic reticulum lumen, where the co-translational machinery ensures the fidelity of protein folding by coupling molecular chaperones, foldases and folding sensors. We seek to investigate the co-translational maturation events of disease-related glycoproteins containing different membrane topology to determine the generality and substrate specificity of this process, to hopefully provide new insight into therapeutic methods by targeting protein maturation at early stages. A variety of cell biological, biochemical, and molecular biological approaches using cell-free assays, isolated organelles and live cells have been applied in this study. The work on co-translational maturation of a type I membrane protein human tyrosinase has shown that Hsp70 family member BiP handed off tyrosinase to the lectin chaperones calnexin/calreticulin as glycans were added. The maturation pathway of the albino mutation of tyrosinase (C71R) diverges from that of the wild type co-translationally through its recognition by the oxidoreductase ERp57. The work on a type II membrane protein influenza neuraminidase (NA) subtype N9 has shown that calnexin co-translationally interacted with NA prior to calreticulin. This sequential manner was found to be a common feature of the ER assembly line determined by the membrane localization and soluble characteristics of calnexin/calreticulin, respectively. These interactions were required for the proper maturation of NA as NA aggregated if calnexin/calreticulin interaction was abolished by glycosylation inhibition or removal of specific glycans. Surprisingly, a subset of NA molecules can form intermolecular disulfides co-translationally supporting NA homodimerization. NA co-translational dimerization also occurs for a NA mutant lacking the critical large loop disulfide bonds, indicating that the dimerization of the stem domain does not require proper folding of the top globular domain of NA. This represents an exception to the general rule that protein oligomerization happens after the folding of individual domains. Future work on a variety of other substrates will help illuminate a global pathway of glycoprotein co-translational maturation.

Nur77, an orphan nuclear receptor, plays a key role in T cell apoptosis. As a transcription factor, Nur77 is assumed to exert its functions by driving the expression of target genes. However, Nur77 targets in T cell apoptosis are unknown. In cancer cell lines, Nur77 can induce apoptosis through the intrinsic apoptotic pathway but it remains controversial how Nur77 kills T cells. In this study, we provide biochemical, pharmacological and genetic evidence demonstrating that Nur77 induces apoptosis through the activation of the intrinsic pathway in T cells. We also show that Nur77 is a physiological substrate of the MEK-ERK-RSK-cascade. Specifically, we demonstrate that RSK phosphorylate Nur77 at serine 354 and this modulates Nur77 nuclear export and intracellular translocation during T cell death. Our data reveal that Nur77 phosphorylation and mitochondrial targeting, regulated by RSK, may define a role for the MEK1/2-ERK1/2 cascade in T cell apoptosis.

The infections agent of Lyme disease, Borrelia Burgdorferi is a complex microorganism with a highly diverse genome. One of the most remarkable aspects of the B. burgdorferi genome is the large number of sequences encoding predicted or known lipoproteins, including outer-surface proteins. The B. burgdorferi genome encodes no recognizable toxins. Instead, this extracellular pathogen causes pathology by migration through tissues, adhesion to host cells, and evasion of immune clearance. Inflammation elicited by infection with B. burgdorferi depends on the ability of the spirochete to survive in the mammalian host, as well as the immune response that arises upon the interaction of the bacterium with phagocytic, T and other cell types. Innate immune responses are critical in recognition and clearance of pathogens, and also play an important role in the outcome of adaptive immune responses. The regulation of innate immune responses to pathogens occurs through the interaction of Toll-like receptors (TLRs) with pathogen-associated molecular patterns (PAMPs) and the activation of several signaling pathways whose contribution to the overall innate immune response to pathogens is poorly understood. In this study we demonstrate a mechanism of control of murine macrophage responses mediated by TLR1/2 heterodimers through c-Jun N-terminal kinase 1 (JNK1) activity. JNK also controls tumor necrosis factor production and TLR-mediated macrophage responses to B. burgdorferi. We also show that the proximal promoter region of the human tlr1 gene contains an AP-1 binding site that is subjected to regulation by the kinase and binds two complexes that involve the JNK substrates c-Jun, JunD, and ATF-2. These results demonstrate that JNK1 regulates the response to TLR1/2 ligands and suggest a positive feedback loop that may serve to increase the innate immune response to the spirochete. MCJ is a newly identified member of the DnaJ protein family of cochaperones that contains unique features different than the normally described DnaJ proteins. However, there is little known about its function and the role it plays in different cells and systems. It has been previously shown that MCJ is required for the repression of the ABCB1 drug transporter expression in breast cancer cells, and that this repression is mediated through the control of c-Jun protein stability. We were therefore interested in determining the role that MCJ plays in macrophages in response to B. burgdorferi antigens. We now provide evidence that MCJ controls inflammatory responses of macrophages through the regulation of c-Jun protein stability, and the expression and release of the inflammatory cytokine TNF, through the regulation of the expression of TNF converting enzyme (TACE) inhibitor tissue inhibitor of metalloproteinase 3 (TIMP-3).

Lymphatic filariasis is a human parasitic disease which causes great physical suffering, while also having a substantial economic impact on the peoples of the tropics and sub-tropics. In order to collect accurate epidemiological data which are crucial for designing effective control programs, reliable diagnostic and taxonomic information is needed. The studies described here were designed to obtain DNA sequence data from specific coding and non-coding loci of the parasite genome, which could augment existing morphological, biochemical, and ecological data, to test hypotheses concerning diagnostic and taxonomic classification. Highly repeated DNA elements, members of the Hha I repeat family, were cloned, sequenced and the data employed in a phylogenetic analysis of species, sub-species and distinct geographic isolates of human filarial parasites from the genus Brugia. Comparative analysis reveals that although the 322 base pair (bp) repeat sequences between Brugia pahangi and Brugia malayi are nearly 90% identical overall, there is a small 70 bp region which contains enough divergence to clearly distinguish between these two major species. Nucleotide differences in this and other regions were exploited to draw distinctions between repeats cloned from B. timori, B. patei and various geographic isolates of B. malayi which differ in biological characteristics such as host range, vector preference and periodicity. In addition to the Hha I repeats, the gene encoding a prominent, stage-specific surface antigen from the animal parasite, Brugia pahangi was also cloned and sequenced. Homologous sequences were obtained from the related human pathogen Brugia malayi and a comparative analysis initiated. The results show that the protein coding, flanking and intervening sequences are highly conserved between the two species. In addition to its utility as a taxonomic and phylogenetic tool, the highly conserved nature of this protein sequence makes it a potential candidate for recombinant vaccine development.

No description available.