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- Dodig-Crnković, Tea
(författare)
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On the application and validation of multiplexed affinity assays
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are essential macromolecules that carry out complex functions in human cells, tissues, and organs. They regulate a diverse set of biological processes and protect against pathogens. However, dysregulation or malformation of proteins can cause disease. By characterizing proteins in health and disease, we can gain insights into disease aetiology and identify druggable targets to treat disorders. By bringing protein discoveries from the research lab into clinical practice, protein assays have been and will continue to be important tools for enabling and improving medical decision-making. The work presented in this thesis concerns both exploratory and targeted affinity-based assays applied for the study of proteins. High-throughput and multiplexed suspension bead arrays have been the primary technology for measuring proteins with antibodies in samples such as human blood. Identification and validation of protein-protein interactions that may provide novel insights into the druggable proteome have also been carried out. Throughout the projects, methods for validating the observations have been pursued and include replication in independent sample sets, as well as the assessment of antibody selectivity via other proteomics assays or orthogonal methods such as genetic associations. In Paper I, we used multiplexed exploratory antibody arrays comprising almost 1.500 affinity binders to study proteins that circulate in plasma. Here, the focus was to determine the longitudinal variability of proteins. We analysed samples from 101 clinically healthy individuals, collected each third month for one year. The protein data provided insights into inter-individual diversity and the unique molecular fingerprint of each participant. We found that 49% of the studied proteins were stable across one year, as these had low variability in each individual. Eight modules, each containing 11-242 proteins, were found to co-vary across one year. We also found genetic variations to influence 15 of the detected protein profiles and confirmed selected indications in an independent set of 3.000 subjects. In summary, we observed the existence of individual-specific protein profiles and found that short-term and continuous changes occurred in almost every participant. In Paper II, we investigated blood-derived serum and plasma to identify age-associated proteins. We started from a large set of exploratory antibody bead arrays to screen 156 individuals aged 50-92 years. We found protein profiles of the histidine-rich glycoprotein (HRG) to be significantly associated with age. This association was further corroborated by the analysis of >4.000 individuals from eight additional and independent sets of blood samples. We further validated the HRG protein profiles by sandwich assays and protein microarrays developed in-house. Comparing genetic data and HRG profiles obtained by two independent antibodies, we observed strong but inverse associations to the genetic variants for two anti-HRG antibodies. In Paper III, we applied multiplexed assays for the detection of autoantibodies against cancer-testis antigens (CTAs) in 133 non-small cell lung cancer (NSCLC) patients. We found reactivity against 29 unique CTAs exclusively in cases, compared to 57 matched controls with benign lung diseases. The presence of six CTAs was further confirmed in an independent set of 34 NSCLC cases. Analysis of longitudinal samples from seven patients demonstrated that the presence of CTA autoantibodies was stable over time for each of the individuals. In Paper IV, we developed a novel multiplexed sandwich-immunoassay for the detection of interaction partners to G-protein coupled receptors (GPCRs). This pharmaceutically important family of membrane proteins is believed to be regulated by another group of receptor activity-modulating proteins (RAMPs) by the formation of protein complexes. We studied cell lysates expressing combinations of 23 GPCRs with three RAMPs. We confirmed most of the previously reported interaction pairs and additionally found evidence for 15 new GPCR-RAMP complexes. All interactions were validated using epitope tags that were engineered onto the proteins. Selected complexes were further validated by in situ proximity ligation assays performed in cell membranes. In summary, the work included in this thesis describes the use of multiplexed affinity-based assays for research within plasma proteomics and the interrogation of protein complexes. The work highlights the method’s potential for the identification of circulating proteins that may aid and add to the current knowledge about human health and disease.
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| 2. |
- Hong, Mun-Gwan
(författare)
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Genomics and bioinformatics strategies in the study of aging and Alzheimer disease
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- To understand complex phenotypes, medical research has evolved from the study of single genes and proteins to approaches that encompass more comprehensive catalogues of molecules. Among the more widely used are genome-wide expression and high-throughput genotyping, the latter primarily making use of single nucleotide polymorphisms (SNPs) in what has been termed genome-wide association studies (GWAS). Because of the scale of the data sets that are being produced, unique problems have emerged that necessitate the extensive use of bioinformatics tools. This thesis has entailed the analysis of several such large data sets in the context of biological pathways and introduces several bioinformatics solutions. Paper III, IV, and V deal with this topic. This thesis is primarily oriented around the study of Alzheimer disease (AD) and aging. The questions about the etiology of AD are often concurrent with questions about the biology of aging. This thesis pursues insight on genomic factors pertaining to both inquiries, acknowledging that both the AD state and aging itself are complex and multi-factorial. Two constituent papers (I and III) address aging and two papers (II and V) deal with genetic models in the study of AD. In paper I, we examined the association of age with several genetic markers in the insulin degrading enzyme (IDE) and explored possible molecular mechanisms. In contrast to women, both age-at-sampling and age-at-death of the males were significantly lower in individuals that were heterozygous at genetic loci spanning the IDE locus. Plasma insulin levels and the expression levels of the gene were found to be higher in those same heterozygous males. In paper II, SNPs in 25 genes involved in cholesterol metabolism were tested for association with AD and dementia. Genetic markers in a large linkage disequilibrium block spanning SREBF1, TOM1L2, and ATPAF2 were significantly associated with disease. Gene expression and gene network analyses supported the findings. In paper III, we investigated the biological pathway basis of age in human brain and lymphocytes. Mitochondrial genes were negatively regulated in both tissue samples, while the protein translation genes appeared to decrease in lymphocytes but increase in brain. Those observations indicated that there are common themes across tissues, but also tissue specific changes in gene regulation. We also examined the genomic architecture of the age-regulated genes, and found that the expression of non-compact genes tend to decrease with advancing age. A large number of genome-wide association studies (GWAS) have now been performed over the past few years. In paper IV, we developed a program that automates the conversion of SNPs to representative gene lists in order to facilitate the exploration of biological pathway in the context of GWAS. In paper V, we employed the software developed in study IV to identify biological pathways enriched among the genes that were significantly associated from a GWAS of AD. Genes involved in intracellular protein transmembrane transport were found to be significantly overrepresented. These results highlighted the possibility that TOMM40 contributes to AD pathology together with other translocases. Through this thesis, several biological relationships have been identified linking AD and aging. Genetic markers in IDE, a gene previously claimed to be associated with AD, also associate with age. With advancing age, mitochondrial gene expression deteriorates significantly. TOMM40 may contribute the AD pathology, together with other genes that encode proteins of the intracellular transmembrane protein transport pathway. Methodologically, pathway analyses were conducted successfully with the program, ProxyGeneLD. This enabled discoveries and discussion of the challenges that face the exploration of GWAS data sets in a pathway context. In the future, more sophisticated bioinformatics tools and enhanced gene annotation may lead to the discovery of the molecular mechanisms that dominate complex diseases and traits.
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