Fueled by ever-growing genomic information and rapid developments of proteomics–the large scale analysis of proteins and mapping its functional role has become one of the most important disciplines for characterizing complex cell function. For building functional linkages between the biomolecules, and for providing insight into the mechanisms of biological processes, last decade witnessed the exploration of combinatorial and chip technology for the detection of bimolecules in a high throughput and spatially addressable fashion. Among the various techniques developed, the protein chip technology has been rapid. Recently we demonstrated a new platform called “Spacially addressable protein array” (SAPA) to profile the ligand receptor interactions. To optimize the platform, the present study investigated various parameters such as the surface chemistry and role of additives for achieving high density and high-throughput detection with minimal nonspecific protein adsorption. In summary the present poster will address some of the critical challenges in protein micro array technology and the process of fine tuning to achieve the optimum system for solving real biological problems. / Singapore-MIT Alliance (SMA)

abstract: Currently in the US, many patients with cancer do not benefit from the population-based screening, due to challenges associated with the existing cancer screening scheme. Blood-based diagnostic assays have the potential to detect diseases in a non-invasive way. Proteins released from small early tumors may only be present intermittently and get diluted to tiny concentrations in the blood, making them difficult to use as biomarkers. However, they can induce autoantibody (AAb) responses, which can amplify the signal and persist in the blood even if the antigen is gone. Circulating autoantibodies is a promising class of molecules that have potential to serve as early detection biomarkers for cancers. This Ph.D thesis aims to screen for autoantibody biomarkers for the early detection of two deadly cancer, basal-like breast cancer and lung adenocarcinoma. First, a method was developed to display proteins in both native and denatured conformation on protein array. This method adopted a novel protein tag technology, called HaloTag, to covalently immobilize proteins on glass slide surface. The covalent attachment allowed these proteins to endure harsh treatment without getting dissociated from slide surface, which enabled the profiling of antibody responses against both conformational and linear epitopes. Next, a plasma screening protocol was optimized to significantly increase signal to noise ratio of protein array based AAb detection. Following this, the AAb responses in basal-like breast cancer were explored using nucleic acid programmable protein arrays (NAPPA) containing 10,000 full-length human proteins in 45 cases and 45 controls. After verification in a large sample set (145 basal-like breast cancer cases / 145 controls / 70 non-basal breast cancer) by ELISA, a 13-AAb classifier was developed to differentiate patients from controls with a sensitivity of 33% at 98% specificity. Similar approach was also applied to the lung cancer study to identify AAbs that distinguished lung cancer patients from computed-tomography positive benign pulmonary nodules (137 lung cancer cases, 127 smoker controls, 170 benign controls). In this study, two panels of AAbs were discovered that showed promising sensitivity and specificity. Six out of eight AAb targets were also found to have elevated mRNA level in lung adenocarcinoma patients using TCGA data. These projects as a whole provide novel insights on the association between AAbs and cancer, as well as general B cell antigenicity against self-proteins. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2015

This thesis is about protein profiling in blood-derived samples using suspension bead ar- rays built with protein affinity reagents, and the evaluation of binding characteristics and potential disease relation of such profiles. A central aim of the presented work was to discover and verify disease associated protein profiles in blood-derived samples such as serum or plasma. This was based on immobiliz- ing antigens or antibodies on color-coded beads for a multiplexed analysis. This concept generally allow for a dual multiplexing because hundreds of samples can be screened for hundreds of proteins in a miniaturized and parallelized fashion. At first, protein antigens were used to study humoral immune responses in cattle suffering from a mycoplasma infec- tion (Paper I). Here, the most immunogenic of the applied antigens were identified based on reactivity profiles from the infected cattle, and were combined into an antigen cocktail to serve as a diagnostic assay in a standard ELISA set-up. Next, antibodies and their em- ployment in assays with directly labeled human samples was initiated. This procedure was applied in a study of kidney disorders where screening of plasma resulted in the discovery of a biomarker candidate, fibulin-1 (Paper II). In parallel to the disease related applica- tions, systematic evaluations of the protein profiles were conducted. Protein profiles from 2,300 antibodies were classified on the bases of binding properties in relation to sample heating and stringent washing (Paper III). With a particular focus on heat dependent de- tectability, a method was developed to visualize those proteins that were captured to the beads in an immunoassay by using Western blotting (Paper IV). In conclusion, this thesis presents examples of the possibilities of comparative plasma profiling enabled by protein bead arrays. / <p>QC 20130208</p>

abstract: Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by progressive autoimmune destruction of insulin-producing pancreatic β-cells. Genetic, immunological and environmental factors contribute to T1D development. The focus of this dissertation is to track the humoral immune response in T1D by profiling autoantibodies (AAbs) and anti-viral antibodies using an innovative protein array platform called Nucleic Acid Programmable Protein Array (NAPPA). AAbs provide value in identifying individuals at risk, stratifying patients with different clinical courses, improving our understanding of autoimmune destructions, identifying antigens for cellular immune response and providing candidates for prevention trials in T1D. A two-stage serological AAb screening against 6,000 human proteins was performed. A dual specificity tyrosine-phosphorylation-regulated kinase 2 (DYRK2) was validated with 36% sensitivity at 98% specificity by an orthogonal immunoassay. This is the first systematic screening for novel AAbs against large number of human proteins by protein arrays in T1D. A more comprehensive search for novel AAbs was performed using a knowledge-based approach by ELISA and a screening-based approach against 10,000 human proteins by NAPPA. Six AAbs were identified and validated with sensitivities ranged from 16% to 27% at 95% specificity. These two studies enriched the T1D “autoantigenome” and provided insights into T1D pathophysiology in an unprecedented breadth and width. The rapid rise of T1D incidence suggests the potential involvement of environmental factors including viral infections. Sero-reactivity to 646 viral antigens was assessed in new-onset T1D patients. Antibody positive rate of EBV was significantly higher in cases than controls that suggested a potential role of EBV in T1D development. A high density-NAPPA platform was demonstrated with high reproducibility and sensitivity in profiling anti-viral antibodies. This dissertation shows the power of a protein-array based immunoproteomics approach to characterize humoral immunoprofile against human and viral proteomes. The identification of novel T1D-specific AAbs and T1D-associated viruses will help to connect the nodes in T1D etiology and provide better understanding of T1D pathophysiology. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2015

Decoding the genomes from organisms that encompass all taxonomies provides the foundation for extensive, large scale studies of biological molecules such as RNA, protein and carbohydrates. The high-throughput studies facilitated by the existence of these genome sequences necessitate the development of new analytic methods for the interpretation of large sets of results. The work herein focuses on the development of a novel clustering method for the analysis of protein array results and examines its utilization in the analysis of integrated interaction data sets. Sets of proteins that interact with a molecule of interest were clustered according to their functional similarity. The simUI distance metric in the statistical analysis package BioConductor was applied to measure the similarity of two proteins utilizing the assembly of their Gene Ontology annotation. Clusters were identified by partitioning around medoids and interpreted using the summary label provided by the Gene Ontology annotation of the medoid. The utility of the method was tested on two published yeast protein array data sets and shown to allow interpretation of the data to yield novel biological hypotheses. We performed a protein array screen using the E3 ubiquitin ligase and PDZ domain-containing protein LNX1. We combined these results with other published LNX1 interactors to produce a set of 220 proteins that was clustered according to Gene Ontology annotation. From the clustering results, 14 proteins were selected for subsequent examination by co-immunoprecipitation, of which 8 proteins were confirmed as LNX1 interactors. Recognition of 6 proteins by specific LNX1 PDZ domains was confirmed by fusion protein pull-downs. This work supports the role of LNX1 as a signalling scaffold. The interpretation of protein array results using our novel clustering method facilitated the identification of candidate molecules for subsequent experimental analysis. Thus our analytical method facilitates identification of biologically relevant molecules within a large data set, making this method an essential component of complex, high-throughput experimentation.

Decoding the genomes from organisms that encompass all taxonomies provides the foundation for extensive, large scale studies of biological molecules such as RNA, protein and carbohydrates. The high-throughput studies facilitated by the existence of these genome sequences necessitate the development of new analytic methods for the interpretation of large sets of results. The work herein focuses on the development of a novel clustering method for the analysis of protein array results and examines its utilization in the analysis of integrated interaction data sets. Sets of proteins that interact with a molecule of interest were clustered according to their functional similarity. The simUI distance metric in the statistical analysis package BioConductor was applied to measure the similarity of two proteins utilizing the assembly of their Gene Ontology annotation. Clusters were identified by partitioning around medoids and interpreted using the summary label provided by the Gene Ontology annotation of the medoid. The utility of the method was tested on two published yeast protein array data sets and shown to allow interpretation of the data to yield novel biological hypotheses. We performed a protein array screen using the E3 ubiquitin ligase and PDZ domain-containing protein LNX1. We combined these results with other published LNX1 interactors to produce a set of 220 proteins that was clustered according to Gene Ontology annotation. From the clustering results, 14 proteins were selected for subsequent examination by co-immunoprecipitation, of which 8 proteins were confirmed as LNX1 interactors. Recognition of 6 proteins by specific LNX1 PDZ domains was confirmed by fusion protein pull-downs. This work supports the role of LNX1 as a signalling scaffold. The interpretation of protein array results using our novel clustering method facilitated the identification of candidate molecules for subsequent experimental analysis. Thus our analytical method facilitates identification of biologically relevant molecules within a large data set, making this method an essential component of complex, high-throughput experimentation.

The overall aim of this thesis is to investigate strategies to aid in the measurement of G-protein coupled receptor (GPCR) activity for high-throughput screening and sensing applications. GPCRs are cell surface receptors which have seven membrane spanning domains. They are the largest family of membrane proteins in the human genome and are involved in a number of physiological and pathophysiological pathways. They are the most widely targeted protein family for therapeutics being the target for over 30% of the currently available prescription drugs (Jacoby et al. 2006). For this reason commercial interest and investment into compound screening using these receptors as targets is of high importance in lead drug discovery. Additionally, the extensive ligand range of the GPCR superfamily, which includes light, odorants/ volatiles, neurotransmitters and hormones, make them an attractive biological recognition element in biosensor applications. This thesis demonstrates the functional expression of the H1-histamine, M2-muscarinic and α₂ₐ-adrenergic receptors of the G-protein coupled receptor family, along with their associated G-proteins (Gα, Gβ and Gγ). Expression was achieved using the Sf9/baculovirus expression system. The G-proteins were successfully incorporated into an assay system using time-resolved fluorescence resonance energy transfer (TRFRET). TR-FRET was used in order to create a homogeneous assay format capable of monitoring GPCR activation through the movement of the G-protein subunits. Fluorescence changes in the TR-FRET assay indicated a change in distance between the Gα subunit and Gβγ dimer. The separation of the Gα subunit and the Gβγ dimer after activation resulted in a significant decrease in TR-FRET measurement. The homogeneous set-up of the TR-FRET assay could potentially be adaptable to an array based format. This thesis describes the capture of vesicles containing functional GPCRs onto a solid substrate via the specific interaction between complementary oligonucleotides. GPCR presence and function within the immobilized vesicles, was demonstrated using fluorescent ligands. Further to this, alternative lipid hosts (to the vesicles), known as cubosomes, were introduced. When tagged with an oligonucleotide, these cubosome particles were also shown to immobilize site specifically onto a complementary oligonucleotide surface. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1369537 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

The overall aim of this thesis is to investigate strategies to aid in the measurement of G-protein coupled receptor (GPCR) activity for high-throughput screening and sensing applications. GPCRs are cell surface receptors which have seven membrane spanning domains. They are the largest family of membrane proteins in the human genome and are involved in a number of physiological and pathophysiological pathways. They are the most widely targeted protein family for therapeutics being the target for over 30% of the currently available prescription drugs (Jacoby et al. 2006). For this reason commercial interest and investment into compound screening using these receptors as targets is of high importance in lead drug discovery. Additionally, the extensive ligand range of the GPCR superfamily, which includes light, odorants/ volatiles, neurotransmitters and hormones, make them an attractive biological recognition element in biosensor applications. This thesis demonstrates the functional expression of the H1-histamine, M2-muscarinic and α₂ₐ-adrenergic receptors of the G-protein coupled receptor family, along with their associated G-proteins (Gα, Gβ and Gγ). Expression was achieved using the Sf9/baculovirus expression system. The G-proteins were successfully incorporated into an assay system using time-resolved fluorescence resonance energy transfer (TRFRET). TR-FRET was used in order to create a homogeneous assay format capable of monitoring GPCR activation through the movement of the G-protein subunits. Fluorescence changes in the TR-FRET assay indicated a change in distance between the Gα subunit and Gβγ dimer. The separation of the Gα subunit and the Gβγ dimer after activation resulted in a significant decrease in TR-FRET measurement. The homogeneous set-up of the TR-FRET assay could potentially be adaptable to an array based format. This thesis describes the capture of vesicles containing functional GPCRs onto a solid substrate via the specific interaction between complementary oligonucleotides. GPCR presence and function within the immobilized vesicles, was demonstrated using fluorescent ligands. Further to this, alternative lipid hosts (to the vesicles), known as cubosomes, were introduced. When tagged with an oligonucleotide, these cubosome particles were also shown to immobilize site specifically onto a complementary oligonucleotide surface. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1369537 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

The overall aim of this thesis is to investigate strategies to aid in the measurement of G-protein coupled receptor (GPCR) activity for high-throughput screening and sensing applications. GPCRs are cell surface receptors which have seven membrane spanning domains. They are the largest family of membrane proteins in the human genome and are involved in a number of physiological and pathophysiological pathways. They are the most widely targeted protein family for therapeutics being the target for over 30% of the currently available prescription drugs (Jacoby et al. 2006). For this reason commercial interest and investment into compound screening using these receptors as targets is of high importance in lead drug discovery. Additionally, the extensive ligand range of the GPCR superfamily, which includes light, odorants/ volatiles, neurotransmitters and hormones, make them an attractive biological recognition element in biosensor applications. This thesis demonstrates the functional expression of the H1-histamine, M2-muscarinic and α₂ₐ-adrenergic receptors of the G-protein coupled receptor family, along with their associated G-proteins (Gα, Gβ and Gγ). Expression was achieved using the Sf9/baculovirus expression system. The G-proteins were successfully incorporated into an assay system using time-resolved fluorescence resonance energy transfer (TRFRET). TR-FRET was used in order to create a homogeneous assay format capable of monitoring GPCR activation through the movement of the G-protein subunits. Fluorescence changes in the TR-FRET assay indicated a change in distance between the Gα subunit and Gβγ dimer. The separation of the Gα subunit and the Gβγ dimer after activation resulted in a significant decrease in TR-FRET measurement. The homogeneous set-up of the TR-FRET assay could potentially be adaptable to an array based format. This thesis describes the capture of vesicles containing functional GPCRs onto a solid substrate via the specific interaction between complementary oligonucleotides. GPCR presence and function within the immobilized vesicles, was demonstrated using fluorescent ligands. Further to this, alternative lipid hosts (to the vesicles), known as cubosomes, were introduced. When tagged with an oligonucleotide, these cubosome particles were also shown to immobilize site specifically onto a complementary oligonucleotide surface. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1369537 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

Prédire la capacité de développement et d'implantation des embryons reste un enjeu majeur pour l’Assistance Médicale à la Procréation (AMP). L’AMP doit répondre au désir du couple d’avoir un enfant en limitant les risques encourus par la mère et l’enfant en cas de grossesse multiple. Tous les laboratoires d’AMP utilisent des critères morphologiques pour évaluer la compétence au développement des embryons en dépit de la faible valeur prédictive de cette analyse. L'interaction ovocyte-cumulus participe à l’acquisition par l'ovocyte de sa compétence au développement. Cette interaction met en jeu l’expression de gènes spécifiques dans les cellules de cumulus (CCs). Notre objectif était d'identifier des marqueurs non invasifs de la compétence ovocytaire au développement. Ainsi nous avons recherché au niveau des CCs des gènes et des protéines exprimés en fonction de l’aptitude de l’ovocyte fécondé à atteindre le stade de blastocyste. L'expression des gènes des CCs a été étudiée par puce à ADN et qPCR haut débit. Après avoir tenu compte de la variabilité des patientes, nous avons identifié les gènes RGS2, POLR3K et CUL4B comme biomarqueurs. L'expression des protéines des CCs a été étudiée par puce à protéines et après validation des anticorps ciblant les protéines d'intérêt, les protéines RGS2, POLR3K et MERTK ont été identifiées comme biomarqueurs de la compétence au développement de l'ovocyte. Ces résultats permettent d’envisager la création d’un modèle prédictif multicritère incluant la morphologie de l’embryon à J2, les gènes et protéines marqueurs. / The ability to predict the developmental and implantation ability of embryos remains a major goal in human assisted reproductive technology (ART).ART should allow couple to become parents while limiting the risks to the mother and the child in case of multiple pregnancy. ART laboratories use morphological criteria to evaluate the oocyte competence despite the poor predictive value of this analysis. The oocyte-cumulus interaction helps the oocyte to acquire its developmental competence partly through the expression of specific genes at the cumulus level. Therefore our aim was to identify at the level of cumulus cells (CCs) genes and proteins related to oocyte developmental competence as non-invasive marker. Gene expression of CCs was studied using microarray and high throughput qPCR according to the developmental competence of the oocyte (ability to reach the blastocyst stage after fertilization). While taking into account the patient variability we identified RGS2, POLR3K and CUL4B as biomarkers at RNA level. Then protein expression of CCs was studied using Reverse Phase Protein Array. After validation of the antibodies targeting the proteins of interests, RGS2, POLR3K and MERTK were identified as protein biomarkers of the developmental competence of the oocyte. These results lead us to consider a multi variables predictive model including the morphology of the embryo at J2, genes and protein markers.