Academic literature on the topic 'Genetic characterization, proteomics'

The study of an organism’s genome, often known as “genomics,” has advanced quickly, producing a wealth of publicly accessible genetic data. Despite how valuable the genome is; proteins essentially control most aspects of cell function. Proteomics, or the comprehensive study of proteins, has emerged as an important technology for disease characterization, diagnosis, prognosis, drug development, and therapy. Proteomics technologies are now used to support the diagnosis and treatment of both infectious and noninfectious diseases. Nevertheless, it is more difficult to describe a proteomic profile since a single gene product may result in a number of unique proteins, and proteins have a wider range of chemical configurations. The proteome profiles of a particular organism, tissue, or cell are impacted by a variety of environmental factors, including those triggered by infectious agents. This review intends to highlight the applications of proteomics in the study of disease diagnosis and treatment. In this review, the different technologies used in proteomics studies, like two-dimensional gel electrophoresis, mass spectrometry, and protein microarray as well as biomarker discovery and drug target identification using proteomics, have also been focused on.

Abstract Identification of protease substrates and detailed characterization of processed sites are essential for understanding the biological function of proteases. Because of inherent complexity reasons, this however remains a formidable analytical challenge, illustrated by the fact that the majority of the more than 500 human proteases are uncharacterized to date. Recently, in addition to conventional genetic and biochemical approaches, diverse quantitative peptide-centric proteomics approaches, some of which selectively recover N-terminal peptides, have emerged. These latter proteomic technologies in particular allow the identification of natural protease substrates and delineation of cleavage sites in a complex, natural background of thousands of different proteins. We here review current biochemical, genetic and proteomic methods for global analysis of substrates of proteases and discuss selected applications.

Proteomics is a new area of study that in recent decades has provided great advances in the field of medicine. However, its enormous potential for the study of proteomes makes it also applicable to other areas of science. Milk is a highly heterogeneous and complex fluid, where there are numerous genetic variants and isoforms with post-translational modifications (PTMs). Due to the vast number of proteins and peptides existing in its matrix, proteomics is presented as a powerful tool for the characterization of milk samples and their products. The technology developed to date for the separation and characterization of the milk proteome, such as two-dimensional gel electrophoresis (2DE) technology and especially mass spectrometry (MS) have allowed an exhaustive characterization of the proteins and peptides present in milk and dairy products with enormous applications in the industry for the control of fundamental parameters, such as microbiological safety, the guarantee of authenticity, or the control of the transformations carried out, aimed to increase the quality of the final product.

Inherited metabolic disorders (IMD) are rare medical conditions caused by genetic defects that interfere with the body’s metabolism. The clinical phenotype is highly variable and can present at any age, although it more often manifests in childhood. The number of treatable IMDs has increased in recent years, making early diagnosis and a better understanding of the natural history of the disease more important than ever. In this review, we discuss the main challenges faced in applying proteomics to the study of IMDs, and the key advances achieved in this field using tandem mass spectrometry (MS/MS). This technology enables the analysis of large numbers of proteins in different body fluids (serum, plasma, urine, saliva, tears) with a single analysis of each sample, and can even be applied to dried samples. MS/MS has thus emerged as the tool of choice for proteome characterization and has provided new insights into many diseases and biological systems. In the last 10 years, sequential window acquisition of all theoretical fragmentation spectra mass spectrometry (SWATH-MS) has emerged as an accurate, high-resolution technique for the identification and quantification of proteins differentially expressed between healthy controls and IMD patients. Proteomics is a particularly promising approach to help obtain more information on rare genetic diseases, including identification of biomarkers to aid early diagnosis and better understanding of the underlying pathophysiology to guide the development of new therapies. Here, we summarize new and emerging proteomic technologies and discuss current uses and limitations of this approach to identify and quantify proteins. Moreover, we describe the use of proteomics to identify the mechanisms regulating complex IMD phenotypes; an area of research essential to better understand these rare disorders and many other human diseases.

Protamines replace histones as the main nuclear protein in the sperm cells of many species and play a crucial role in compacting the paternal genome. Human spermatozoa contain protamine 1 (P1) and the family of protamine 2 (P2) proteins. Alterations in protamine PTMs or the P1/P2 ratio may be associated with male infertility. Top-down proteomics enables large-scale analysis of intact proteoforms derived from alternative splicing, missense or nonsense genetic variants or PTMs. In contrast to current gold standard techniques, top-down proteomics permits a more in-depth analysis of protamine PTMs and proteoforms, thereby opening up new perspectives to unravel their impact on male fertility. We report on the analysis of two normozoospermic semen samples by top-down proteomics. We discuss the difficulties encountered with the data analysis and propose solutions as this step is one of the current bottlenecks in top-down proteomics with the bioinformatics tools currently available. Our strategy for the data analysis combines two software packages, ProSight PD (PS) and TopPIC suite (TP), with a clustering algorithm to decipher protamine proteoforms. We identified up to 32 protamine proteoforms at different levels of characterization. This in-depth analysis of the protamine proteoform landscape of normozoospermic individuals represents the first step towards the future study of sperm pathological conditions opening up the potential personalized diagnosis of male infertility.

Abstract Pediatric Central Nervous System (CNS) tumors are the leading cause of cancer-related death among children. Identifying new targeted therapies necessitates the use of pediatric cancer models that faithfully recapitulate the patient’s disease. However, the generation and characterization of pediatric cancer models has significantly lagged adult cancers, underscoring the urgent need to develop and characterize pediatric CNS models of disease. Herein, we establish a single-site collection of 233 CNS tumour cell lines, representing 14 distinct brain childhood tumor types. We subjected >200 cell lines to multi-omics analyses (DNA-sequencing, RNA-sequencing, DNA methylation, proteomics, phospho-proteomics), and in parallel performed pharmacological and genetic CRISPR-Cas9 loss of function screens to identify pediatric-specific treatment opportunities and biomarkers. Our work provides insight into specific pathway vulnerabilities in molecularly defined pediatric tumor classes and uncovers biomarker-linked therapeutic opportunities of clinical relevance. Cell line data and resources are provided in an open access portal (vicpcc.org.au/dashboard).

Abstract Comprehensive assessment of the flow of genetic information through multi-omic data integration can reveal the molecular consequences of genetic variation underlying human disease. Next generation sequencing (NGS) is used to identify genetic variants and characterize gene function (e.g. transcriptome and epigenome), while mass spectrometry is used to assess the proteome through characterization of protein abundances, modifications, and interactions. A new plasma profiling platform, the Proteograph࣪ Product Suite, leverages multiple nanoparticles with distinct physiochemical properties to enable deep plasma proteome analyses at scale. Here, we present a cloud-based, data analysis software platform called Proteograph Analysis Suite (PAS) for proteogenomic data analyses through the integration of proteomics data derived from the Proteograph with genomic variant information derived from NGS experiments. PAS features include an experiment data management system, analysis protocols, an analysis setup wizard, and tools for reviewing and visualizing results. PAS can support both Data Independent Analysis (DIA) and Data Dependent Analysis (DDA) proteomics workflows and is compatible with variant call format (vcf) files from NGS workflows to enable personalized database searches. To assess quality of the resulting data PAS includes various metrics like peptide/protein group intensity, protein sequence coverage, relative protein abundance distribution, peptide and protein group counts. Visualizations including principal component analysis, hierarchical clustering, and heatmaps allow intuitive identification of experimental trends. To enable biological insights, differential expression analyses results are reported with interactive visualizations such as volcano plots, protein interaction maps, and protein-set enrichment. From data to insight, PAS provides an easy-to-use and efficient suite of functionality to enable proteogenomic data analysis. Integration of proteomics and genomics data require a variety of tools, many of which require command-line interfaces and operating system-specific requirements that can act as a barrier for researchers to adapt new data analysis tools. Here, we demonstrate the utility of PAS by analyzing samples from the Proteograph NSCLC plasma dataset1. PAS can analyze VCF files generated from NGS pipelines in combination with mass spec data to identify peptide variants using personalized libraries. Using the cloud-based architecture computational tasks are distributed for rapid analysis. The integrated proteogenomics viewers allow variant IDs to be interpreted in the context of genomic coordinates, protein sequence, functional domains and features. Together, these results show the utility of PAS for seamless and fast proteomic data analysis. Reference: 1 Blume, J. E. et al. Nature Communications, 2020 Citation Format: Aaron S. Gajadhar, Margaret K. Donovan, Harsharn Auluck, Yan Berk, Yuandan Lou, Theo Platt, Serafim Batzoglou. A cloud-scalable software suite for large-cohort proteogenomics data analysis and visualization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6348.

Temperature-sensitive (TS) missense mutants have been foundational for characterization of essential gene function. However, an unbiased approach for analysis of biochemical and biophysical changes in TS missense mutants within the context of their functional proteomes is lacking. We applied MS-based thermal proteome profiling (TPP) to investigate the proteome-wide effects of missense mutations in an application that we refer to as mutant thermal proteome profiling (mTPP). This study characterized global impacts of temperature sensitivity–inducing missense mutations in two different subunits of the 26S proteasome. The majority of alterations identified by RNA-Seq and global proteomics were similar between the mutants, which could suggest that a similar functional disruption is occurring in both missense variants. Results from mTPP, however, provide unique insights into the mechanisms that contribute to the TS phenotype in each mutant, revealing distinct changes that were not obtained using only steady-state transcriptome and proteome analyses. Computationally, multisite λ-dynamics simulations add clear support for mTPP experimental findings. This work shows that mTPP is a precise approach to measure changes in missense mutant–containing proteomes without the requirement for large amounts of starting material, specific antibodies against proteins of interest, and/or genetic manipulation of the biological system. Although experiments were performed under permissive conditions, mTPP provided insights into the underlying protein stability changes that cause dramatic cellular phenotypes observed at nonpermissive temperatures. Overall, mTPP provides unique mechanistic insights into missense mutation dysfunction and connection of genotype to phenotype in a rapid, nonbiased fashion.

Biotechnology can be defined broadly as a set of tools that allows scientists to genetically characterize or improve living organisms. Several emerging technologies, such as molecular characterization and genetic transformation, are already being used extensively for the purpose of plant improvement. Other emerging sciences, including genomics and proteomics, are also starting to impact plant improvement. Tools provided by biotechnology will not replace classical breeding methods, but rather will help provide new discoveries and contribute to improved nutritional value and yield enhancement through greater resistance to disease, herbicides and abiotic factors. In soybeans, biotechnology has and will continue to play a valuable role in public and private soybean breeding programs. Based on the availability and combination of conventional and molecular technologies, a substantial increase in the rate of genetic gain for economically important soybean traits can be predicted in the next decade. In this paper, a short review of technologies for molecular markers analysis in soybean is given as well as achievements in the area of genetic transformation in soybean.

[Formulae and special characters can only be approximated. Please see the pdf version of this abstract for an accurate reproduction.] Tumor necrosis factor (TNF) is a pleiotropic cytokine that mediates a long list of immunological and pathophysiological processes. TNF is produced by a wide variety of cells including immune and non-immune cells, however in most cell types TNF is not expressed prior to stimulation. The function of TNF is mediated via its trimeric domain by binding to TNF receptors that are found on most types of cells, especially of the haematopoietic systems, hence transpiring its effects on a wide variety of cells and organ systems. The cytotoxic (apoptosis) and pro-inflammatory (differentiation, proliferation and activation) functions of TNF are protective but can also result in pathological or deleterious consequences. A biallelic G to A transition polymorphism in the promoter region of TNF at nucleotide position 308 from the transcription start site is suggested to be involved in differential transcriptional regulation of TNF expression. The high TNF producing 308A allele is associated with susceptibility to or worse outcome of many infectious diseases in addition to autoimmune and other pathophysiological conditions. A previous study in our laboratory observed a selective affinity towards the polymorphic 308A allele by an EMSA protein(s) complex, named E. Several other protein complexes were found along with complex E and one of them was identified as Sp1. The identification of complex E was unsuccessful but it was hypothesized to play a major role as transcriptional activator in 308A allele individuals hence transpiring its effect in various pathophysiological states. In this study, the EMSA complexes observed in the TNF promoter region between nucleotides 322 to 283, encompassing the 308 polymorphism, is characterized. EMSA using mutated oligonucleotides mapped the binding sites of complexes B, C, D and E. TRANSFAC database search in addition to previous work revealed the identity of complex C as Sp1 but the rest of complexes remained unknown. Moreover, in contrast to our previous study, the protein(s) in the complex E was found to preferentially bind 308G nucleotide hence posing as a transcriptional repressor, resulting in decreased production state of TNF in 308G allele individuals than 308A allele individuals. In order to characterize putative transcription factors binding to the promoter region, first the biochemical characteristics such as the effects of temperature, salts and cations on DNA binding ability of EMSA complexes were studied. EMSA complexes B, C, DI and E required cations, probably Zn+2, to bind DNA. By optimizing a technique that couples EMSA with SDS-PAGE, the molecular weight of C, DI and E was determined. A novel technique that couples EMSA with IEF determined the pI of complexes B, C, D, DI and E. Although a commonly used technique of identifying unknown DNA-binding protein of interest, Yeast One-Hybrid assay, did not identify complex E, the novel identification method involving chromatography, two-dimensional electrophoresis, EMSA, mass spectrometry and database interrogation successfully identified TNF EMSA complex E as transcription factor Ying Yang 1 (YY1). Supershift EMSA confirmed complex E as YY1. In addition, the supershift assay showed presence of Sp1 and Sp3 in complex C. Similarly, complex DI is identified as Sp3. The novel method in identifying DNA-binding proteins is particularly useful as this technique allows identification of protein seen in EMSA without the need of extensive identification process. YY1 binds to a 6 base pair sequence, 5? TTGAGG 3?, from nt 295 to 290 of TNF promoter. The loss of affinity in 308A allele is caused by transition of underlined G nucleotide to A. The determined and described molecular weight of YY1 in literature is 60 kDa while the theoretical weight is 45 kDa. Both the determined and theoretical pI of YY1 is 5.8. YY1 is a multifunctional transcription factor implicated in both positive and negative regulation of gene expression as well as in initiation of transcription. It is ubiquitously expressed in growing, differentiated, and growth-arrested cells. Although future experiment is yet to establish in vivo presence of YY1 in TNF promoter, our study so far provides convincing evidence that the putative transcription factor that has selective affinity towards 308G allele is indeed YY1.

Spinal muscular atrophy (SMA) is a genetic disorder characterized by muscle weakness and atrophy and death of motor neurons in humans. Although almost all cases of SMA occur due to mutations in a gene called survival motor neuron 1 (SMN1), SMA patients present with a wide range of severities of the symptoms. The most severe cases never achieve any developmental motor milestone and die within a few years after birth. On the other hand, mild cases of SMA have a normal life span and show trivial motor deficits. This suggests the role of other factors (rather than the function of SMN1) in the outcome of the disease. Indeed, the copy number of an almost identical gene, called SMN2, is the main determining factor for the severity of SMA. In addition, a few other genes (e.g. Plastin 3) are proposed as disease modifiers in SMA. SMN1 is a housekeeping gene, but due to unknown reasons, the most prominent pathologies in SMA are atrophy of myofibers and death of motor neurons. However, recent studies showed that some other cell types are also affected in the course of SMA disease. We investigated the alterations of central synapses in Smn2B/- mice, a model of SMA. We did not observe any degeneration of central synapses in these mice until a post symptomatic stage. However, mass spectrometry (MS) analysis on isolated synaptosomes from spinal cords of these animals revealed widespread alterations in the proteome of their central synapses at a presymptomatic stage. Functional cluster analysis on MS results suggested that several molecular pathways are affected within synapses of spinal cords of Smn2B/- mice prior to the onset of any obvious pathology in their motor units. The affected molecular pathways are involved in basic cell biological functions including energy production, protein synthesis, cytoskeleton regulation and intracellular trafficking. We showed that the levels of several proteins involved in actin cytoskeleton regulation are altered in synaptosomes isolated from spinal cords of Smn2B/- mice. More investigations are required to determine the exact functional abnormalities of affected pathways in central synapses of these mice. We also generated congenic Smn2B/- mice in two different mouse genetic backgrounds; FVB and BL6. Using a systematic approach, we showed that congenic Smn2B/- mice in the FVB background show a more severe SMA phenotype than Smn2B/- mice in a BL6 background. Smn2B/- mice in the FVB background had a shorter survival, higher rate of weight loss, earlier and more severe pathologic changes compared to Smn2B/- mice in the BL6 background. We investigated the levels of several actin binding proteins in spinal cords of these animals and found higher induction of plastin 3 in Smn2B/- mice in the BL6 background. More investigations are underway to determine the role of plastin 3 in the severity of the phenotype of Smn2B/- mice, and to find other possible SMA modifier genes in these animals.

Fusarium head blight (FHB) caused by Fusarium graminearum is a dreadful disease of wheat (Triticum aestivum L.). Host resistance to FHB in wheat is quantitatively inherited. Though more than 100 QTLs have been identified, only a few have been validated. However, the resistance mechanisms governed by these QTLs are poorly understood. A type II FHB resistance QTL Fhb1 is the most consistent and largest effect QTL in wheat against FHB spread in wheat. Non-targeted metabolic and proteomic profiling of wheat near isogenic lines (NILs) with resistant and susceptible Fhb1 alleles was used to functionally characterize Fhb1 using a high resolution LC-MS. The Fhb1 from a moderately resistant cultivar Nyubai was associated with cell wall thickening, mainly at the rachis, due to deposition of hydroxycinnamic acid amides (HCAAs), phenolic glucosides and flavonoids. A hypothetical protein coding gene (GenBank: CBH32656.1) near Fhb1 locus was putatively identified as hydroxycinnamoyl transferase, which catalyzes the biosynthesis of HCAAs. Deoxynivalenol (DON) accumulation was high in both the NILs, eliminating DON detoxification as a mechanism associated with Fhb1 (Chapter III). For additional confirmation, the Fhb1 resistant allele, from a highly FHB resistant cultivar Sumai-3 was profiled. Even though the DON accumulation was low in resistant NIL, the detoxification of DON by host UDP-glycosyltransferase was moderately high in both the NILs, with no significant difference. Interestingly, unlike in the resistant NIL, constitutively present glycerophospholipids were absent in the susceptible NIL following pathogen inoculation due to degradation of membrane. Membrane degradation was caused due to programmed cell death as evidenced by DNA laddering in the susceptible NIL. A locus TAA_ctg0954b.00390.1 was identified as an Fhb1 candidate gene that contains a calmodulin binding motif and two nucleolar localization signal motifs and hence re-annotated as calmodulin binding protein (TaCaMBP_Fhb1). The TaCaMBP_Fhb1 is induced following pathogen infection, binds to Ca2+ bound calmodulin, and triggers Ca2+ signalling cascade including transcriptional activation of endonucleases that cleaves the genomics DNA and cause programmed cell death. The resistant allele of TaCaMBP_Fhb1 lacks part of the promoter region and is non-functional in triggering Ca2+ signalling. While the susceptible allele of TaCaMBP_Fhb1, with functional promoter region is capable of triggering Ca2+ signalling and programmed cell death. The necrotrophic pathogen F. graminearum feeds on the dead tissue, multiply in the host and produce more DON, following a repeated cycle in the susceptible genotype (Chapter IV). The wheat resistance mechanisms against FHB were further confirmed, based on metabolic profiling of rachis, from a resistant cultivar Sumai-3 and a susceptible cultivar Roblin, for resistance against spread of a trichothecene producing (Wild: FgTri5+) and a trichothecene non- producing (mutant: FgTri5-) isolates of F. graminearum. The wild isolate repressed several host resistance mechanisms in both the cultivars due to production of DON. The FHB resistance to spread in Sumai-3 was mainly because of increased cell wall thickening, especially at rachis, due to deposition of lignin, HCAAs and flavonoids, and partially, due to induced RR metabolites which in turn reduced the fungal biomass and toxin biosynthesis. The resistance was not attributed to DON detoxification by UDP-glycosyltransferase, as it was not significant in both the cultivars confirming our previous studies (Chapter V). The resistant alleles of two Fhb1 candidate genes, identified in this study, can be suitably stacked into genome of elite cultivars to enhance FHB resistance in wheat.
La fusariose de l'épi est une maladie fongique attaquant le blé (Triticum aestivum) induite par Fusarium graminearum. La fusariose cause de sévères pertes économiques dues à la réduction de la qualité et des rendements suite à la contamination par les mycotoxines trichothecene. La résistance du blé face à la fusariose est un trait quantitatif. Plus de 100 LCQ on été identifiés et un petit nombre a été validé. Cependant, les mécanismes de résistance gouvernés par ces LCQ sont peu connus. Fhb1 est le LCQ le plus consistent qui produit le plus grand effet face à la fusariose du blé. Une caractérisation fonctionnelle à l'aide d'un LC-MS à haute résolution de lignées isogéniques avec ou sans l'allèle susceptible Fhb1 a générée des profils de métabolites non ciblés ainsi que protéomique. Le Fhb1 d'un cultivar modérément résistant, Nyubai, a été associé avec le développement de la paroi cellulaire plus épaisse, surtout au niveau du rachis due à la déposition d'acides amides hydroxycinnamic (HCAAs), de glucosides phénolique ainsi que de flavonoïdes. Le gène codant pour une protéine hypothétique (GenBank: CBH32656.1) située près du locus Fhb1 a été identifiée comme étant possiblement une hydroxycinnamoyle transférase. Cette protéine déclencherait la biosynthèse de HCAAs. L'accumulation de DON a été plus élevée dans les deux lignes isogéniques. La détoxification de DON est un mécanisme associé avec Fhb1 (Chapitre III). Pour confirmer, l'allèle Fhb1 la résistance du cultivar Sumai-3 a été profilé. Contrairement aux lignes iso géniques, aucune présence constitutive de glycérophospholipides, n'a été détectée chez les lignées susceptibles en raison de la dégradation des membranes. La dégradation de membrane s'est avérée être causée par la mort cellulaire programmée comme le démontre le patron de dégradation de l'ADN de la variété susceptible NIL. Le locus TAA_ctg0954b.00390.1 fut identifié comme candidat pour le gène Fhb1 qui contient un domaine de liaison à la calmoduline et deux signaux de localisation nucléaire. Ce dernier fut donc annoté en tant que protéine de liaison à la calmoduline (TaCaMBP_Fhb1). TaCaMBP_Fhb1 est induit suite à l'infection du pathogène pour ensuite se lier à la calmoduline liée au Ca2+ pour ensuite initier une cascade de signaux qui inclut l'activation transcriptionnelle d'endonucléases qui clivent l'ADN génomique causant ainsi la mort cellulaire programmée. L'allèle résistante de TaCaMBP_Fhb1 présente une délétion au niveau du promoteur ce qui la rend non fonctionnel pour l'activation du signalement Ca2+ impliqué dans la mort cellulaire programmée. Le pathogène nécrotrophe F. graminearum se nourrit des tissus morts, se multiplie et produit plus de DON pour faciliter l'infection; perpétuant ainsi un cercle vicieux chez le génotype susceptible (Chapitre IV). C'est résultats on été confirmés à l'aide d'un profilage métabolique des rachis de la lignée résistante Sumai-3 et la lignée susceptible Roblin lors de l'infection avec (Wild : FgTri5+) trichothécène producteur et (Mutant :FgTri5- ) trichothécène non producteur qui sont deux isolats de F. graminearum. L'isolat producteur est parveu à inhiber plusieurs mécanismes de résistance de l'hôte dans les deux cultivars grâce à la production de DON. La résistance FHB à l'infection dans Sumai-3 était principalement lié à l'augmentation des parois cellulaires particulièrement au niveau des rachis à cause de la déposition de lignine, HCAAs et de flavonoïdes et partiellement due à l'augmentation des métabolite RR qui réduisent la biomasses des champignons ainsi que la synthèse des toxines. La résistance n'a pas été attribuée à détoxification de DON par l'UDP-glycosyltransferase, puisque les résultats étaient similaires dans les deux cultivars (Chapitre V). Les allèles résistants, des deux gènes candidats Fhb1 identifiés dans cette étude, pourraient-être ajoutés au génome de cultivars élites de blé pour augmenter leur résistance au FHB.

A sorghum gene SbLRR2, which is predicted to encode a simple extracellular leucine-rich repeat (LRR) protein, was previously isolated among a collection of fungal pathogen-induced sorghum cDNA clones generated by suppression subtractive hybridization. Phylogenetic analysis revealed that they are distinct from the simple extracellular LRR proteins reported previously. Subcellular localization analysis demonstrated that the SbLRR2-EYFP fusion protein was targeted to the extracellular space in tobacco leaf cells. Peptide N-Glycosidase F treatment revealed that the SbLRR2 is N-glycosylated with non-fucosylated oligosaccharides when transiently expressed in Nicotiana benthamiana leaves. Functional analysis was performed in SbLRR2 over-expression (OE) Arabidopsis plants which showed enhanced resistance against the necrotrophic pathogens Botrytis cinerea and Alternaria brassicicola. In addition, the OE lines were found to have elevated expression of several jasmonate acid (JA)-associated genes and higher endogenous JA contents. Hence, the SbLRR2-mediated defense responses in transgenic Arabidopsis are likely to be dependent on JA-signaling through increased JA production. On the other hand, the OE lines remained susceptible to Pseudomonas syringae pv. tomato as the wild type plants. Consistently, there was no up-regulation of salicylic acid (SA) defense marker gene expression or SA levels in the OE lines. Using yeast two-hybrid analysis, SbLRR2 was further shown to interact with Arabidopsis hypersensitive-induced response protein 1. Such interaction may suppress hypersensitive response which is known to enhance necrotrophic pathogen invasion. These data suggested a positive regulatory role of SbLRR2 in plant defense. Further phenotypic analysis of transgenic SbLRR2 revealed its novel role in enhancing lead [Pb(II)] tolerance in Arabidopsis. OE-lines were showed to alleviate Pb(II)-induced root inhibition, reduce the accumulation of Pb(II), and enhance transcription of AtPDR12 which was previously shown to function as a potential Pb(II) efflux pump contributing to Pb(II) detoxification. However, all the Pb(II) tolerance responses were abolished when SbLRR2 was transformed into the atpdr12 mutant. Meanwhile, the extracellular localization of SbLRR2 was shown to be essential for the enhanced Pb(II) tolerance in transgenic Arabidopsis. Together, these results indicated that SbLRR2-mediated Pb(II) tolerance was dependent on AtPDR12 via Pb(II) extrusion. Further investigations revealed the Pb(II)-induced transcriptional activation of SbLRR2 and several highly conserved AtPDR12 homologs in sorghum seedlings, suggesting the possibilities of a common molecular mechanism for Pb(II) tolerance in diverse plant species. Finally, an iTRAQ-based LC-MS/MS quantitative proteomics approach was used to investigate of lead responses in Arabidopsis. A total of 114 proteins showed significant changes in protein abundance with 58 up-regulated and 56 down-regulated proteins. Analysis of changes in the protein profile revealed that the photosynthesis, photorespiration and protein biosynthesis in Arabidopsis were inhibited under lead toxicity. On the other hand, abundances of proteins involved in the antioxidant system, glucosinolate-myrosinase system and JA biosynthesis pathway were elevated upon Pb(II) treatment. Further investigation revealed that Pb(II) stress induced a rapid increase of JA contents in Arabidopsis whereas a JA biosynthesis deficient mutant (AOS) showed hypersensitivity to Pb(II) toxicity, strongly implicating a significant role of JA in Pb(II) response.
published_or_final_version
Biological Sciences
Doctoral
Doctor of Philosophy

DYRK1A is a protein kinase encoded by a gene implicated in Down syndrome pathogenesis. Loss of DYRK1A could promote oncogenic transformation. However, the regulation and substrates of DYRK1A are not fully understood. MudPIT proteomic analysis revealed novel DYRK1A interacting proteins with poorly characterized or even unknown functions. Therefore, the aim of this thesis was to understand the function of DYRK1A through the characterization of its interacting proteins. To achieve this aim, we established stable cell lines expressing these proteins and confirmed the interactions between DYRK1A and seven candidate binding partners. Furthermore, we found that all novel DYRK1A-interacting proteins also bind DCAF7, a previously reported DYRK1A-binding scaffold protein that binds to the N-terminus of DYRK1A. Using cyto-nuclear fractionation and immunostaining we found that DYRK1A-interacting proteins were present in different cellular compartments, suggesting that DYRK1A could play distinct roles in the cell depending on its localization. DYRK1A has been shown to regulate cell proliferation and actin cytoskeleton therefore we used cell proliferation assays and actin staining to determine the role of DYRK1A-interacting proteins in these processes. Here we report functional characterization of the interacting partners of DYRK1A and present cell-based models that will help to understand the function and regulation of this important protein kinase.

L'Océan Pacifique sud-ouest et les eaux de la Nouvelle-Calédonie sont caractérisés par de fortes abondances en cyanobactéries. Parmi ces cyanobactéries, certaines ont la capacité de fixer l'azote atmosphérique (N2), et sont appelées cyanobactéries diazotrophes. Ces organismes sont connus pour contenir en proportions variables des métabolites et nutriments à haute valeur ajoutée qui leur confèrent un potentiel de valorisation économique possiblement intéressants pour la Nouvelle-Calédonie. Plusieurs de ces cyanobactéries ont été isolées en culture depuis les eaux côtières et hauturières du Pacifique·Sud-Ouest mais la caractérisation précise de leur diversité et de leur potentiel de valorisation restent encore inconnus. Dans l 'optique d'une meilleure connaissance de la diversité et d'une éventuelle valorisation économique, les objectifs de ce travail doctoral étaient (i) d'étudier la variabilité saisonnière de la diversité/activité des cyanobactéries diazotrophes dans le lagon de Nouméa, (i) d'effectuer une caractérisation morpho-génétique et protéomique de souches autochtones récemment isolées en culture et (iii) d'évaluer leurs potentiels de valorisation
The southwest Pacific Ocean and the waters of New Caledonia are characterized by high abundances of cyanobacteria. Among these cyanobacteria, some have the ability to fix atmospheric nitrogen (N2), and are called diazotrophic cyanobacteria. These organisms are known to contain high added value metabolites and nutrients in varying proportions, which give them potential for economic development that may be of interest to New Caledonia. Several of these cyanobacteria have been isolated in culture from the coastal and offshore waters of the Southwest Pacific, but the precise characterization of their diversity and their potential for recovery are still unknown. With a view to a better knowledge of diversity and a possible economic valuation, the objectives of this doctoral work were (i) to study the seasonal variability of the diversity / activity of diazotrophic cyanobacteria in the lagoon of Noumea, (i) to carry out a morphogenetic and proteomic characterization of indigenous strains recently isolated in culture and (iii) to evaluate their potential for valorization

Abstract The introduction and development of genomics and proteomics during the past decade have significantly changed the way in which biomedical, agricultural, and biological research is conducted. Genomics and proteomics, which represent the identification and sequencing of genes and the systematic analysis and documentation of proteins in a biological sample, have been recently combined to give rise to functional genomics. Functional genomics encompasses assigning function to genes identified by genomics as well as investigating the organization and control of genetic pathways and the modification of their resulting proteins. This approach is breaking down the distinction be- tween classic areas of study such as biochemistry, genetics, and physiology and bringing aspects of each field together to advance the understanding of biological systems. In this chapter we address the genomics aspect of functional genomics and its application to mammalian preimplantation-stage embryos by introducing and discussing methods designed to investigate and identify “new” genes involved in preimplantation development.

Multiomics also described as integrative omics is an analytical approach that combines data from multiple ‘omics’ approaches including genomics, transcriptomics, proteomics, metabolomics, epigenomics, metagenomics and Meta transcriptomics to answer the complex biological processes involved in rare genetic disorders. This omics approach is particularly helpful since it identifies biomarkers of disease progression and treatment progress by collective characterization and quantification of pools of biological molecules within and among the various types of cells to better understand and categorize the Mendelian and non- Mendelian forms of rare diseases. As compared to studies of a single omics type, multi-omics offers the opportunity to understand the flow of information that underlies the disease. A range of omics software and databases, for example WikiPathways, MixOmics, MONGKIE, GalaxyP, GalaxyM, CrossPlatform Commander, and iCluster are used for multi-omics data exploration and integration in rare disease analysis. Recent advances in the field of genetics and translational research have opened new treatment avenues for patients. The innovation in the next generation sequencing and RNA sequencing has improved the ability from diagnostics to detection of molecular alterations like gene mutations in specific disease types. In this chapter, we provide an overview of such omics technologies and focus on methods for their integration across multiple omics layers. The scrupulous understanding of rare genetic disorders and their treatment at the molecular level led to the concept of a personalized approach, which is one of the most significant advancements in modern research which enable researchers to better comprehend the flow of knowledge which underpins genetic diseases.

Genetic, transcriptional, and clinical heterogeneity of disease has remained to be a prominent obstacle to the development of a targeted therapeutic approach against TNBC. So far, based on tumor size, lymph node status, and histologic features TNBC subtypes were stratified. Insights into inter and intratumoral heterogeneity of TNBC were gained by next-generation sequencing, genomic, transcriptomic, proteomic, and clinicopathological characterization. To depict tumor response to neoadjuvant chemotherapy, radiological characterization may also a play significant role. Biomarkers for subtyping TNBC were highly needed to depict the survival outcome. This chapter discussed the available and possible molecular and pathological diagnostic approaches to TNBC. Furthermore, the integration of morphological and genomic data may emerge as a promising approach for the identification of new therapeutic and prognostic markers to predict the likely outcome of the disease. This chapter aims to highlight the molecular and pathological diagnostic approaches to depict both metastatic and non-metastatic TNBC.

In recent years, an explosion has occurred in the acquisition of biological data through the use of so-called ‘omics’ techniques. Whilst many different omics technologies are now featured in the literature, the most frequently used omics are genomics, transcriptomics, proteomics and metabolomics. A transcriptome is the complete set and quantity of all RNA molecules produced by the genome at any given time for a particular developmental stage or physiological state in a cell, a population of cells, or an organism. Transcriptomics is regarded as a highthroughput technology concerned with determining how the transcriptome changes with respect to various factors at a certain time point and at a given biological state. The study of the transcriptome is essential for understanding the functional elements of the genome and revealing the molecular constituents of cells. Nowadays, transcriptomics have come to the forefront of international scientific attention due to the rapid development of RNA-sequencing (RNA-Seq) methods. The key aim of transcriptomics is precise, comprehensive characterization of the transcriptome; the annotation of all species of transcript, including mRNAs, non-coding RNAs, such as small RNAs, the determination of the transcriptional structure of genes, and the quantification of differential expression levels of each transcript under different conditions. Generally, the goal of transcriptome analysis is to identify genes differentially expressed among different conditions, leading to a new understanding of the genes or pathways associated with the conditions. This chapter provides fundamental information into the promising technology of transcriptomics.

Crop improvement for nitrogen (N) use efficiency (NUE) has been complicated by too many definitions for NUE, different forms of N inputs and different harvested outputs, apart from the large number of genes and processes involved. The recent characterization of the NUE phenotype in rice and the availability of N-responsive transcriptomic and proteomic datasets have enabled systematic identification and shortlisting of genetic and epigenetic targets for crop improvement. Transgenic validation of several candidate genes is in progress, including by genome editing. Further, genome-wide association studies (GWAS), identification of quantitative trait loci (QTL) and development of NUE associated markers are in progress, spanning N-uptake, -assimilation, -local and -systemic translocation, -remobilization as well as associated regulatory networks. This chapter summarizes and critically evaluates the progress made so far on these aspects and highlights the opportunities and challenges for the improvement of NUE in rice.

Hepatocellular carcinoma (HCC) is one of the third leading and common lethal cancers worldwide. Early detection of tumorigenesis of hepatocellular carcinoma is through ultrasonography, computerized tomography (CT) scans, and magnetic resonance imaging (MRI) scans; however, these methods are not up to the mark, so a search for an efficient biomarker for early diagnosis and treatment of hepatocarcinogenesis is important. Proteomic and genomic approaches aid to develop new promising biomarkers for the diagnosis of HCC at the early stages. These biomarkers not only help in prognosis but also provide better therapeutic intervention against HCC. Among the different biomarker candidates, liquid biopsy [including circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA)] has recently emerged as a noninvasive detection technique for the characterization of circulating cells, providing a strong basis and early diagnosis for the individualized treatment of patients. This review provides the current understanding of HCC biomarkers that predict the risk of HCC recurrence.

Potato (Solanum tuberosum L.) skin is composed of suberized phellem cells, the outer component of the tuber periderm. The focus of the proposed research was to apply genomic approaches to identify genes that control tuber skin appearance - smooth and shiny skin is highly preferred by the customers while russeted/netted skin potatoes are rejected. The breeding program (at Cornell University) seeks to develop smooth-skin varieties but has encountered frequent difficulties as inheritance of russeting involves complementary action by independently segregating genes, where a dominant allele at each locus is required for any degree of skin russeting. On the other hand, smooth-skin varieties frequently develop unsightly russeting in response to stress conditions, mainly high soil temperatures. Breeding programs in Israel aimed towards the improvement of heat tolerant varieties include skin quality as one of the desired characteristics. At the initiation of the present project it was unclear whether heat induced russeting and genetically inherited russeting share the same genes and biosynthesis pathways. Nevertheless, it has been suggested that russeting might result from increased periderm thickness, from strong cohesion between peridermal cells that prevents the outer layers from sloughing off, or from altered suberization processes in the skin. Hence, the original objectives were to conduct anatomical study of russet skin development, to isolate skin and russeting specific genes, to map the loci that determine the russet trait, and to compare with map locations the candidate russet specific genes, as well as to identify marker alleles that associated with russet loci. Anatomical studies suggested that russet may evolve from cracking at the outer layers of the skin, probably when skin development doesn’t meet the tuber expansion rate. Twodimensional gel electrophoresis and transcript profiling (cDNA chip, potato functional genomic project) indicated that in comparison to the parenchyma tissue, the skin is enriched with proteins/genes that are involved in the plant's responses to biotic and abiotic stresses and further expand the concept of the skin as a protective tissue containing an array of plantdefense components. The proteomes of skin from heat stressed tubers and native skin didn’t differ significantly, while transcript profiling indicated heat-related increase in three major functional groups: transcription factors, stress response and protein degradation. Exceptional was ACC synthase isogene with 4.6 fold increased level in the heat stressed skin. Russeting was mapped to two loci: rusB on chromosome 4 and rusC on chromosome 11; both required for russeting. No evidence was found for a third locus rusA that was previously proposed to be required for russeting. In an effort to find a link between the russeting character and the heat-induced russeting an attempt was made to map five genes that were found in the microarray experiment to be highly induced in the skin under heat stress in the segregating russet population. Only one gene was polymorphic; however it was localized to chromosome 2, so cannot correspond to rusB or rusC. Evaluation of AFLP markers tightly linked to rusB and rusC showed that these specific alleles are not associated with russeting in unrelated germplasm, and thus are not useful for MAS per se. To develop markers useful in applied breeding, it will be necessary to screen alleles of additional tightly linked loci, as well as to identify additional russet (heat-induced and/or native) related genes.

Plant cuticle development and metabolism are still poorly understood, partly due to the chemical complexity of the cuticular layer. The overall research objective was to broaden and deepen our understanding of tomato fruit cuticle development by analyzing novel germplasm with cuticular malformations and by studying the transcriptome and proteome of the fruit epidermal tissues, as strategies to overcome the challenges posed by the recalcitrance of the biological system. During the project we succeeded in identifying two genes with major impact on cuticle development. One of these encoded the first cutin synthase to be identified in plants, a metabolic step that had been a black box in cutin synthesis. In addition genes controlling the triterpenoid components of the cuticle were identified and, most interestingly, genetic variability for this component was identified among the wild tomato species germplasm. Additional germplasm was developed based on interspecific crosses that will allow for the future characterization of modifier genes that interact with the microfissuring gene (CWP) to promote or inhibit fruit cracking. One of the major accomplishments of the joint project was the integrated transcriptomic and proteomic analysis of the fruit cuticle and underlying tissues which allows for the identification of the pericarp cell layers responsible for the extracellular, cuticle-localized protein component. The results of the project have expanded our understanding of tomato fruit cuticle development and its genetic control. In addition, germplasm developed will be useful in developing tomato varieties resistant to cracking, on the one hand, and varieties useful for the dehydration industry on the other.