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1. Способ диагностики предрасположенности к посттравматическому гонартрозу, характеризующийся тем, что проводят генотипирование полиморфизма гена путем выделения геномной ДНК из периферической крови и проведения повторяющихся циклов полимеразной цепной реакции (ПЦР), каждый из которых включает денатурацию ДНК-матрицы, отжиг праймеров и элонгацию ДНК при заданных температурах с последующим разделением продуктов ПЦР (ампликонов) посредством электрофореза в агарозном геле, отличающийся тем, что при генотипировании полиморфизма гена определяют полиморфизм rs5743708, G2258A (Arg753Gln) гена TLR 2 (Toll-подобного рецептора) и в полиморфных локусах выявляют аллель A и генотипы АА и AG и при их наличии диагностируют высокую генетическую предрасположенность к развитию посттравматического гонартроза. 2. Способ по п. 1, отличающийся тем, что ПЦР цикл повторяют 35 раз. 3. Способ по п. 1 или 2, отличающийся тем, что денатурацию двухцепочечной ДНК-матрицы проводят в течение 70 с при температуре 93°С. 4. Способ по п. 1 или 2, отличающийся тем, что отжиг праймеров проводят в течение 10 с при температуре 64°С. 5. Способ по п. 1 или 2, отличающийся тем, что время элонгации ДНК составляет 80 с при 72°С. 6. Способ по п. 1, отличающийся тем, что для выявления полиморфных аллелей гена толл-подобного рецептора TLR2, G2258A проводят одновременно две реакции амплификации с двумя парами аллель-специфических праймеров, комплементарных нормальному и полиморфному аллелю.

1. Способ диагностики предрасположенности к посттравматическому гонартрозу, характеризующийся тем, что проводят генотипирование полиморфизма гена путем выделения геномной ДНК из периферической крови и проведения повторяющихся циклов полимеразной цепной реакции (ПЦР), каждый из которых включает денатурацию ДНК-матрицы, отжиг праймеров и элонгацию ДНК при заданных температурах с последующим разделением продуктов ПЦР (ампликонов) посредством электрофореза в агарозном геле, отличающийся тем, что при генотипировании полиморфизма гена определяют полиморфизм rs5743708, G2258A (Arg753Gln) гена TLR 2 (Toll-подобного рецептора) и в полиморфных локусах выявляют аллель A и генотипы АА и AG и при их наличии диагностируют высокую генетическую предрасположенность к развитию посттравматического гонартроза. 2. Способ по п. 1, отличающийся тем, что ПЦР цикл повторяют 35 раз. 3. Способ по п. 1 или 2, отличающийся тем, что денатурацию двухцепочечной ДНК-матрицы проводят в течение 70 с при температуре 93°С. 4. Способ по п. 1 или 2, отличающийся тем, что отжиг праймеров проводят в течение 10 с при температуре 64°С. 5. Способ по п. 1 или 2, отличающийся тем, что время элонгации ДНК составляет 80 с при 72°С. 6. Способ по п. 1, отличающийся тем, что для выявления полиморфных аллелей гена толл-подобного рецептора TLR2, G2258A проводят одновременно две реакции амплификации с двумя парами аллель-специфических праймеров, комплементарных нормальному и полиморфному аллелю.

This thesis examines the roles of two crucial gene expression regulators- collectively called “Mediator kinases”- in the normal and malignant intestines. It identifies new functions of these genes in maintaining intestinal homeostasis and establishes that these genes function as tumour suppressors in the pre-malignant stage of colorectal cancer.

Shikonin is 1,4-naphthoquinones produced exclusively in Boraginaceae species. The compound and its derivatives are predominantly made in roots where they function in mediating plant-plant (allelopathic) and plant-microbe interactions. Moreover, this compound has been a target for drug development due to its strong anti-cancer properties. Our genome assembly and analysis of Lithospermum erythrorhizon uncovered metabolic innovation events that contributed to the evolution of the shikonin biosynthesis. This metabolic innovation also reveals the evolutionary link between shikonin biosynthesis and ubiquinone biosynthesis, one of the central metabolism functions in aerobic cellular respiration. To explore additional links between these two pathways, we used a transcriptome-based network analysis which uncovered a shikonin gene network model that predicts strong associations between primary metabolic pathway genes and known shikonin biosynthesis genes, as well as links with uncharacterized genes. L. erythrorhizon geranyldiphosphate (GPP) synthase (LeGPPS) is one of the candidates predicted by the network analysis, of which encodes a cytoplasmic enzyme shown in vitro to produce GPP. Knocking down of LeGPPS in L. erythrorhizon hairy roots (LeGPPSi lines) results in reduced shikonin content. This result provides functional evidence that cytoplasmic LeGPPS supplies GPP precursor to the shikonin biosynthesis. LeGPPSi lines also increased ubiquinone content, further supporting our hypothesis on the metabolic and evolutionary connection between shikonin and ubiquinone biosynthesis. Further RNA-seq analysis of the LeGPPSi line showed that downregulating LeGPPS significantly reduces the expression of benzenoid/phenylpropanoid genes, indicating the presence of factors that coordinately regulate the pathways providing the 4-hydroxybenzoic acid and GPP precursors to the shikonin pathway. In addition to LeGPPS, we also found ubiquinone biosynthesis protein COQ4-like gene (LeCOQ4-L) which provided another evolutionary link between shikonin and ubiquinone biosynthesis. The enzymatic activity of canonical COQ4 is unknown. In yeast, the protein is essential for ubiquinone biosynthesis and its metabolon formation. With the existing connections between shikonin and ubiquinone biosynthesis, if LeCOQ4 functions in the same manner as yeast COQ4, it is possible that LeCOQ4-L has an analogous function in shikonin biosynthesis as a structural protein for stabilizing biosynthesis metabolon. This leads us to the characterization of COQ4 ortholog in Arabidopsis (AtCOQ4) to gain insight into its functional mechanism. Characterization of atcoq4 T-DNA mutant line showed that reduced AtCOQ4 expression resulted in reduced ubiquinone. Further subcellular localization study revealed that AtCOQ4 and LeCOQ4-L localize in mitochondria without conventional transit peptide. We also performed pull-down assay to identify AtCOQ4 interactors which might be the missing enzymes that cannot be identified based on homology. 80 potential AtCOQ4 interactors were found including proteins like AtCHLM, GRIM-19, and AtSSLs. However, further study is needed to verify the protein interactions captured by pull-down assay. Taken all together, our study sheds light on the metabolic innovations that give rise to shikonin biosynthesis from ubiquinone biosynthesis and provide insight into the dynamics of the metabolic networks.

“Perioperative genomics” is an emerging field of research in perioperative medicine. The body of work contained in this thesis describes the utilisation of analyses that explore the human genome to better understand the nature of the inflammatory and immune responses to surgical injury. It provides greater insight into how potentially harmful levels of inflammation affect recovery after surgery. Several analyses, and a review are presented that better define the nature and impact of different levels of inflammation on recovery after major surgery. The work has demonstrated the value of integrating genomic research into clinical trials in perioperative medicine.

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system. Vitamin D deficiency is an established risk factor but the mechanisms by which it contributes to MS pathogenesis and pathophysiology are unclear. MS disproportionately affects women more than men. Family planning is an important management consideration. However, how modern MS treatments interact with disease activity during and after pregnancy is not well understood. This thesis investigates these factors of vitamin D and pregnancy in the context of MS by transcriptomic analyses of immune cells and through retrospective analysis of a large dataset from the MSBase Registry, respectively.

The JAK2V617F mutation triggers constitutive activation of the JAK2 signalling pathway via STAT3 and STAT5 to drive expression of genes that govern disease phenotypes in myeloproliferative neoplasms (MPNs). The current JAK2 inhibitors (JAK2i) are effective in alleviating symptoms but do not reduce the JAK2 mutant allele burden and may lead to re-activation of compensatory resistance mechanisms. This thesis sought to identify novel targets by deciphering the transcriptional regulation of the central JAK-STAT signalling pathway in human erythroid and JAK2V617F-positive MPN cell models. The findings will aid in the identification of better biomarkers and targeted therapies for MPNs.

Microbiome studies are varied and involve the examination of microorganisms at different levels: individual cells to determine individual functions, populations of specific microorganisms to determine interactions between organisms, and/or communities of microorganisms for a broader investigation of interactions between organism and environment. These studies are typically done within the context of a particular niche or environment. There are two parts to this dissertation, separated by the types of research involved. First, the analysis of bacterial communities using 16S rRNA sequencing and analysis. In this first part the bacterial communities of the reproductive tract of bulls and the gastrointestinal tract of weanling pigs were studied. The reproductive organs of the male, domestic species had not been studied from an ecological perspective prior to the study. As such, the research was mainly focused on characterizing the bacterial communities found within the prepuce of bulls that were considered to be healthy, or that the breeding soundness exam was satisfactory and the bulls had no clinical disease in the urogenital tract. Through this study two distinct types of bacterial communities were found based on the diversity of the observed taxa; the groups were split into a low diversity group identified by the presence of Bradyrhizobium and a high diversity group distinguished by the abundance of mucosal-associated bacteria found in oral, respiratory, and vaginal communities of cattle. Second, the effects of supplementary, soluble fiber on the intestinal bacterial communities of piglets pre- and/or post-weaning were studied. The rationale behind this study was to determine if pre-weaning fiber could alter the microbiome prior to weaning and the change of diet from liquid to solid. Pre-weaning, supplementary, soluble fiber was found to increase short-chain fatty acid concentrations and bacterial taxa potentially involved in their production. Additionally, bacterial taxa implicated in an increased inflammatory response were reduced in groups fed supplementary fiber. Taken together, the two bacterial community studies highlight the gaps in knowledge for reproductive communities in male animals as well as the potential for reducing weaning stress in pigs. Part two of this dissertation focuses on whole genome sequence analysis as a way to study bacterial populations associated with bovine respiratory disease (BRD), a common and potentially fatal disease in cattle. Identification of BRD has low accuracy and the presence of antibiotic resistant bacteria increases the chance of treatment failure. Using machine learning, the prediction of antibiotic resistance in bacterial isolates from animals with BRD was performed to find potential sequences for use in future molecular assays. While using known resistance genes was helpful for some antibiotics, several of the antibiotics used in treating BRD were better predicted using the machine learning models. Model output sequences should be further tested using molecular methods to determine function and importance before using as an assay target. Put together, the contents of this dissertation should serve as an introduction to bacterial ecology as well as how the concepts can be applied to food animal production systems.

Slides presented at the 'Handling Pandemic Omics Data in the Nordics' event on 19th May 2022. Event organised by SciLifeLab Data Centre, ELIXIR Norway, ELIXIR Estonia, and ELIXIR Sweden. The talk focused on data brokering of SARS-Cov-2 data to European Nucleotide Archive. The challenges that were faced with brokering omics data throughout the COVID-19 pandemic, included sharing the metadata to different repositories. Getting rich metadata was one of the challenges faced.

The unpredictable Type IV drug-induced hypersensitivity reactions to the anti-seizure medication carbamazepine result in skin pathology ranging from a mild rash to life-threatening Stevens-Johnson syndrome. I undertook a multi-omics approach, that included genomics and transcriptomics, which resulted in the identification of novel biomarkers that differentiate disease severity and highlight the propensity of drug treated individuals to develop more severe drug reactions. This analysis also highlighted a role for non-conventional T cells in the more severe skin pathologies. Overall, these insights will inform future clinical management of drug-induced hypersensitivity reactions including more accurate rash classification, prognosis and direct novel treatment options.

NP-C is a neurodegenerative Inherited Metabolic Disease with a heterogeneous clinical presentation, and due to mutations in either the NPC1 or NPC2 genes.We have studied a patient with clinical diagnosis of NP-C but presenting inconclusive results regarding molecular analysis.To better characterize this patient, we have performed NGS-based technologies (targeted DNA sequencing and single cell-RNA sequencing).For the molecular diagnosis we used a NGS gene panel followed by the analysis of cDNA.Latter, we used massively parallel single cell RNA-seq (MARS-Seq) to address gene profiling changes and characterize the pathomechanisms related to specific disease-causing mutations. Using our targeted NGS panel we identified two novel mutations in NPC1 gene.Next, through cDNA analysis of one of the patient parents we were able to understand the impact of the silent mutation.This mutation leads to exon skipping giving origin to an out-of-frame transcript and eliciting the nonsense-mediated decay pathway.Thus, we were not able to easily detect the mutant transcript which turned the molecular diagnosis more challenging. Apparently the presence of the other mutation (a missense mutation) impairs the NPC protein folding leading to its ER retention.The MARS-Seq analysis of this patient showed that a number of upregulated genes are related to the unfold protein response (UPR) and ER stress, which deserves further studies. N/A

En la industria hortofrutícola las bajas temperaturas se utilizan para retrasar la maduración y el decaimiento de los frutos. A pesar de tener un uso extendido, los frutos de melocotón sometidos a almacenamiento prolongado en frío (CS) a menudo desarrollan una forma de daño por frío (CI) genéticamente controlada llamada harinosidad (mealiness/woolliness [WLT]): un desorden textural de la pulpa del fruto caracterizado por la perdida de suculencia Aunque durante el frío se han observado algunas alteraciones microscópicas, los síntomas visibles o macroscópicos de daño se desarrollan, no obstante, cuando los frutos se transfieren a temperatura ambiente (temperaturas de simulación de vida útil, SLR) para inducir la maduración después del almacenamiento. Con el objetivo de analizar los mecanismos moleculares subyacentes al desarrollo de la harinosidad durante el almacenamiento en frío (estadio pre-sintomático) y maduración en condiciones de simulación (estadio sintomático), diversos estudios transcriptómicos y proteómicos se han realizado. Estos estudios han resultado en la identificación de diferentes funciones celulares probablemente importantes para el desarrollo de los síntomas de la harinosidad, aunque no profundizan en el análisis y algunos resultados son aparentemente contradictorios (ya que en ellos se usan diferentes variedades de melocotón y protocolos de CS/SLR). Para proporcionar una visión más comprehensiva del desorden de la harinosidad en esta tesis se ha utilizado una aproximación genómica-genética que consiste en la combinación de grupos de líneas hermanas de la población de melocotón Pop-DG (con sensibilidad contrastante para desarrollar harinosidad) con una micromatriz de cDNA (Chillpeach microarray) dedicada al daño por frío para perfilar la expresión génica. Esta estrategia nos ha permitido identificar un gran número de genes asociados a la sensibilidad/tolerancia a desarrollar la harinosidad tanto a en estadios pre-sintomático (fruto maduro y CS) como sintomático, mientras que se compensan otras diferencias fenotípicas que segregan al azar en la población. Mediante la el análisis Fluidigm RT PCR, que se podría definir como qRT-PCR de media capacidad, hemos confirmado la validez de esos marcadores de expresión en los grupos de líneas hermanas, pero más importante, estos marcadores de expresión han resultado ser validos y reproducibles en lineas hermanas individuales de la población Pop-DG y en otros genotipos de melocotón (Oded y Hermoza) con diferentes grados de tolerancia al daño por frío. Además los perfiles de expresión de Od, Hz y los dos grupos de la población Pop-DG se han analizado también con la herramienta bioinformática ROSMETER, que proporciona información de la especificidad de la respuesta transcriptómica al estrés oxidativo. La anotación funcional de los genes diferencialmente expresados nos ha permitido proponer algunas hipótesis sobre los procesos moleculares que ocurren en el fruto de melocotón mientras está almacenado a 5ºC y cuando, posteriormente se deja madurar a temperatura ambiente, que extienden nuestra hipótesis de trabajo sobre el desorden de la harinosidad más allá de los cambios en la pared celular. Low temperatures are commonly used in the horticultural industry to delay peach ripening s and fruit decay. Despite widespread use, peach fruits subjected to prolonged cold storage (CS) periods often develop a genetically controlled form of chilling injury (CI) called mealiness/woolliness (WLT): a flesh textural disorder characterized by a lack of juiciness. Although some microscopic alterations had been observed during CS, visual symptoms (or macroscopic) of WLT appeared however upon transferring the fruits to the room temperature conditions (shelf life ripening [SLR] temperatures,) that are used for inducing ripening after CS. To dissect the molecular mechanisms underlying the WLT development during CS (pre-symptomatic stage) and SLR (symptomatic stage), several transcriptomic and proteomic studies have been reported. These studies have resulted in the identification of different cellular functions as important for the development of the WLT but they did not go deep in the analysis and some results were apparently contradictory (as they used different peach varieties and CS/SLR protocols). To give a more comprehensive view of WLT disorder we have used a genetical genomic approach consisting in the combination of pools of siblings of the Pop-DG population, with contrasting sensitivity to develop WLT, with a CI-dedicated Chillpeach microarray to profile gene expression. This strategy have enable us to identify a large number of genes associated to the sensitivity /tolerance to develop WLT both at pre-symptomatic (mature and CS) and symptomatic (SLR) stages, while compensating for other phenotypic differences randomly segregating in the population. By medium throughput qRT PCR analysis we confirmed the validity of these gene expression markers in the pools, but most important, there were proved to be reliable in individual siblings of the Pop-DG population and other peach genotypes (Oded and Hermoza) with different tolerance degree to chilling injury. Further the expression profiles of Oded, Hermoza and of the two pools from the Pop‐DG population were also analyzed with the bioinformatic tool ROSMETER that provides information on the specificity of the transcriptomic response to oxidative stress. Functional annotation of genes differentially expressed has enabled us to propose some hypotheses of the molecular processes occurring in the peach fruit while stored at 5ºC and later when let to ripen at room temperature that extends our working hypothesis about WLT disorder beyond the cell wall changes

Background Mesorhizobium metallidurans STM 2683T and Mesorhizobium sp. strain STM 4661 were isolated from nodules of the metallicolous legume Anthyllis vulneraria from distant mining spoils. They tolerate unusually high Zinc and Cadmium concentrations as compared to other mesorhizobia. This work aims to study the gene expression profiles associated with Zinc or Cadmium exposure and to identify genes involved in metal tolerance in these two metallicolous Mesorhizobium strains of interest for mine phytostabilization purposes. Results The draft genomes of the two Mezorhizobium strains were sequenced and used to map RNAseq data obtained after Zinc or Cadmium stresses. Comparative genomics and transcriptomics allowed the rapid discovery of metal-specific or/and strain-specific genes. Respectively 1.05% (72/6,844) and 0.97% (68/6,994) predicted Coding DNA Sequences (CDS) for STM 2683 and STM 4661 were significantly differentially expressed upon metal exposure. Among these, a significant number of CDS involved in transport (13/72 and 13/68 for STM 2683 and STM 4661, respectively) and sequestration (15/72 and 16/68 for STM 2683 and STM 4661, respectively) were identified. Thirteen CDS presented homologs in both strains and were differentially regulated by Zinc and/or Cadmium. For instance, several PIB-type ATPases and genes likely to participate in metal sequestration were identified. Among the conserved CDS that showed differential regulation in the two isolates, we also found znuABC homologs encoding for a high affinity ABC-type Zinc import system probably involved in Zinc homeostasis. Additionally, global analyses suggested that both metals also repressed significantly the translational machinery. Conclusions The comparative RNAseq-based approach revealed a relatively low number of genes significantly regulated in the two Mesorhizobium strains. Very few of them were involved in the non-specific metal response, indicating that the approach was well suited for identifying genes that specifically respond to Zinc and Cadmium. Among significantly up-regulated genes, several encode metal efflux and sequestration systems which can be considered as the most widely represented mechanisms of rhizobial metal tolerance. Downstream functional studies will increase successful phytostabilization strategies by selecting appropriate metallicolous rhizobial partners.