Novel approaches for SER spectroscopic analysis of protein cofactors

Biomimetic systems employed for biotechnological applications i.e. as biosensors or bio fuel cells, require initial formation of conducting support/protein complexes with controlled properties. The specific interaction of the protein with the support determines important qualities of the device such as electrical communication, long-term stability and catalytic efficiency. In this respect the system parameters have to be chosen in a way that high protein loading on the support is achieved while protein denaturation upon adsorption is prevented. The conditions on the surface have to be adjusted in such a way that the desired surface reaction of the protein i.e. electron transfer to either the electrode or a second redox partner, is still guaranteed. Hence the choice of support, its functionlisation as well as the right adjustment of solution parameters play a crucial role in the rational design of these support/protein constructs.

In silico analyses reveal common cellular pathways affected by loss of heterozygosity (LOH) events in the lymphomagenesis of Non-Hodgkin’s lymphoma (NHL)

Background The analysis of cellular networks and pathways involved in oncogenesis has increased our knowledge about the pathogenic mechanisms that underlie tumour biology and has unmasked new molecular targets that may lead to the design of better anti-cancer therapies. Recently, using a high resolution loss of heterozygosity (LOH) analysis, we identified a number of potential tumour suppressor genes (TSGs) within common LOH regions across cases suffering from two of the most common forms of Non-Hodgkin’s lymphoma (NHL), Follicular Lymphoma (FL) and Diffuse Large B-cell Lymphoma (DLBCL). From these studies LOH of the protein tyrosine phosphatase receptor type J (PTPRJ) gene was identified as a common event in the lymphomagenesis of these B-cell lymphomas. The present study aimed to determine the cellular pathways affected by the inactivation of these TSGs including PTPRJ in FL and DLBCL tumourigenesis. Results Pathway analytical approaches identified that candidate TSGs located within common LOH regions participate within cellular pathways, which may play a crucial role in FL and DLBCL lymphomagenesis (i.e., metabolic pathways). These analyses also identified genes within the interactome of PTPRJ (i.e. PTPN11 and B2M) that when inactivated in NHL may play an important role in tumourigenesis. We also detected genes that are differentially expressed in cases with and without LOH of PTPRJ, such as NFATC3 (nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 3). Moreover, upregulation of the VEGF, MAPK and ERBB signalling pathways was also observed in NHL cases with LOH of PTPRJ, indicating that LOH-driving events causing inactivation of PTPRJ, apart from possibly inducing a constitutive activation of these pathways by reduction or abrogation of its dephosphorylation activity, may also induce upregulation of these pathways when inactivated. This finding implicates these pathways in the lymphomagenesis and progression of FL and DLBCL. Conclusions The evidence obtained in this research supports findings suggesting that FL and DLBCL share common pathogenic mechanisms. Also, it indicates that PTPRJ can play a crucial role in the pathogenesis of these B-cell tumours and suggests that activation of PTPRJ might be an interesting novel chemotherapeutic target for the treatment of these B-cell tumours.

LacO-LacI interaction in affinity adsorption of plasmid DNA

Current approaches for purifying plasmids from bacterial production systems exploit the physiochemical properties of nucleic acids in non-specific capture systems. In this study, an affinity system for plasmid DNA (pDNA) purification has been developed utilizing the interaction between the lac operon (lacO) sequence contained in the pDNA and a 64mer synthetic peptide representing the DNA-binding domain of the lac repressor protein, LacI. Two plasmids were evaluated, the native pUC19 and pUC19 with dual lacO3/lacOs operators (pUC19lacO3/lacOs), where the lacOs operator is perfectly symmetrical. The DNA-protein affinity interaction was evaluated by surface plasmon resonance using a Biacore system. The affinity capture of DNA in a chromatography system was evaluated using LacI peptide that had been immobilized to Streamline™ adsorbent. The KD-values for double stranded DNA (dsDNA) fragments containing lacO1 and lacO3 and lacOs and lacO3 were 5.7 ± 0.3 × 10 -11 M and 4.1 ± 0.2 × 10-11 M respectively, which compare favorably with literature reports of 5 × 10-10 - 1 × 10-9 M for native laCO1 and 1-1.2 × 10-10 M for lacO1 in a saline buffer. Densitometric analysis of the gel bands from the affinity chromatography run clearly showed a significant preference for capture of the supercoiled fraction from the feed pDNA sample. The results indicate the feasibility of the affinity approach for pDNA capture and purification using native protein-DNA interaction.

Green-fluorescent protein facilitates rapid in vivo detection of genetically transformed plant cells

Early detection of plant transformation events is necessary for the rapid establishment and optimization of plant transformation protocols. We have assessed modified versions of the green fluorescent protein (GFP) from Aequorea victoria as early reporters of plant transformation using a dissecting fluorescence microscope with appropriate filters. Gfp-expressing cells from four different plant species (sugarcane, maize, lettuce, and tobacco) were readily distinguished, following either Agrobacterium-mediated or particle bombardment-mediated transformation. The identification of gfp-expressing sugarcane cells allowed for the elimination of a high proportion of non-expressing explants and also enabled visual selection of dividing transgenic cells, an early step in the generation of transgenic organisms. The recovery of transgenic cell clusters was streamlined by the ability to visualize gfp-expressing tissues in vitro.

Short-term nutritional supplementation during management of pulmonary exacerbations in cystic fibosis: A controlled study, including effects of protein turnover

Effects of nutritional supplements on minimizing weight loss and abnormalities of protein turnover during pulmonary exacerbations in cystic fibrosis (CF) were studied by controlled trial. Patients received pulmonary therapy and either standard diet (n = 10) or adjunctive enteral supplements (n = 12). Initial protein turnover, measured by [15N]glycine kinetics, showed alterations of protein synthesis (P Syn) and catabolism (P Cat), which correlated with the degree of underweight, and negligible net protein deposition (P Dep). With treatment both groups had significant increases in mean body weight and forced expiratory volume in 1 s, expressed as percent predicted value for height (FEV1) by 3 wk, but a significant correlation between initial underweight and subsequent weight gain was observed only in supplemented patients. Mean P Syn and P Dep increased significantly (p < 0.001) only in the supplemented group. Pulmonary exacerbations in CF have important adverse effects on body-protein metabolism, similar to changes in protein-energy malnutrition and infection. These effects are reversed by short-term nutritional support. Strategic nutritional intervention should thus be considered in management, especially in malnourished patients.

Preparation and in vitro release of drug-loaded microparticles for oral delivery using wholegrain sorghum kafirin protein

Kafirin microparticles have been proposed as an oral nutraceutical and drug delivery system. This study investigates microparticles formed with kafirin extracted from white and raw versus cooked red sorghum grains as an oral delivery system. Targeted delivery to the colon would be beneficial for medication such as prednisolone, which is used in the management of inflammatory bowel disease. Therefore, prednisolone was loaded into microparticles of kafirin from the different sources using phase separation. Differences were observed in the protein content, in vitro protein digestibility, and protein electrophoretic profile of the various sources of sorghum grains, kafirin extracts, and kafirin microparticles. For all of the formulations, the majority of the loaded prednisolone was not released in in vitro conditions simulating the upper gastrointestinal tract, indicating that most of the encapsulated drug could reach the target area of the lower gastrointestinal tract. This suggests that these kafirin microparticles may have potential as a colon-targeted nutraceutical and drug delivery system.

In vitro and in vivo examination of the cell surface glycoprotein CDCP1

A number of reports have demonstrated the importance of the CUB domaincontaining protein 1 (CDCP1) in facilitating cancer progression in animal models and the potential of this protein as a prognostic marker in several malignancies. CDCP1 facilitates metastasis formation in animal models by negatively regulating anoikis, a type of apoptosis triggered by the loss of attachment signalling from cell-cell contacts or cell-extra cellular matrix (ECM) contacts. Due to the important role CDCP1 plays in cancer progression in model systems, it is considered a potential drug target to prevent the metastatic spread of cancers. CDCP1 is a highly glycosylated 836 amino acid cell surface protein. It has structural features potentially facilitating protein-protein interactions including 14 N-glycosylation sites, three CUB-like domains, 20 cysteine residues likely to be involved in disulfide bond formation and five intracellular tyrosine residues. CDCP1 interacts with a variety of proteins including Src family kinases (SFKs) and protein kinase C ä (PKCä). Efforts to understand the mechanisms regulating these interactions have largely focussed on three CDCP1 tyrosine residues Y734, Y743 and Y762. CDCP1-Y734 is the site where SFKs phosphorylate and bind to CDCP1 and mediate subsequent phosphorylation of CDCP1-Y743 and -Y762 which leads to binding of PKCä at CDCP1-Y762. The resulting trimeric protein complex of SFK•CDCP1•PKCä has been proposed to mediate an anti-apoptotic cell phenotype in vitro, and to promote metastasis in vivo. The effect of mutation of the three tyrosines on interactions of CDCP1 with SFKs and PKCä and the consequences on cell phenotype in vitro and in vivo have not been examined. CDCP1 has a predicted molecular weight of ~90 kDa but is usually detected as a protein which migrates at ~135 kDa by Western blot analysis due to its high degree of glycosylation. A low molecular weight form of CDCP1 (LMWCDCP1) of ~70 kDa has been found in a variety of cancer cell lines. The mechanisms leading to the generation of LMW-CDCP1 in vivo are not well understood but an involvement of proteases in this process has been proposed. Serine proteases including plasmin and trypsin are able to proteolytically process CDCP1. In addition, the recombinant protease domain of the serine protease matriptase is also able to cleave the recombinant extracellular portion of CDCP1. Whether matriptase is able to proteolytically process CDCP1 on the cell surface has not been examined. Importantly, proteolytic processing of CDCP1 by trypsin leads to phosphorylation of its cell surface-retained portion which suggests that this event leads to initiation of an intracellular signalling cascade. This project aimed to further examine the biology of CDCP1 with a main of focus on exploring the roles played by CDCP1 tyrosine residues. To achieve this HeLa cells stably expressing CDCP1 or the CDCP1 tyrosine mutants Y734F, Y743F and Y762F were generated. These cell lines were used to examine: • The roles of the tyrosine residues Y734, Y743 and Y762 in mediating interactions of CDCP1 with binding proteins and to examine the effect of the stable expression on HeLa cell morphology. • The ability of the serine protease matriptase to proteolytically process cell surface CDCP1 and to examine the consequences of this event on HeLa cell phenotype and cell signalling in vitro. • The importance of these residues in processes associated with cancer progression in vitro including adhesion, proliferation and migration. • The role of these residues on metastatic phenotype in vivo and the ability of a function-blocking anti-CDCP1 antibody to inhibit metastasis in the chicken embryo chorioallantoic membrane (CAM) assay. Interestingly, biochemical experiments carried out in this study revealed that mutation of certain CDCP1 tyrosine residues impacts on interactions of this protein with binding proteins. For example, binding of SFKs as well as PKCä to CDCP1 was markedly decreased in HeLa-CDCP1-Y734F cells, and binding of PKCä was also reduced in HeLa-CDCP1-Y762F cells. In contrast, HeLa-CDCP1-Y743F cells did not display altered interactions with CDCP1 binding proteins. Importantly, observed differences in interactions of CDCP1 with binding partners impacted on basal phosphorylation of CDCP1. It was found that HeLa-CDCP1, HeLa-CDCP1-Y743F and -Y762F displayed strong basal levels of CDCP1 phosphorylation. In contrast, HeLa-CDCP1-Y734F cells did not display CDCP1 phosphorylation but exhibited constitutive phosphorylation of focal adhesion kinase (FAK) at tyrosine 861. Significantly, subsequent investigations to examine this observation suggested that CDCP1-Y734 and FAK-Y861 are competitive substrates for SFK-mediated phosphorylation. It appeared that SFK-mediated phosphorylation of CDCP1- Y734 and FAK-Y861 is an equilibrium which shifts depending on the level of CDCP1 expression in HeLa cells. This suggests that the level of CDCP1 expression may act as a regulatory mechanism allowing cells to switch from a FAK-Y861 mediated pathway to a CDCP1-Y734 mediated pathway. This is the first time that a link between SFKs, CDCP1 and FAK has been demonstrated. One of the most interesting observations from this work was that CDCP1 altered HeLa cell morphology causing an elongated and fibroblastic-like appearance. Importantly, this morphological change depended on CDCP1- Y734. In addition, it was observed that this change in cell morphology was accompanied by increased phosphorylation of SFK-Y416. This suggests that interactions of SFKs with CDCP1-Y734 increases SFK activity since SFKY416 is critical in regulating kinase activity of these proteins. The essential role of SFKs in mediating CDCP1-induced HeLa cell morphological changes was demonstrated using the SFK-selective inhibitor SU6656. This inhibitor caused reversion of HeLa-CDCP1 cell morphology to an epithelial appearance characteristic of HeLa-vector cells. Significantly, in vitro studies revealed that certain CDCP1-mediated cell phenotypes are mediated by cellular pathways dependent on CDCP1 tyrosine residues whereas others are independent of these sites. For example, CDCP1 expression caused a marked increase in HeLa cell motility that was independent of CDCP1 tyrosine residues. In contrast, CDCP1- induced decrease in HeLa cell proliferation was most prominent in HeLa- CDCP1-Y762F cells, potentially indicating a role for this site in regulating proliferation in HeLa cells. Another cellular event which was identified to require phosphorylation of a particular CDCP1 tyrosine residue is adhesion to fibronectin. It was observed that the CDCP1-mediated strong decrease in adhesion to fibronectin is mostly restored in HeLa-CDCP1-Y743F cells. This suggests a possible role for CDCP1-Y743 in causing a CDCP1-mediated decrease in adhesion. Data from in vivo experiments indicated that HeLa-CDCP1-Y734F cells are more metastic than HeLa-CDCP1 cells in vivo. This indicates that interaction of CDCP1 with SFKs and PKCä may not be required for CDCP1-mediated metastasis formation of HeLa cells in vivo. The metastatic phenotype of these cells may be caused by signalling involving FAK since HeLa-CDCP1- Y734F cells are the only CDCP1 expressing cells displaying constitutive phosphorylation of FAK-Y861. HeLa-CDCP1-Y762F cells displayed a very low metastatic ability which suggests that this CDCP1 tyrosine residue is important in mediating a pro-metastatic phenotype in HeLa cells. More detailed exploration of cellular events occurring downstream of CDCP1-Y734 and -Y762 may provide important insights into the mechanisms altering the metastatic ability of CDCP1 expressing HeLa cells. Complementing the in vivo studies, anti-CDCP1 antibodies were employed to assess whether these antibodies are able to inhibit metastasis of CDCP1 and CDCP1 tyrosine mutants expressing HeLa cells. It was found that HeLa- CDCP1-Y734F cells were the only cell line which was markedly reduced in the ability to metastasise. In contrast, the ability of HeLa-CDCP1, HeLa- CDCP1-Y743F and -Y762F cells to metastasise in vivo was not inhibited. These data suggest a possible role of interactions of CDCP1 with SFKs, occurring at CDCP1-Y734, in preventing an anti-metastatic effect of anti- CDCP1 antibodies in vivo. The proposal that SFKs may play a role in regulating anti-metastatic effects of anti-CDCP1 antibodies was supported by another experiment where differences between HeLa-CDCP1 cells and CDCP1 expressing HeLa cells (HeLa-CDCP1-S) from collaborators at the Scripps Research Institute were examined. It was found that HeLa-CDCP1-S cells express different SFKs than CDCP1 expressing HeLa cells generated for this study. This is important since HeLa-CDCP1-S cells can be inhibited in their metastatic ability using anti-CDCP1 antibodies in vivo. Importantly, these data suggest that further examinations of the roles of SFKs in facilitating anti-metastatic effects of anti-CDCP1 antibodies may give insights into how CDCP1 can be blocked to prevent metastasis in vivo. This project also explored the ability of the serine protease matriptase to proteolytically process cell surface localised CDCP1 because it is unknown whether matriptase can cleave cell surface CDCP1 as it has been reported for other proteases such as trypsin and plasmin. Furthermore, the consequences of matriptase-mediated proteolysis on cell phenotype in vitro and cell signalling were examined since recent reports suggested that proteolysis of CDCP1 leads to its phosphorylation and may initiate cell signalling and consequently alter cell phenotype. It was found that matriptase is able to proteolytically process cell surface CDCP1 at low nanomolar concentrations which suggests that cleavage of CDCP1 by matriptase may facilitate the generation of LWM-CDCP1 in vivo. To examine whether matriptase-mediated proteolysis induced cell signalling anti-phospho Erk 1/2 Western blot analysis was performed as this pathway has previously been examined to study signalling in response to proteolytic processing of cell surface proteins. It was found that matriptase-mediated proteolysis in CDCP1 expressing HeLa cells initiated intracellular signalling via Erk 1/2. Interestingly, this increase in phosphorylation of Erk 1/2 was also observed in HeLa-vector cells. This suggested that initiation of cell signalling via Erk 1/2 phosphorylation as a result of matriptase-mediated proteolysis occurs by pathways independent of CDCP1. Subsequent investigations measuring the flux of free calcium ions and by using a protease-activated receptor 2 (PAR2) agonist peptide confirmed this hypothesis. These data suggested that matriptase-mediated proteolysis results in cell signalling via a pathway induced by the activation of PAR2 rather than by CDCP1. This indicates that induction of cell signalling in HeLa cells as a consequence of matriptase-mediated proteolysis occurs via signalling pathways which do not involve phosphorylation of Erk 1/2. Consequently, it appears that future attempts should focus on the examination of cellular pathways other than Erk 1/2 to elucidate cell signalling initiated by matriptase-mediated proteolytic processing of CDCP1. The data presented in this thesis has explored in vitro and in vivo aspects of the biology of CDCP1. The observations summarised above will permit the design of future studies to more precisely determine the role of CDCP1 and its binding partners in processes relevant to cancer progression. This may contribute to further defining CDCP1 as a target for cancer treatment.

Multiscale analysis of protein functions and stochastic modelling of gene transcriptional regulatory networks

Genomic and proteomic analyses have attracted a great deal of interests in biological research in recent years. Many methods have been applied to discover useful information contained in the enormous databases of genomic sequences and amino acid sequences. The results of these investigations inspire further research in biological fields in return. These biological sequences, which may be considered as multiscale sequences, have some specific features which need further efforts to characterise using more refined methods. This project aims to study some of these biological challenges with multiscale analysis methods and stochastic modelling approach. The first part of the thesis aims to cluster some unknown proteins, and classify their families as well as their structural classes. A development in proteomic analysis is concerned with the determination of protein functions. The first step in this development is to classify proteins and predict their families. This motives us to study some unknown proteins from specific families, and to cluster them into families and structural classes. We select a large number of proteins from the same families or superfamilies, and link them to simulate some unknown large proteins from these families. We use multifractal analysis and the wavelet method to capture the characteristics of these linked proteins. The simulation results show that the method is valid for the classification of large proteins. The second part of the thesis aims to explore the relationship of proteins based on a layered comparison with their components. Many methods are based on homology of proteins because the resemblance at the protein sequence level normally indicates the similarity of functions and structures. However, some proteins may have similar functions with low sequential identity. We consider protein sequences at detail level to investigate the problem of comparison of proteins. The comparison is based on the empirical mode decomposition (EMD), and protein sequences are detected with the intrinsic mode functions. A measure of similarity is introduced with a new cross-correlation formula. The similarity results show that the EMD is useful for detection of functional relationships of proteins. The third part of the thesis aims to investigate the transcriptional regulatory network of yeast cell cycle via stochastic differential equations. As the investigation of genome-wide gene expressions has become a focus in genomic analysis, researchers have tried to understand the mechanisms of the yeast genome for many years. How cells control gene expressions still needs further investigation. We use a stochastic differential equation to model the expression profile of a target gene. We modify the model with a Gaussian membership function. For each target gene, a transcriptional rate is obtained, and the estimated transcriptional rate is also calculated with the information from five possible transcriptional regulators. Some regulators of these target genes are verified with the related references. With these results, we construct a transcriptional regulatory network for the genes from the yeast Saccharomyces cerevisiae. The construction of transcriptional regulatory network is useful for detecting more mechanisms of the yeast cell cycle.

Correlations between designability and various structural characteristics of protein lattice models

Using six kinds of lattice types (4×4 ,5×5 , and6×6 square lattices;3×3×3 cubic lattice; and2+3+4+3+2 and4+5+6+5+4 triangular lattices), three different size alphabets (HP ,HNUP , and 20 letters), and two energy functions, the designability of proteinstructures is calculated based on random samplings of structures and common biased sampling (CBS) of proteinsequence space. Then three quantities stability (average energy gap),foldability, and partnum of the structure, which are defined to elucidate the designability, are calculated. The authors find that whatever the type of lattice, alphabet size, and energy function used, there will be an emergence of highly designable (preferred) structure. For all cases considered, the local interactions reduce degeneracy and make the designability higher. The designability is sensitive to the lattice type, alphabet size, energy function, and sampling method of the sequence space. Compared with the random sampling method, both the CBS and the Metropolis Monte Carlo sampling methods make the designability higher. The correlation coefficients between the designability, stability, and foldability are mostly larger than 0.5, which demonstrate that they have strong correlation relationship. But the correlation relationship between the designability and the partnum is not so strong because the partnum is independent of the energy. The results are useful in practical use of the designability principle, such as to predict the proteintertiary structure.

CDKN2A mutation in a non-FAMMM kindred with cancers at multiple sites results in a functionally abnormal protein

The CDKN2A gene encodes p16 (CDKN2A), a cell-cycle inhibitor protein which prevents inappropriate cell cycling and, hence, proliferation. Germ-line mutations in CDKN2A predispose to the familial atypical multiple-mole melanoma (FAMMM) syndrome but also have been seen in rare families in which only 1 or 2 individuals are affected by cutaneous malignant melanoma (CMM). We therefore sequenced exons 1alpha and 2 of CDKN2A using lymphocyte DNA isolated from index cases from 67 families with cancers at multiple sites, where the patterns of cancer did not resemble those attributable to known genes such as hMLH1, hMLH2, BRCA1, BRCA2, TP53 or other cancer susceptibility genes. We found one mutation, a mis-sense mutation resulting in a methionine to isoleucine change at codon 53 (M531) of exon 2. The individual tested had developed 2 CMMs but had no dysplastic nevi and lacked a family history of dysplastic nevi or CMM. Other family members had been diagnosed with oral cancer (2 persons), bladder cancer (1 person) and possibly gall-bladder cancer. While this mutation has been reported in Australian and North American melanoma kindreds, we did not observe it in 618 chromosomes from Scottish and Canadian controls. Functional studies revealed that the CDKN2A variant carrying the M531 change was unable to bind effectively to CDK4, showing that this mutation is of pathological significance. Our results have confirmed that CDKN2A mutations are not limited to FAMMM kindreds but also demonstrate that multi-site cancer families without melanoma are very unlikely to contain CDKN2A mutations.

Deprivation is an independent predictor of 1-year mortality in outpatients with chronic obstructive pulmonary disease

Deprivation is linked to increased incidence in a number of chronic diseases but its relationship to chronic obstructive pulmonary disease (COPD) is uncertain despite suggestions that the socioeconomic gradient seen in COPD is as great, if not greater, than any other disease (Prescott and Vestbo).1 There is also a need to take into account the confounding effects of malnutrition which have been shown to be independently linked to increased mortality (Collins et al).2 The current study investigated the influence of social deprivation on 1-year survival rates in COPD outpatients, independently of malnutrition. 424 outpatients with COPD were routinely screened for malnutrition risk using the ‘Malnutrition Universal Screening Tool’; ‘MUST’ (Elia),3 between July and May 2009; 222 males and 202 females; mean age 73 (SD 9.9) years; body mass index 25.8 (SD 6.3) kg/m2. Each individual's deprivation was calculated using the index of multiple deprivation (IMD) which was established according to the geographical location of each patient's address (postcode). IMD includes a number of indicators covering economic, housing and social issues (eg, health, education and employment) into a single deprivation score (Nobel et al).4 The lower the IMD score, the lower an individual's deprivation. The IMD was assigned to each outpatient at the time of screening and related to1-year mortality from the date screened. Outpatients who died within 1-year of screening were significantly more likely to reside within a deprived postcode (IMD 19.7±SD 13.1 vs 15.4±SD 10.7; p=0.023, OR 1.03, 95% CI 1.00 to 1.06) than those that did not die. Deprivation remained a significant independent risk factor for 1-year mortality even when adjusted for malnutrition as well as age, gender and disease severity (binary logistic regression; p=0.008, OR 1.04, 95% CI 1.04 to 1.07). Deprivation was not associated with disease-severity (p=0.906) or body mass index, kg/m2 (p=0.921) using ANOVA. This is the first study to show that deprivation, assessed using IMD, is associated with increased 1-year mortality in outpatients with COPD independently of malnutrition, age and disease severity. Deprivation should be considered in the targeted management of these patients.

Prediction of transcriptional regulatory interactions in bacteria : a comparative genomics approach

Exponential growth of genomic data in the last two decades has made manual analyses impractical for all but trial studies. As genomic analyses have become more sophisticated, and move toward comparisons across large datasets, computational approaches have become essential. One of the most important biological questions is to understand the mechanisms underlying gene regulation. Genetic regulation is commonly investigated and modelled through the use of transcriptional regulatory network (TRN) structures. These model the regulatory interactions between two key components: transcription factors (TFs) and the target genes (TGs) they regulate. Transcriptional regulatory networks have proven to be invaluable scientific tools in Bioinformatics. When used in conjunction with comparative genomics, they have provided substantial insights into the evolution of regulatory interactions. Current approaches to regulatory network inference, however, omit two additional key entities: promoters and transcription factor binding sites (TFBSs). In this study, we attempted to explore the relationships among these regulatory components in bacteria. Our primary goal was to identify relationships that can assist in reducing the high false positive rates associated with transcription factor binding site predictions and thereupon enhance the reliability of the inferred transcription regulatory networks. In our preliminary exploration of relationships between the key regulatory components in Escherichia coli transcription, we discovered a number of potentially useful features. The combination of location score and sequence dissimilarity scores increased de novo binding site prediction accuracy by 13.6%. Another important observation made was with regards to the relationship between transcription factors grouped by their regulatory role and corresponding promoter strength. Our study of E.coli ��70 promoters, found support at the 0.1 significance level for our hypothesis | that weak promoters are preferentially associated with activator binding sites to enhance gene expression, whilst strong promoters have more repressor binding sites to repress or inhibit gene transcription. Although the observations were specific to �70, they nevertheless strongly encourage additional investigations when more experimentally confirmed data are available. In our preliminary exploration of relationships between the key regulatory components in E.coli transcription, we discovered a number of potentially useful features { some of which proved successful in reducing the number of false positives when applied to re-evaluate binding site predictions. Of chief interest was the relationship observed between promoter strength and TFs with respect to their regulatory role. Based on the common assumption, where promoter homology positively correlates with transcription rate, we hypothesised that weak promoters would have more transcription factors that enhance gene expression, whilst strong promoters would have more repressor binding sites. The t-tests assessed for E.coli �70 promoters returned a p-value of 0.072, which at 0.1 significance level suggested support for our (alternative) hypothesis; albeit this trend may only be present for promoters where corresponding TFBSs are either all repressors or all activators. Nevertheless, such suggestive results strongly encourage additional investigations when more experimentally confirmed data will become available. Much of the remainder of the thesis concerns a machine learning study of binding site prediction, using the SVM and kernel methods, principally the spectrum kernel. Spectrum kernels have been successfully applied in previous studies of protein classification [91, 92], as well as the related problem of promoter predictions [59], and we have here successfully applied the technique to refining TFBS predictions. The advantages provided by the SVM classifier were best seen in `moderately'-conserved transcription factor binding sites as represented by our E.coli CRP case study. Inclusion of additional position feature attributes further increased accuracy by 9.1% but more notable was the considerable decrease in false positive rate from 0.8 to 0.5 while retaining 0.9 sensitivity. Improved prediction of transcription factor binding sites is in turn extremely valuable in improving inference of regulatory relationships, a problem notoriously prone to false positive predictions. Here, the number of false regulatory interactions inferred using the conventional two-component model was substantially reduced when we integrated de novo transcription factor binding site predictions as an additional criterion for acceptance in a case study of inference in the Fur regulon. This initial work was extended to a comparative study of the iron regulatory system across 20 Yersinia strains. This work revealed interesting, strain-specific difierences, especially between pathogenic and non-pathogenic strains. Such difierences were made clear through interactive visualisations using the TRNDifi software developed as part of this work, and would have remained undetected using conventional methods. This approach led to the nomination of the Yfe iron-uptake system as a candidate for further wet-lab experimentation due to its potential active functionality in non-pathogens and its known participation in full virulence of the bubonic plague strain. Building on this work, we introduced novel structures we have labelled as `regulatory trees', inspired by the phylogenetic tree concept. Instead of using gene or protein sequence similarity, the regulatory trees were constructed based on the number of similar regulatory interactions. While the common phylogentic trees convey information regarding changes in gene repertoire, which we might regard being analogous to `hardware', the regulatory tree informs us of the changes in regulatory circuitry, in some respects analogous to `software'. In this context, we explored the `pan-regulatory network' for the Fur system, the entire set of regulatory interactions found for the Fur transcription factor across a group of genomes. In the pan-regulatory network, emphasis is placed on how the regulatory network for each target genome is inferred from multiple sources instead of a single source, as is the common approach. The benefit of using multiple reference networks, is a more comprehensive survey of the relationships, and increased confidence in the regulatory interactions predicted. In the present study, we distinguish between relationships found across the full set of genomes as the `core-regulatory-set', and interactions found only in a subset of genomes explored as the `sub-regulatory-set'. We found nine Fur target gene clusters present across the four genomes studied, this core set potentially identifying basic regulatory processes essential for survival. Species level difierences are seen at the sub-regulatory-set level; for example the known virulence factors, YbtA and PchR were found in Y.pestis and P.aerguinosa respectively, but were not present in both E.coli and B.subtilis. Such factors and the iron-uptake systems they regulate, are ideal candidates for wet-lab investigation to determine whether or not they are pathogenic specific. In this study, we employed a broad range of approaches to address our goals and assessed these methods using the Fur regulon as our initial case study. We identified a set of promising feature attributes; demonstrated their success in increasing transcription factor binding site prediction specificity while retaining sensitivity, and showed the importance of binding site predictions in enhancing the reliability of regulatory interaction inferences. Most importantly, these outcomes led to the introduction of a range of visualisations and techniques, which are applicable across the entire bacterial spectrum and can be utilised in studies beyond the understanding of transcriptional regulatory networks.

Timing and distribution of protein ingestion during prolonged recovery from resistance exercise alters myofibrillar protein synthesis

Quantity and timing of protein ingestion are major factors regulating myofibrillar protein synthesis (MPS). However, the effect of specific ingestion patterns on MPS throughout a 12 h period is unknown. We determined how different distributions of protein feeding during 12 h recovery after resistance exercise affects anabolic responses in skeletal muscle. Twenty-four healthy trained males were assigned to three groups (n = 8/group) and undertook a bout of resistance exercise followed by ingestion of 80 g of whey protein throughout 12 h recovery in one of the following protocols: 8 × 10 g every 1.5 h (PULSE); 4 × 20 g every 3 h (intermediate: INT); or 2 × 40 g every 6 h (BOLUS). Muscle biopsies were obtained at rest and after 1, 4, 6, 7 and 12 h post exercise. Resting and post-exercise MPS (l-[ring-(13)C6] phenylalanine), and muscle mRNA abundance and cell signalling were assessed. All ingestion protocols increased MPS above rest throughout 1-12 h recovery (88-148%, P < 0.02), but INT elicited greater MPS than PULSE and BOLUS (31-48%, P < 0.02). In general signalling showed a BOLUS>INT>PULSE hierarchy in magnitude of phosphorylation. MuRF-1 and SLC38A2 mRNA were differentially expressed with BOLUS. In conclusion, 20 g of whey protein consumed every 3 h was superior to either PULSE or BOLUS feeding patterns for stimulating MPS throughout the day. This study provides novel information on the effect of modulating the distribution of protein intake on anabolic responses in skeletal muscle and has the potential to maximize outcomes of resistance training for attaining peak muscle mass.

Epidermal growth factor receptors and cyclooxygenase-2 in the pathogenesis of non-small cell lung cancer : potential targets for chemoprevention and systemic therapy

The epidermal growth factor receptor (EGFR) is part of a family of plasma membrane receptor tyrosine kinases that control many important cellular functions, from growth and proliferation to cell death. Cyclooxygenase (COX)-2 is an enzyme which catalyses the conversion of arachidonic acid to prostagladins and thromboxane. It is induced by various inflammatory stimuli, including the pro-inflammatory cytokines, Interleukin (IL)-1β, Tumour Necrosis Factor (TNF)-α and IL-2. Both EGFR and COX-2 are over-expressed in non-small cell lung cancer (NSCLC) and have been implicated in the early stages of tumourigenesis. This paper considers their roles in the development and progression of lung cancer, their potential interactions, and reviews the recent progress in cancer therapies that are directed toward these targets. An increasing body of evidence suggests that selective inhibitors of both EGFR and COX-2 are potential therapeutic agents for the treatment of NSCLC, in the adjuvant, metastatic and chemopreventative settings. © 2002 Elsevier Science Ireland Ltd. All rights reserved.