Computational Methods on Study of Differentially Expressed Proteins in Maize Proteomes Associated with Resistance to Aflatoxin Accumulation

Plant breeders have focused on improving maize resistance to Aspergillus flavus infection and aflatoxin accumulation by breeding with genotypes having the desirable traits. Various maize inbred lines have been developed for the breeding of resistance. Identification of differentially expressed proteins among such maize inbred lines will facilitate the development of gene markers and expedite the breeding process. Computational biology and proteomics approaches on the investigation of differentially expressed proteins were explored in this research. The major research objectives included 1) application of computational methods in homology and comparative modeling to study 3D protein structures and identify single nucleotide polymorphisms (SNPs) involved in changes of protein structures and functions, which can in turn increase the efficiency of the development of DNA markers; 2) investigation of methods on total protein profiling including purification, separation, visualization, and computational analysis at the proteome level. Special research goals were set on the development of open source computational methods using Matlab image processing tools to quantify and compare protein expression levels visualized by 2D protein electrophoresis gel techniques

The Bronchoalveolar Lavage Proteome- Phenotypic associations to smoking and divergence towards development of COPD

Proteomic analysis of bronchoalveolar lavage (BAL) fluid from smokers at risk of developing chronic obstructive pulmonary disease (COPD) and never smokers is described. COPD is currently the world's fourth leading cause of death and its prevalence is increasing. The leading cause of COPD is smoking and an estimated 600 million people in the world suffer from COPD which makes it the world's most common chronic disease. The aim of this thesis was to explore and characterize the BAL proteome of never smokers and smokers. The hypotheses were that the BAL proteome reflect smoking habits in subjects, and that smokers susceptible to COPD development have a specific proteome. In order to relate the measurement of protein expression with clinical phenotypes we have developed and utilized an interdisciplinary toolbox that includes protein separation (two-dimensional gel electrophoresis and liquid chromatography), mass spectrometry identification and statistical methods for multivariate analysis. The study material used in this thesis consisted of age matched men all born in 1933, living in one city differing by lifelong smoking history. These were compared by clinical function measurements and histological assessment at the same relative time points. A follow up study after 6-7 years identified a group of subjects who had progressed to COPD GOLD stage 2. Those with COPD shared a distinct protein expression profile in the baseline BAL sample which could be identified using multivariate analysis. This pattern was not observed in BAL samples of asymptomatic smokers free of COPD at the 6-7 year follow-up. The results suggest that specific patterns of protein expression occur in the airways of smokers susceptible to COPD disease progression, before the disease is clinically measurable

Proteomics of Toxoplasma gondii

The Apicomplexan parasite Toxoplasma gondii is an obligate intracellular parasite. Infection by T .gondii causes the disease toxoplasmosis, which is one of the most prevalent parasitic diseases of animals and humans. It has been 100 years since the first discovery of the parasite in 1908; research on T. gondii has been carried out in many scientific disciplines consistently expanding the understanding of this parasite. In the last ten years, the developments of EST, microarray, genome sequencing and continuing efforts towards genome annotation has centralized the focus of T. gondii research on the understanding of gene expression and gene functions on the genome scale. Equipped with the technical advances in mass spectrometry and bioinformatics, proteomics has become established as an integral component in the post-genomics era by providing first-hand data on the functional products of gene expression. In this study, three complementary proteomic strategies, 1-DE, 2-DE and MudPIT, have been used to characterise the proteome of T. gondii tachyzoites. Protein identifications have been acquired for more than two thousand (2252) unique release 4 genes, representing almost one third (29%) of the predicted proteome of all life cycle stages. Functional predictions for each protein were carried out, which provided valuable insights into the composition of the expressed proteome and their potential biological roles. The T. gondii proteomic data has been integrated into the publically accessible ToxoDB, where 2477 intron-spanning peptides provided supporting evidence for correct splice site annotation of the release 4 genome annotation. The incompleteness of the release 4 genome annotation has been highlighted using peptide evidence, confirming 421 splice sites that are only predicted by alternative gene models. Analysis has also been carried out on the proteomic data in the light of other genome wide expression data. The comparison of the proteome and transcriptome of Toxoplasma and other Apicomplexa parasites has revealed important discrepancies between protein and mRNA expression where interesting candidates have been highlighted for further investigation. A preliminary DIGE study has been developed to characterize protein expression changes in T. gondii grown in the presence or absence of glucose. In conclusion, this study has demonstrated the importance of proteomic applications in understanding gene expression profiles and regulation in T. gondii and highlighted the importance and potential of proteogenomic approaches in genome annotation process. The importance of temporal and quantitative proteomics as well as the future of systems biology has been discussed

Proteomic analysis of the heat shock and acclimation responses of Cyanobacteria

The cyanobacterium Synechocystis sp. PCC 6803 is a model experimental organism for proteomic research because its entire genomic sequence is available and cyanobacteria have high adaptive potential towards a variety of environmental stresses. Heat shock characteristically induces expression of the heat shock proteins (Hsps) and photosynthetic organisms have demonstrated the ability to acclimatise their photosynthetic apparatus to milder elevated temperatures. In this study the proteomic methodology of two dimension gel electrophoresis and peptide mass fingerprinting mass spectrometry (PMF MS) was developed for the analysis of Synechocystis proteins. High resolution was attained with the application of narrow acidic pH range 'zoom' gels and of 192 individual soluble protein spots analysed via MALDI ToF, 105 were identified. A 2-D difference gel electrophoresis (2D DIGE) based proteomic approach has been applied to characterise the heat shock response in the soluble protein fraction and determine protein factors involved in the thermal acclimation of the thylakoid membrane and its associated photosynthetic machinery in Synechocystis. These analyses together with PMF MS for protein identification characterised 176 and 108 heat shock and heat acclimation responsive protein spots, respectively. In both analyses, molecular chaperones displayed the highest heat elevated level, demonstrating a dual role in stabilisation and refolding of both soluble and membrane-bound proteins. Other proteins identified in the heat shock response included those involved in photosynthesis, carbon fixation, translation, amino acid biosynthesis and several hypothetical proteins. Proteins involved in heat acclimation of the thylakoid membrane included constituents of photosynthesis, respiration, hopene biosynthesis and several hypothetical proteins. Furthermore, a candidate heat sensor involved in the regulation of heat shock gene expression has been characterised through analysis of the heat shock response in a histidine kinase knock out mutant strain of Synechocystis, namely Мік34, which displays increased thermal tolerance. The gene product of hik34 has a possible dual role in both the suppression of hsp gene expression under normal growth temperature and enhancement under heat shock

Molecular mechanisms of drug resistance, invasion and metastasis in pancreatic carcinoma

The research outlined in this thesis aims to further our knowledge of the biological characteristics underlying the aggressive behaviour by which pancreatic cancer invades and metastasises at an early stage and is refractory to most chemotherapeutic drugs. Investigation into drug resistance in pancreatic cancer involved pulse-selection of three cell lines with epirubicin, taxotere and gemcitabine. The drug-selected cell lines, in general, displayed low changes in sensitivity to their respective selecting drugs and no obvious cross-resistance profile. However, drug treatment modulated the invasive potential of the pulse-selected cell lines. Expression of multi-drug resistant proteins (P-gp and MRP-1) was also determined by IHC in 45 pancreatic tumour specimens, results suggested a contributory role for P-gp expression in pancreatic cancer. An in vitro pancreatic invasion model was established by single cell cloning of MiaPaCa-2, yielding sub-clones displaying diverse invasive properties. The malignant phenotypes of MiaPaCa-2, Clone #3 (highly invasive) and Clone #8 (poorly invasive) were characterised, comparative proteomic profiling by 2-D DIGE, MALDI-TOF MS and RNAi revealed three targets, ALDH1A1, VIM and STIP1 with functional involvement in the invasion process. ALDH1A1 appeared to play a role in the aggressive invasive phenotype; expression was associated with increased invasion, drug resistance and decreased adhesion. VIM and STIP expression was also linked to increased invasion and decreased adhesion, with STIP1 displaying a role in proliferation, which has not previously been described. Factors secreted into conditioned media of Clone #3 and Clone #8 was also examined by proteomics. Targets, GSN and ALDH1A1 were chosen for functional analysis and their potential involvement in cancer cell invasion assessed. EGFR/HER2 expression was analysed in 5 pancreatic cancer cell lines, and two cell lines which co-expressed EGFR and HER2 were tested with lapatinib. Lapatinib, a dual EGFR/HER2 inhibitor in combination with chemotherapeutic drugs showed additive interactions, suggesting the possibility of a therapeutic role for lapatinib in combination with chemotherapy in pancreatic cancer patients co-expressing EGFR/HER2

Development of microbial technologies for the soluble phosphorus production by phosphate rock solubilization = Desarrollo de tecnologías microbianas para la producción de fósforo soluble por solubilización de roca fosfórica

210 p.El fósforo (P) es, después del nitrógeno (N), el segundo nutriente más limitante del desarrollo de los cultivos agrícolas. Por este motivo, ambos son añadidos periódicamente junto con el potasio (K), en los conocidos como fertilizantes NPK. El P juega un importante papel en numerosos procesos metabólicos como la fotosíntesis, la síntesis de ácidos nucleicos, o incluso la resistencia a diversas infecciones. Sin embargo, a pesar de ser abundante en suelos, el P no se encuentra disponible para las plantas. De hecho, se estima que tan sólo entre el 10 y el 15% del P añadido en los fertilizantes es realmente consumido por las plantas. El resto reacciona con elementos presentes en el suelo como el aluminio (Al), el hierro (Fe) o el calcio (Ca), dando lugar a formas insolubles del P que no son asimilables por los cultivos. Debido al bajo rendimiento de los fertilizantes, éstos se añaden en exceso para proporcionar a los cultivos el P necesario para su desarrollo, acumulándose poco a poco en el suelo. Este fenómeno de precipitación del P da lugar al conocido como “legacy phosphorus” (literalmente traducido como “fósforo heredado”), sobre el cual, algunos expertos afirman que podría ser suficiente como para continuar la producción agrícola al ritmo actual durante 100 años, si se encontrara accesible para los cultivos. El problema del P no acaba aquí, ya que es importante destacar que se trata de un recurso no renovable. De hecho, a pesar de que podemos encontrar reservas de roca fosfórica alrededor de todo el mundo, algunos expertos defienden que estas reservas podrían agotarse durante los próximos 50 años. Actualmente, la fuente principal de P es la roca fosfórica: grandes depósitos de roca sedimentaria con un elevado contenido en fosfatos. La extracción del P presente en la roca fosfórica se lleva a cabo, principalmente, mediante el empleo de ácido sulfúrico concentrado. Éste reacciona con los fosfatos de la roca, generando ácido fosfórico, el cual es recogido y neutralizado posteriormente con amonio para ser utilizado en los fertilizantes químicos. No obstante, un 20% del P total contenido en la roca fosfórica no puede ser extraído mediante estos procedimientos, ya que el consumo necesario de ácido para su procesado sería tan elevado que haría que no fuese rentable. De modo que este P queda retenido en unos residuos llamados schlams, los cuales son apilados en enormes escombreras, por lo que una gran cantidad de P es desperdiciada durante el proceso. Por estos motivos, en los últimos años se han propuesto diferentes técnicas para incrementar el rendimiento del P existente. Por ejemplo, se ha estudiado su extracción a partir de fuentes menos ricas en P como el guano, o incluso recuperar el P suministrado en los aditivos alimenticios del ganado a partir de los excrementos de ganadería. De entre todas las propuestas, la estrategia en la que más se ha ahondado es el desarrollo de biofertilizantes que contengan microorganismos solubilizadores de P (PSM, por sus siglas en inglés) con el objetivo de que éstos incrementen el rendimiento de los fertilizantes, lo que permitiría disminuir tanto el contenido de P de los mismos, como la cantidad de fertilizantes utilizados. A lo largo de este trabajo, no sólo se aborda el estudio de PSM para su posible aplicación en bioabonos, si no que se plantea la posibilidad de desarrollar un proceso de biominería para la extracción del P presente en los schlams de forma ecológica y económica. Los procesos de biominería emplean sistemas biológicos para facilitar la extracción de compuestos de interés industrial, generalmente a partir de productos de desecho y residuos de minería. Estos residuos presentan un bajo contenido de los compuestos de interés, por lo que su tratamiento no resultaría rentable mediante tecnologías convencionales. En cambio, los procesos biomineros son, en general, poco contaminantes y tienen bajo coste de producción. En la gran mayoría de los procesos de biominería se emplean bacterias oxidadoras del azufre (SOB, por sus siglas en inglés), cuya principal habilidad es la biosíntesis de ácido sulfúrico a partir de la oxidación de compuestos reducidos del azufre (especialmente azufre elemental y tiosulfatos). De este modo, se plantea la posibilidad de desarrollar un proceso de biominería que permita extraer el P residual presente en los schlams de manera rentable. Así, el ácido sulfúrico producido por las SOB actuaría disolviendo el P de los schlams, que se extraería como ácido fosfórico del mismo modo que en la producción de P a partir de roca fosfórica. Con este objetivo, se aislaron 5 cepas diferentes de SOB, y se analizó su capacidad de solubilizar el P presente en los schlams. Para ello, se elaboraron unas mini-escombreras sobre unos embudos Büchner. A lo largo de diferentes ensayos, se pudo comprobar que la cepa que mejores resultados mostró fue Acidithiobacillus thiooxidans. Se trata de una bacteria gram negativa, del grupo de las γ proteobacterias, extremófila, quimiolitoautótrofa, que utiliza el azufre como fuente de energía para llevar a cabo la fijación de CO2, el cual utiliza como fuente de carbono para su desarrollo. Durante este proceso, lleva a cabo la síntesis de ácido sulfúrico, que es secretado al exterior celular, creando un entorno extremadamente ácido que puede llegar a alcanzar un pH cercano a 2. Así, A. thiooxidans fue la bacteria seleccionada para llevar a cabo un primer proceso de escalado. Se elaboraron unas pequeñas escombreras de 25 toneladas suplementadas con azufre mineral (un residuo procedente de la industria del petróleo que presenta un gran contenido en azufre y que puede adquirirse de manera muy económica), y se inocularon con A. thiooxidans. Al cabo de 6 meses, se tomaron muestras en puntos aleatorios de la escombrera y se analizó el contenido de P soluble, P insoluble y P total presente en los schlams. Se observó que la cepa es capaz de solubilizar el P de los schlams sin necesidad de intervención humana en el proceso. De modo que se pudo demostrar la viabilidad del proceso de extracción del P residual de los schlams mediante biominería, utilizando, además, un segundo residuo procedente de la refinería del petróleo. Por lo tanto, se ha podido comprobar que se trata de un proceso limpio, ecológico y de bajo coste. Sin embargo, los procesos metabólicos involucrados en la síntesis de ácido sulfúrico y en la capacidad del microorganismo para sobrevivir en ambientes tan hostiles es aún una incógnita. Por este motivo, se llevó a cabo un estudio del proteoma de la bacteria bajo diferentes condiciones de pH que permitieran dilucidar sus mecanismos de supervivencia. Debido al pobre conocimiento que se tiene acerca del genoma y proteoma de esta bacteria, no fue posible determinar con exactitud qué ocurre en el interior de A. thiooxidans en condiciones de pH extremas, aunque el estudio nos acerca un poco más a comprender a estos microorganismos tan especiales. Por otro lado, con el objetivo de aislar y estudiar cepas para su posible aplicación en bioabonos, se llevó a cabo el aislamiento y selección de 9 bacterias solubilizadoras de fosfato (PSB): Bacillus megaterium, Pseudomonas plecoglossicida, Bacillus sp., Pantoea eucrina, Achromobacter xylosoxidans, Pseudomonas koreensis, Burkholderia fungorum, Enterobacter cloacae y Advenella mimigardefordensis. De todas ellas se analizaron los posibles mecanismos de solubilización de P, además de otras habilidades promotoras del crecimiento de la planta, como la solubilización de minerales como el zinc (Zn) y el K, la actividad antifúngica frente a diversos patógenos del cereal, o la producción de hormonas vegetales como el ácido indolacético. Finalmente, las bacterias se aplicaron a cultivos de cebada en un ensayo en invernadero, en el cual se analizó su efecto sobre la asimilación de P y el crecimiento de la planta, y sobre el desarrollo de espigas y su contenido en almidón. Se observó que dos cepas destacaban por su efecto sobre el desarrollo de los cultivos de cebada: B. megaterium y A. mimigardefordensis. En el caso de B. megaterium, han sido previamente descritos tanto su capacidad solubilizadora de P, como su efecto en el crecimiento de algunos cultivos, aunque aún no ha sido comercializada como biofertilizante. Sin embargo, no hemos podido encontrar constancia de que A. mimigardefordensis haya sido previamente descrita como bacteria solubilizadora de fosfato ni se haya analizado su capacidad promotora del crecimiento de las plantas. Por lo que podemos concluir que esta es la primera referencia de A. mimigardefordensis como cepa solubilizadora de P, y como bacteria promotora del crecimiento de plantas (en este caso de cebada). 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Application of Different Bioanalytical Workflows for Proteomics of Prostate Cancer

In the current dissertation, we focused on the development and application of multiple mass spectrometry-based bioanalytical platforms for phosphoproteomic characterization in cell culture and clinical specimens of prostate cancer; and on the application of optimized methods to analysis of differential protein expression to reveal molecular mechanism of drug action in animal model of prostate cancer. Characterization of the phosphoproteome in prostate cancer Our study in phosphoprotein signatures on a large scale in prostate cancer focused on the LNCaP human prostate cancer cell line, and on human prostate cancer tissue. For the LNCaP prostate cancer cell line, we applied a combination of analytical platforms: (1) a novel in-gel isoelectric focusing (IEF) LC-MS/MS analytical platform; (2) a 2-DE based platform combined with phospho-specific staining. The in-gel IEF LC-MS/MS analytical methodology used in the study included separation of the LNCaP proteins by in-gel isoelectric focusing; digestion of the proteins with trypsin; enrichment of the digests for phosphopeptides with immobilized metal ion affinity chromatography (IMAC); analysis of the enriched digests by LC-MS/MS; and identification of the phosphorylated peptides/proteins through searches of the Swiss-Prot protein sequence database. With in-gel IEF based analytical platform, we have characterized over 600 different phosphorylation sites in 296 phosphoproteins in the LNCaP prostate cancer cell line. This panel of the LNCaP phosphoproteins was 3-fold larger than the panel obtained in our previous work, and is the largest phosphoprotein panel in prostate cancer reported to date. The phosphoproteins identified in this study belonged to various locations within the cell and were involved in various processes including cell differentiation, transcription regulation, and intercellular signal transduction. We also developed a 2-DE based platform, in combination with multiplexed staining and LC-MS/MS, for the identification of LNCaP phosphoproteins. In this study, we applied 2-DE as separation technique, Pro-QTM Diamond stain as phosphoprotein detection method, LC-MS/MS and database searches for protein identification to investigate the phosphoproteins in the LNCaP prostate cancer cell line. Proteins identified from spots of interest were shown to be highly relevant to prostate cancer. We demonstrated the feasibility of using 2-DE with phospho-specific stain and mass spectrometry to investigate the phosphoproteins in the LNCaP cell line. This methodology complements the in-gel IEF LC-MS/MS platform that we used for phosphoproteomics study; it will be of particular value for future comparative studies of phosphoproteins in various physiological states. For prostate cancer tissue, a gel-free approach was performed to analyze five prostate cancer tissue specimens to obtain phosphoproteomic signature of prostate cancer for biomarker discovery. Proteins were extracted with Trizol reagent, and then in-solution digested with trypsin. Phosphopeptides were enriched with IMAC, and analyzed the phosphorylated peptides/proteins by LC-MS/MS with identification through searches of the Swiss-Prot protein sequence database. The panels obtained for prostate cancer tissue contain 15-24 phosphoproteins. Some of the characterized phosphoproteins were present in all five specimens; in addition, each specimen also produced a unique set of phosphoproteins. The findings provided a direct glimpse into the phosphoprotein machinery operating within the human prostate cancer tissue. This pilot study focused on a small set of specimens. The phosphoprotein panels that were obtained contained a number of proteins that were unique to a particular specimen. Comparative proteomics study of drug effects in prostate cancer We carried out the first comparative proteomics study for the examination of the effects of bicalutamide/embelin combination treatment on prostate tumors by characterizing the alterations in protein expression that was induced upon treatment of mice bearing prostate tumors with anticancer combination therapy. A comparative proteomic strategy based on 2-DE coupled with LC-MS/MS was performed on mouse prostate tumor tissue. Proteins from the mouse prostate tumors were extracted with Trizol, and the protein mixtures were separated by 2-DE. Differences in the protein profiles for the different treatment groups were evaluated by computer-assisted analysis of SYPRO Ruby stained 2-DE gels. LC-MS/MS and database searches were used to identify differentially expressed proteins. Pathway analysis was carried out on the dataset of identified proteins with the Ingenuity bioinformatics tool. Out of the 33 differentially expressed protein spots, 30 protein spots were identified and grouped into various functional classes. The major protein categories were metabolism (52%), stress response (12%), protein biosynthesis (13%) and apoptosis (11%), suggesting that alterations in these processes may be involved in the mechanism of drug action. Proteins associated with oxidative stress were up-regulated, which indicated that treatment with bicalutamide/embelin may affect the redox balance within the prostate tumor, and this effect may contribute to tumor suppression

Biochemical and proteomic investigation of bovine nasal secretion

The principal aims of the work presented in this thesis were to investigate the biochemical properties and protein compositions of nasal secretion (NS) from healthy cattle and to document changes in the protein expression in NS from diseased and vaccinated cattle using advanced proteomic methods. Bovine respiratory disease (BRD) is the principal source of economic loss for the cattle industry throughout the world. Even though it has been studied extensively, it still remains the number one cause of disease and death in cattle. Although the etiopathogenesis of BRD is multifactorial and complex, all of the pathogens have the common route of infection which is intranasal. Thus, NS is potentially a valuable source of biological sample in the detection of biomarkers for BRD. Therefore, a method for the collection of substantial volumes of NS from cattle was developed in this study to establish a reference range of analytes that are present in the NS of healthy cattle. Biochemical profiles of NS from a group of 38 healthy Holstein-Friesian cows revealed high alkaline phosphatase (AP) activity of up to 2392 IU/L which is 15.7 fold higher compared to AP activity in serum reference ranges. This study further investigated the source of the high AP activity in bovine NS. Histochemical analysis using AP specific staining confirmed the localization of the AP enzyme activity to epithelial cells and serous glands of the nasal mucosa. Advanced molecular methods were used to determine the characterization of nasal AP. The analysis at mRNA levels from nasal mucosa by endpoint RT-PCR and PCR product sequencing confirmed that the AP was locally produced and is identical at the nucleotide level to the non-specific AP splice variant gene (ALPL) found in bovine liver, bone and kidney. Investigation using isoelectric focussing (IEF) confirmed that AP was produced locally at a high level in nasal epithelium demonstrating that novel AP isoenzymes in NS had pIs in the same range as those of the nasal mucosa extracts (pH 4.8-6.2) but were clearly different from the extracts of other tissues. The differences in IEF migration of the AP extracts is likely to be due to post translational modifications (PTMs) such as glycosylation or phosphorylation which would be areas worthy of future research. Preliminary proteomic investigation of NS from healthy cattle using 1D gel electrophoresis, 2D gel electrophoresis and ESI-MS/MS analysis putatively identified 10 major proteins in NS consisting of 7 vascular proteins and other glandular and cellular proteins such as lactoferrin, an anti-bacterial protein commonly present in mucosal secretions, odorant binding protein known to have a role in scent recognition and glutathione-S-transferase, an enzyme capable of detoxifying noxious compounds. The final objective was therefore to compare the nasal proteins from healthy, disease and vaccinated group of animals using 2D difference gel electrophoresis (DiGE). The experimental model used for this investigation was an immunisation study against bovine malignant catarrhal fever (MCF). It was concluded that quantitative proteomics technology such as DiGE identified and measured changes in nasal protein expression in response to MCF and following vaccine protection. In conclusion, this research has contributed to the scientific knowledge regarding the biochemical properties and protein compositions that is present in bovine NS. In addition, the research also explored the use of proteomic technologies as a novel tool to analyse protein expression and identify possible biomarkers in NS. This thesis is an important step forward for a better understanding of bovine NS, and thus provides a basis for future studies involving bovine NS by way of providing reference data and alternative source of biological sample

Investigation of the intrinsic mechanism of drug resistance in multiple myeloma

The focus of this thesis was to evaluate the mechanisms whereby myeloma cells develop intrinsic resistance with a focus on resistance in the context of bortezomib treatment. The aims of this thesis were to examine multidrug resistance pumps as a mechanism of resistance in MM, to investigate the contribution of p53 signalling perturbations in resistance mechanism in MM, to study the AMPK pathway as an alternative target to overcome MM resistance and finally to characterise myeloma resistance to bortezomib treatment using 2D-DIGE analysis. Focussing on bortezomib resistance models, we found that that overexpression of P-gp attenuates bortezomib activity. Bortezomib is a P-gp substrate and a combination of P-gp inhibitor and bortezomib is able to overcome resistance. Bortezomib is also able to downregulate the expression and function of P-gp. Our findings therefore suggest that combination of a P-gp inhibitor and bortezomib in P-gp positive myeloma would be a reasonable treatment combination to extend use of the drug. We have shown that p53 apoptotic signalling pathways can be accentuated when bortezomib is combined with a Mdm2 inhibitor. In p53 WT cells, nutlin-3 in combination with bortezomib generates additive toxicity in MM cells but is highly synergistic in epithelial models and p53-mutated cell lines. This synergy persists in the presence of BMSCs. This observation has implications more so in epithelial cancers and p53 mutated cancers where single agent bortezomib activity is mild. We have also shown that bortezomib-treated patients who had high expression of nutlin-3-suppressed genes had significantly shorter progression-free (p=0.001, log-rank test) and overall survival (p=0.002, log-rank test) compared to those with low expression levels. AMPK activation is promising as an anticancer pathway and may also be a chemoprevention target. Metformin and AICAR, which activate this pathway, both have demonstrated useful preclinical anticancer properties and have a good therapeutic index in patients. We explored mechanism of cell death and showed that AICAR was able to activate the apoptotic pathway. These agents also synergise with glycolysis inhibitors to further increase cytotoxicity in cancer cells. Identification of proteins whose expression is altered in differing states of sensitivity and resistance provides candidates for better understanding of resistance mechanisms so we also investigated bortezomib resistance in cellular models using proteomic techniques and isolated and identified several novel proteins which may play a role in this phenomenon. Our findings are mechanistically consistent since two of the identified proteins Hsp70 and caspase-3 are known in the literature to be affected by bortezomib treatment

Acute phase proteins and biomarkers for health in chickens

Acute phase proteins (APPs) are proteins synthesised predominantly in the liver, whose plasma concentrations increase (positive APP) or decrease (negative APP) as a result of infection, inflammation, trauma and tissue injury. They also change as a result of the introduction of immunogens such as bacterial lipopolysaccharide (LPS), turpentine and vaccination. While publications on APPs in chickens are numerous, the limited availability of anti-sera and commercial ELISAs has resulted in a lot of information on only a few APPs. Disease is a threat to the poultry industry, as pathogens have the potential to evolve, spread and cause rapid onset of disease that is detrimental to the welfare of birds. Low level, sub-acute disease with non-specific, often undiagnosed causes can greatly affect bird health and growth and impact greatly on productivity and profitability. Developing and validating methods to measure and characterise APPs in chickens will allow these proteins to be used diagnostically for monitoring flock health. Using immune parameters such as APPs that correlate with disease resistance or improvements in production and welfare will allow the use of APPs as selection parameters for breeding to be evaluated. For APPs to be useful parameters on which to evaluate chicken health, information on normal APP concentrations is required. Ceruloplasmin (Cp) and PIT54 concentrations were found to be much lower in healthy birds form commercial production farms than the reported normal values obtained from the literature. These APPs were found to be significantly higher in culled birds from a commercial farm and Cp, PIT54 and ovotransferrin (Ovt) were significantly higher in birds classified as having obvious gait defects. Using quantitative shotgun proteomics to identify the differentially abundant proteins between three pools: highly acute phase (HAP), acute phase (AP) and non-acute phase (NAP), generated data from which a selection of proteins, based on the fold difference between the three pools was made. These proteins were targeted on a individual samples alongside proteins known to be APPs in chickens or other species: serum amyloid A (SAA), C-reactive protein (CRP), Ovt, apolipoprotein A-I (apo-AI), transthyretin (Ttn), haemopexin (Hpx) and PIT54. Together with immunoassay data for SAA, Ovt, alpha-1-acid glycoprotein (AGP) and Cp the results of this research reveal that SAA is the only major APP in chickens. Ovotransferrin and AGP behave as moderate APPs while PIT54 and Cp are minor APPs. Haemopexin was not significantly different between the three acute phase groups. Apolipoprotein AI and Ttn were significantly lower in the HAP and AP groups and as such can be classed as negative APPs. In an effort to identify CRP, multiple anti-sera cross reacting with CRP from other species were used and a phosphorylcholine column known to affinity purify CRP were used. Enriched fractions containing low molecular weight proteins, elutions from the affinity column together with HAP, AP and NAP pooled samples were applied to a Q-Exactive Hybrid Quadrupole–Orbitrap mass spectrometer (Thermo Scientific) for Shotgun analysis and CRP was not identified. It would appear that CRP is not present as a plasma protein constitutively or during an APR in chickens and as such is not an APP in this species. Of the proteins targeted as possible novel biomarkers of the APR in chickens mannan binding lectin associated serine protease-2, α-2-HS-glycoprotein (fetuin) and major facilitator superfamily domain-containing protein 10 were reduced in abundance in the HAP group, behaving as negative biomarkers. Myeloid protein and putative ISG(12)2 were positively associated with the acute phase being significantly higher in the HAP and AP groups. The protein cathepsin D was significantly higher in both HAP and AP compared to the NAP indicating that of all the proteins targeted, this appears to have the most potential as a biomarker of the acute phase, as it was significantly increased in the AP as well as the HAP group. To evaluate APPs and investigate biomarkers of intestinal health, a study using re-used poultry litter was undertaken. The introduction of litter at 12 days of age did not significantly increase any APPs measured using immunoassays and quantitative proteomics at 3, 6 and 10 days post introduction. While no APP was found to be significantly different between the challenged and control groups at anytime point, the APPs AGP, SAA and Hpx did increase over time in all birds. The protein apolipoprotein AIV (apo-AIV) was targeted as a possible APP and because of its reported role in controlling satiety. An ELISA was developed, successfully validated and used to measure apo-AIV in this study. While no significant differences in apo-AIV plasma concentrations between challenged and control groups were identified apo-AIV plasma concentrations did change significantly between certain time points in challenged and control groups. Apoliporotein AIV does not appear to behave as an APP in chickens, as it was not significantly different between acute phase groups. The actin associated proteins villin and gelsolin were investigated as possible biomarkers of intestinal health. Villin was found not to be present in the plasma of chickens and as such not a biomarker target. Gelsolin was found not to be differentially expressed during the acute phase or as a result of intestinal challenge. Finally a proteomic approach was undertaken to investigate gastrocnemius tendon (GT) rupture in broiler chickens with a view of elucidating to and identify proteins associated with risk of rupture. A number of proteins were found to be differentially expressed between tendon pools and further work would enable further detailing of these findings. In conclusion this work has made a number of novel findings and addressed a number of data poor areas. The area of chicken APPs research has stagnated over the last 15 years with publications becoming repetitive and reliant on a small number of immunoassays. This work has sought to characterise the classic APPs in chickens, and use a quantitative proteomic approach to measure and categorise them. This method was also used to take a fresh approach to biomarker identification for both the APR and intestinal health. The development and validation of assays for Ovt and apo-AIV and the shotgun data mean that these proteins can be further characterised in chickens with a view of applying their measurement to diagnostics and selective breeding programs