Molecular characterization of Osh6p, an oxysterol binding protein homolog in the yeast Saccharomyces cerevisiae

Oxysterol binding protein (OSBP) and its homologs have been shown to regulate lipid metabolism and vesicular transport. However, the exact molecular function of individual OSBP homologs remains uncharacterized. Here we demonstrate that the yeast OSBP homolog, Osh6p, bound phosphatidic acid and phosphoinositides via its N-terminal half containing the conserved OSBP-related domain (ORD). Using a green fluorescent protein fusion chimera, Osh6p was found to localize to the cytosol and patch-like or punctate structures in the vicinity of the plasma membrane. Further examination by domain mapping demonstrated that the N-terminal half was associated with FM4-64 positive membrane compartments; however, the C-terminal half containing a putative coiled-coil was localized to the nucleoplasm. Functional analysis showed that the deletion of OSH6 led to a significant increase in total cellular ergosterols, whereas OSH6 overexpression caused both a significant decrease in ergosterol levels and resistance to nystatin. Oleate incorporation into sterol esters was affected in OSH6 overexpressing cells. However, Lucifer yellow internalization, and FM4-64 uptake and transport were unaffected in both OSH6 deletion and overexpressing cells. Furthermore, osh6Δ exhibited no defect in carboxypeptidase Y transport and maturation. Lastly, we demonstrated that both the conserved ORD and the putative coiled-coil motif were indispensable for the in vivo function of Osh6p. These data suggest that Osh6p plays a role primarily in regulating cellular sterol metabolism, possibly stero transport.

The very C-terminus of PRK1/PKN is essential for its activation by RhoA and downstream signaling

PRK1 is a lipid- and Rho GTPase-activated serine/threonine protein kinase implicated in the regulation of receptor trafficking, cytoskeletal dynamics and tumorigenesis. Although Rho binding has been mapped to the HR1 region in the regulatory domain of PRK1, the mechanism involved in the control of PRK1 activation following Rho binding is poorly understood. We now provide the first evidence that the very C-terminus beyond the hydrophobic motif in PRK1 is essential for the activation of this kinase by RhoA. Deletion of the HR1 region did not completely abolish the binding of PRK1-ΔHR1 to GTPγS-RhoA nor the activation of this mutant by GTPγS-RhoA in vitro. In contrast, removing of the last six amino acid residues from the C-terminus of PRK1 or truncating of a single C-terminal residue from PRK1-ΔHR1 completely abrogated the activation of these mutants by RhoA both in vitro and in vivo. The critical dependence of the very C-terminus of PRK1 on the signaling downstream of RhoA was further demonstrated by the failure of the PRK1 mutant lacking its six C-terminal residues to augment lisophosphatidic acid-elicited neurite retraction in neuronal cells. Thus, we show that the HR1 region is necessary but not sufficient in eliciting a full activation of PRK1 upon binding of RhoA. Instead, such activation is controlled by the very C-terminus of PRK1. Our results also suggest that the very C-terminus of PRK1, which is the least conserved among members of the protein kinase C superfamily, is a potential drug target for pharmacological intervention of RhoA-mediated signaling pathways

Measuring the summary informativeness of orders and trades

Improved preservation of order flow history from the automation of derivative trading platforms suggests that traders are potentially learning from the recent history of both order and trade parameters. Consequently, a model to measure price discovery should encapsulate the dynamic interaction between the price-size coordinates of orders and trades. The Hasbrouck (1991) model is extended to measure the summary informativeness of order size and trade size. The two models are used to test for price discovery improvements in the FTSE 100 index futures market from order flow consolidation post deletion of its E-mini counterpart. The informativeness of trades has declined sharply, while the informativeness of orders has risen significantly in the post deletion sample.

Development of DAL and DAPL languages for building distributed applications

A common characteristic among parallel/distributed programming languages is that the one language is used to specify not only the overall organisation of the distributed application, but also the functionality of the application. That is, the connectivity and functionality of processes are specified within a single program. Connectivity and functionality are independent aspects of a distributed application. This thesis shows that these two aspects can be specified separately, therefore allowing application designers to freely concentrate on either aspect in a modular fashion. Two new programming languages have been developed for specifying each aspect. These languages are for loosely coupled distributed applications based on message passing, and have been designed to simplify distributed programming by completely removing all low level interprocess communication. A suite of languages and tools has been designed and developed. It includes the two new languages, parsers, a compilation system to generate intermediate C code that is compiled to binary object modules, a run-time system to create, manage and terminate several distributed applications, and a shell to communicate with the run-tune system. DAL (Distributed Application Language) and DAPL (Distributed Application Process Language) are the new programming languages for the specification and development of process oriented, asynchronous message passing, distributed applications. These two languages have been designed and developed as part of this doctorate in order to specify such distributed applications that execute on a cluster of computers. Both languages are used to specify orthogonal components of an application, on the one hand the organisation of processes that constitute an application, and on the other the interface and functionality of each process. Consequently, these components can be created in a modular fashion, individually and concurrently. The DAL language is used to specify not only the connectivity of all processes within an application, but also a cluster of computers for which the application executes. Furthermore, sub-clusters can be specified for individual processes of an application to constrain a process to a particular group of computers. The second language, DAPL, is used to specify the interface, functionality and data structures of application processes. In addition to these languages, a DAL parser, a DAPL parser, and a compilation system have been designed and developed (in this project). This compilation system takes DAL and DAPL programs to generate object modules based on machine code, one module for each application process. These object modules are used by the Distributed Application System (DAS) to instantiate and manage distributed applications. The DAS system is another new component of this project. The purpose of the DAS system is to create, manage, and terminate many distributed applications of similar and different configurations. The creation procedure incorporates the automatic allocation of processes to remote machines. Application management includes several operations such as deletion, addition, replacement, and movement of processes, and also detection and reaction to faults such as a processor crash. A DAS operator communicates with the DAS system via a textual shell called DASH (Distributed Application SHell). This suite of languages and tools allowed distributed applications of varying connectivity and functionality to be specified quickly and simply at a high level of abstraction. DAL and DAPL programs of several processes may require a few dozen lines to specify as compared to several hundred lines of equivalent C code that is generated by the compilation system. Furthermore, the DAL and DAPL compilation system is successful at generating binary object modules, and the DAS system succeeds in instantiating and managing several distributed applications on a cluster.

Convex hulls in concept induction

Classification learning is dominated by systems which induce large numbers of small axis-orthogonal decision surfaces. This strongly biases such systems towards particular hypothesis types but there is reason believe that many domains have underlying concepts which do not involve axis orthogonal surfaces. Further, the multiplicity of small decision regions mitigates against any holistic appreciation of the theories produced by these systems, notwithstanding the fact that many of the small regions are individually comprehensible. This thesis investigates modeling concepts as large geometric structures in n-dimensional space. Convex hulls are a superset of the set of axis orthogonal hyperrectangles into which axis orthogonal systems partition the instance space. In consequence, there is reason to believe that convex hulls might provide a more flexible and general learning bias than axis orthogonal regions. The formation of convex hulls around a group of points of the same class is shown to be a usable generalisation and is more general than generalisations produced by axis-orthogonal based classifiers, without constructive induction, like decision trees, decision lists and rules. The use of a small number of large hulls as a concept representation is shown to provide classification performance which can be better than that of classifiers which use a large number of small fragmentary regions for each concept. A convex hull based classifier, CH1, has been implemented and tested. CH1 can handle categorical and continuous data. Algorithms for two basic generalisation operations on hulls, inflation and facet deletion, are presented. The two operations are shown to improve the accuracy of the classifier and provide moderate classification accuracy over a representative selection of typical, largely or wholly continuous valued machine learning tasks. The classifier exhibits superior performance to well-known axis-orthogonal-based classifiers when presented with domains where the underlying decision surfaces are not axis parallel. The strengths and weaknesses of the system are identified. One particular advantage is the ability of the system to model domains with approximately the same number of structures as there are underlying concepts. This leads to the possibility of extraction of higher level mathematical descriptions of the induced concepts, using the techniques of computational geometry, which is not possible from a multiplicity of small regions.

Diversity and clonotypic composition of influenza-specific CD8+ TCR repertoires remain unaltered in the absence of Aire

TCR repertoire diversity is important for the protective efficacy of CD8⁺ T cells, limiting viral escape and cross-reactivity between unrelated epitopes. The exact mechanism for selection of restricted versus diverse TCR repertoires is far from clear, although one thought is that the epitopes resembling self-peptides might select a limited array of TCR due to the deletion of autoreactive TCR. The molecule Aire promotes the expression of tissue-specific Ag on thymic medullary epithelial cells and the deletion of autoreactive cells, and in the absence of Aire autoreactive cells persist. However, the contribution of Aire-dependent peptides to the selection of the Ag-specific TCR repertoire remains unknown. In this study, we dissect restricted (D^bNP₃₆₆%⁺CD8⁺) and diverse (D^bPA₂₂₄%⁺CD8⁺, K^dNP₁₄₇%⁺CD8⁺) TCR repertoires responding to three influenza-derived peptides in Aire-deficient mice on both B6 and BALB/c backgrounds. Our study shows that the number, qualitative characteristics and TCR repertoires of all influenza-specific, D^bNP₃₆₆%⁺CD8⁺, D^bPA₂₂₄%⁺CD8⁺ and K^dNP₁₄₇%⁺CD8⁺ T cells are not significantly altered in the absence of Aire. This provides the first demonstration that the selection of an Ag-specific T-cell repertoire is not significantly perturbed in the absence of Aire.

Structural and functional characterization of the oxidoreductase a-DsbA1 from wolbachia pipientis

The α-proteobacterium Wolbachia pipientis is a highly successful intracellular endosymbiont of invertebrates that manipulates its host's reproductive biology to facilitate its own maternal transmission. The fastidious nature of Wolbachia and the lack of genetic transformation have hampered analysis of the molecular basis of these manipulations. Structure determination of key Wolbachia proteins will enable the development of inhibitors for chemical genetics studies. Wolbachia encodes a homologue (α-DsbA1) of the Escherichia coli dithiol oxidase enzyme EcDsbA, essential for the oxidative folding of many exported proteins. We found that the active-site cysteine pair of Wolbachia α-DsbA1 has the most reducing redox potential of any characterized DsbA. In addition, Wolbachia α-DsbA1 possesses a second disulfide that is highly conserved in α-proteobacterial DsbAs but not in other DsbAs. The α-DsbA1 structure lacks the characteristic hydrophobic features of EcDsbA, and the protein neither complements EcDsbA deletion mutants in E. coli nor interacts with EcDsbB, the redox partner of EcDsbA. The surface characteristics and redox profile of α-DsbA1 indicate that it probably plays a specialized oxidative folding role with a narrow substrate specificity. This first report of a Wolbachia protein structure provides the basis for future chemical genetics studies.

Yeast Rnt1p is required for cleavage of the pre-ribosomal RNA in the 3' ETS but not the 5' ETS

We have reexamined the role of yeast RNase III (Rnt1p) in ribosome synthesis. Analysis of pre-rRNA processing in a strain carrying a complete deletion of the RNT1 gene demonstrated that the absence of Rnt1p does not block cleavage at site A0 in the 5' external transcribed spacers (ETS), although the early pre-rRNA cleavages at sites A0, A1, and A2 are kinetically delayed. In contrast, cleavage in the 3' ETS is completely inhibited in the absence of Rnt1p, leading to the synthesis of a reduced level of a 3' extended form of the 25S rRNA. The 3' extended forms of the pre-rRNAs are consistent with the major termination at site T2 (+210). We conclude that Rnt1p is required for cleavage in the 3' ETS but not for cleavage at site A0. The sites of in vivo cleavage in the 3' ETS were mapped by primer extension. Two sites of Rnt1p-dependent cleavage were identified that lie on opposite sides of a predicted stem loop structure, at +14 and +49. These are in good agreement with the consensus Rnt1p cleavage site. Processing of the 3' end of the mature 25S rRNA sequence in wild-type cells was found to occur concomitantly with processing of the 5' end of the 5.8S rRNA, supporting previous proposals that processing in ITS1 and the 3' ETS is coupled.

Cordycepin-hypersensitive growth links elevated polyphosphate levels to inhibition of poly(A) polymerase in Saccharomyces cerevisiae

To identify genes involved in poly(A) metabolism, we screened the yeast gene deletion collection for growth defects in the presence of cordycepin (3′-deoxyadenosine), a precursor to the RNA chain terminating ATP analog cordycepin triphosphate. Δpho80 and Δpho85 strains, which have a constitutively active phosphate-response pathway, were identified as cordycepin hypersensitive. We show that inorganic polyphosphate (poly P) accumulated in these strains and that poly P is a potent inhibitor of poly(A) polymerase activity in vitro. Binding analyses of poly P and yeast Pap1p revealed an interaction with a k_D in the low nanomolar range. Poly P also bound mammalian poly(A) polymerase, however, with a 10-fold higher k_D compared to yeast Pap1p. Genetic tests with double mutants of Δpho80 and other genes involved in phosphate homeostasis and poly P accumulation suggest that poly P contributed to cordycepin hypersensitivity. Synergistic inhibition of mRNA synthesis through poly P-mediated inhibition of Pap1p and through cordycepin-mediated RNA chain termination may thus account for hypersensitive growth of Δpho80 and Δpho85 strains in the presence of the chain terminator. Consistent with this, a mutation in the 3′-end formation component rna14 was synthetic lethal in combination with Δpho80. Based on these observations, we suggest that binding of poly P to poly(A) polymerase negatively regulates its activity.

A critical role for the short intracellular C terminus in receptor activity-modifying protein function

Receptor activity-modifying proteins (RAMPs) interact with and modify the behavior of the calcitonin receptor (CTR) and calcitonin receptor-like receptor (CLR). We have examined the contribution of the short intracellular C terminus, using constructs that delete the last eight amino acids of each RAMP. C-Terminal deletion of individual RAMPs had little effect on the signaling profile induced when complexed with CLR in COS-7 or human embryonic kidney (HEK)293 cells. Likewise, confocal microscopy revealed each of the mutant RAMPs translocated hemagglutinin-tagged CLR to the cell surface. In contrast, a pronounced effect of RAMP C-terminal truncation was seen for RAMP/CTRa complexes, studied in COS-7 cells, with significant attenuation of amylin receptor phenotype induction that was stronger for RAMP1 and -2 than RAMP3. The loss of amylin binding upon C-terminal deletion could be partially recovered with overexpression of Gαs, suggesting an impact of the RAMP C terminus on coupling of G proteins to the receptor complex. In HEK293 cells the c-Myc-RAMP1 C-terminal deletion mutant showed high receptor-independent cell surface expression; however, this construct showed low cell surface expression when expressed alone in COS-7 cells, indicating interaction of RAMPs with other cellular components via the C terminus. This mutant also had reduced cell surface expression when coexpressed with CTR. Thus, this study reveals important functionality of the RAMP C-terminal domain and identifies key differences in the role of the RAMP C terminus for CTR versus CLR-based receptors.

Transcriptional regulation of the yghJ-pppA-yghG-gspCDEFGHIJKLM cluster, encoding the type II secretion pathway in Enterotoxigenic Escherichia coli

The gene cluster gspCDEFGHIJKLM codes for various structural components of the type II secretion pathway which is responsible for the secretion of heat-labile enterotoxin by enterotoxigenic Escherichia coli (ETEC). In this work, we used a variety of molecular approaches to elucidate the transcriptional organization of the ETEC type II secretion system and to unravel the mechanisms by which the expression of these genes is controlled. We showed that the gspCDEFGHIJKLM cluster and three other upstream genes, yghJ, pppA, and yghG, are cotranscribed and that a promoter located in the upstream region of yghJ plays a major role in the expression of this 14-gene transcriptional unit. Transcription of the yghJ promoter was repressed 168-fold upon a temperature downshift from 37°C to 22°C. This temperature-induced repression was mediated by the global regulatory proteins H-NS and StpA. Deletion mutagenesis showed that the promoter region encompassing positions −321 to +301 relative to the start site of transcription of yghJ was required for full repression. The yghJ promoter region is predicted to be highly curved and bound H-NS or StpA directly. The binding of H-NS or StpA blocked transcription initiation by inhibiting promoter open complex formation. Unraveling the mechanisms of regulation of type II secretion by ETEC enhances our understanding of the pathogenesis of ETEC and other pathogenic varieties of E. coli.

Association between 5-HTTLPR genotypes and persisting patterns of anxiety and alcohol use : results from a 10-year longitudinal study of adolescent mental health

The serotonin transporter gene (5-HTT) encodes a transmembrane protein that plays an important role in regulating serotonergic neurotransmission and related aspects of mood and behaviour. The short allele of a 44 bp insertion/deletion polymorphism (S-allele) within the promoter region of the 5-HTT gene (5-HTTLPR) confers lower transcriptional activity relative to the long allele (L-allele) and may act to modify the risk of serotonin-mediated outcomes such as anxiety and substance use behaviours. The purpose of this study was to determine whether (or not) 5-HTTLPR genotypes moderate known associations between attachment style and adolescent anxiety and alcohol use outcomes. Participants were drawn from an eight-wave study of the mental and behavioural health of a cohort of young Australians followed from 14 to 24 years of age (Victorian Adolescent Health Cohort Study, 1992 - present). No association was observed within low-risk attachment settings. However, within risk settings for heightened anxiety (ie, insecurely attached young people), the odds of persisting ruminative anxiety (worry) decreased with each additional copy of the S-allele (B30% per allele: OR 0.77, 95% CI 0.62–0.97, P¼0.029). Within risk settings for binge drinking (ie, securely attached young people), the odds of reporting persisting high-dose alcohol consumption (bingeing) decreased with each additional copy of the S-allele (B35% per allele: OR 0.74, 95% CI 0.64–0.86, Po0.001). Our data suggest that the S-allele is likely to be important in psychosocial development, particularly in those settings that increase risk of anxiety and alcohol use problems.

Alleles of RUNX2/CBFA1 gene are associated with differences in bone mineral density and risk of fracture

The aim of this study was to determine if DNA polymorphism within runt-related gene 2 (RUNX2)/core binding factor A1 (CBFA1) is related to bone mineral density (BMD). RUNX2 contains a glutamine-alanine repeat where mutations causing cleidocranial dysplasia (CCD) have been observed. Two common variants were detected within the alanine repeat: an 18-bp deletion and a synonymous alanine codon polymorphism with alleles GCA and GCG (noted as A and G alleles, respectively). In addition, rare mutations that may be related to low BMD were observed within the glutamine repeat. In 495 randomly selected women of the Geelong Osteoporosis Study (GOS), the A allele was associated with higher BMD at all sites tested. The effect was maximal at the ultradistal (UD) radius (p = 0.001). In a separate fracture study, the A allele was significantly protective against Colles' fracture in elderly women but not spine and hip fracture. The A allele was associated with increased BMD and was protective against a common form of osteoporotic fracture, suggesting that RUNX2 variants may be related to genetic effects on BMD and osteoporosis.

Online audio background determination for complex audio environments

We present a method for foreground/background separation of audio using a background modelling technique. The technique models the background in an online, unsupervised, and adaptive fashion, and is designed for application to long term surveillance and monitoring problems. The background is determined using a statistical method to model the states of the audio over time. In addition, three methods are used to increase the accuracy of background modelling in complex audio environments. Such environments can cause the failure of the statistical model to accurately capture the background states. An entropy-based approach is used to unify background representations fragmented over multiple states of the statistical model. The approach successfully unifies such background states, resulting in a more robust background model. We adaptively adjust the number of states considered background according to background complexity, resulting in the more accurate classification of background models. Finally, we use an auxiliary model cache to retain potential background states in the system. This prevents the deletion of such states due to a rapid influx of observed states that can occur for highly dynamic sections of the audio signal. The separation algorithm was successfully applied to a number of audio environments representing monitoring applications.

HIV infection of dendritic cells subverts the IFN induction pathway via IRF-1 and inhibits type 1 IFN production

Many viruses have developed mechanisms to evade the IFN response. Here, HIV-1 was shown to induce a distinct subset of IFN-stimulated genes (ISGs) in monocyte-derived dendritic cells (DCs), without detectable type I or II IFN. These ISGs all contained an IFN regulatory factor 1 (IRF-1) binding site in their promoters, and their expression was shown to be driven by IRF-1, indicating this subset was induced directly by viral infection by IRF-1. IRF-1 and -7 protein expression was enriched in HIV p24 antigen-positive DCs. A HIV deletion mutant with the IRF-1 binding site deleted from the long terminal repeat showed reduced growth kinetics. Early and persistent induction of IRF-1 was coupled with sequential transient up-regulation of its 2 inhibitors, IRF-8, followed by IRF-2, suggesting a mechanism for IFN inhibition. HIV-1 mutants with Vpr deleted induced IFN, showing that Vpr is inhibitory. However, HIV IFN inhibition was mediated by failure of IRF-3 activation rather than by its degradation, as in T cells. In contrast, herpes simplex virus type 2 markedly induced IFNβ and a broader range of ISGs to higher levels, supporting the hypothesis that HIV-1 specifically manipulates the induction of IFN and ISGs to enhance its noncytopathic replication in DCs.