Dissecting out the mechanisms of hypoxia-dependent responses in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease with a significant impact on patients’ quality of life. One of the well-described features in RA is hypoxia. It has been shown that both RA synovial fluid and synovial tissue are characterised by insufficient amount of oxygen. Abnormality in hypoxia-inducible factor (HIF) expression is a marker of decreased oxygen tension in RA and in other pathological conditions. HIFs, together with the proteins which regulate their stabilisation and transactivation, namely prolyl hydroxylase domain (PHD) enzymes and factor inhibiting HIF-1 (FIH-1), were the main focus of this thesis. The first aim of the study was to investigate the specific roles of HIF isoforms in RA fibroblast-like synoviocytes (FLS), which are the key cells in RA pathogenesis. Expression of HIF-1α, HIF-2α and a panel of genes involved in angiogenesis (ANGPTL-4, ephrin-A3, VEGF), glycolysis (GLUT-1, ENO-1), pH regulation (CA9) and apoptosis (BNIP-3) were significantly increased by hypoxia. Successful silencing of both HIFs using short interfering RNA (siRNA) was achieved in RA FLS, and was followed by examination of the HIF-dependence of the candidate genes. The majority of these genes were found to be HIF-1α- dependent, whereas only ANGPTL-4 and VEGF were regulated by both HIF-1α and HIF-2α. Subsequently, the mRNA and protein expression of PHDs and FIH-1 in RA FLS, their response to hypoxic conditions and their HIF-dependence were examined. Specific knockdown of the hydroxylases was performed and the effects on HIFs and genes of interest were assessed. The most significant changes were noticed after silencing PHD-2, which led to HIF-α stabilisation and upregulation of HIF-dependent genes. In addition, PHD-2 depletion increased a number of pro-angiogenic genes and also contributed to new tubule formation in a functional angiogenesis assay. Comparison of RA, osteoarthritis (OA) FLS and normal human dermal fibroblasts (NHDF) revealed that PHD-2 has similar functions in promoting neovascularisation in arthritic cells (RA and OA), but not in non-arthritic cells (NHDF). Finally, the last chapter describes preliminary data on the expression of the least investigated HIF-3α subunit and its negative splice variant, HIF-3α4, in RA FLS. This research contributes to a better understanding of the upstream and downstream regulation of HIF signalling in RA, showing that PHD-2 has a key role in regulating hypoxic responses via HIFs in RA FLS. Because this pathway controls the expression of so many genes relevant to the disease, it may be an important target for RA therapy. One of the most critical challenges would be to target the correct HIF/PHDs molecule in the right tissue/cell, ensuring that it will not affect angiogenesis in healthy tissues

Exploring the biological basis of residual feed intake in beef cattle using multi-Omics analysis.

Efficient feed utilization is critical for both economic sustainability and environmental responsibility in modern livestock production. While there has been extensive research, the multifaceted nature of feed efficiency remains complex, with many biological factors still unexplored. This dissertation examines the physiological foundations of feed efficiency by elucidating some of the complex biological mechanisms associated with residual feed intake (RFI) phenotype in beef cattle, using a range of Omics approaches. We hypothesized that metabolites related to amino acids, carbohydrates, and fatty acids could act as potential biomarkers for RFI. Through a chemical group-based metabolomics method, we identified enriched pathways in feed-efficient steers, notably in arginine biosynthesis and histidine metabolism. This led to the identification of five potential metabolite biomarkers mainly linked to amino acid metabolism, emphasizing a relationship between blood amino acid profiles and RFI. This led us to investigate the expression of genes and associated pathways related to nutrient and energy metabolism, especially in liver tissue, where hepatic metabolism is driven by transcriptional regulation. Low-RFI steers showed upregulation of genes involved in fatty acid transport, β-oxidation, and mitochondrial ATP production. In contrast, a crucial gene in amino acid metabolism responsible for aminoadipate aminotransferase activity exhibited a significant decrease in expression in low-RFI steers. These results indicate that alteration in expression of hepatic genes regulating lipid and amino acid metabolism, and mitochondrial ATP generation is associated with RFI phenotype. We also investigated potential differences in the rumen microbiome and immune gene expression of beef steers with low or high RFI. We observed increased mRNA expression of immune-related genes in both blood and liver tissues of low-RFI beef steers, especially those linked to pathogen detection and phagocytosis. Low-RFI steers also displayed variation in the relative abundance of microbial taxa compared to high-RFI. Lastly, detailed statistical analysis indicated that plasma amino acids such as tyrosine, glycine, and dimethyl sulfone may be promising economic prospects as cost-efficient predictors of RFI in beef cattle. In conclusion, this dissertation provides invaluable insights into some of the intricate biological processes associated with RFI in crossbred beef cattle, enhancing our grasp of the involved biological mechanisms and laying the groundwork for refining feed utilization in the beef cattle sector of livestock production

Molecular and computational approach to the link between nutrition and cancer

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Química Física Aplicada. Fecha de lectura: 22-11-201

Prenatal Choline Supplementation During Maternal Obesity Alters Offspring Response to Western Diets

Maternal obesity has led to an increase in adverse offspring developmental outcomes and a greater risk for long-term metabolic diseases. Choline, a semi-essential nutrient, can be incorporated into phosphatidylcholine (PC) as well as sphingomyelin (SM) and donate its labile methyl group for the remethylation of homocysteine after choline is oxidized to betaine. Prenatal choline insufficiency has been related to maternal obesity and metabolic diseases, such as metabolic associated fatty liver disease (MAFLD). Choline may interact with maternal obesity to influence the programming offspring. Chapter 1 presents an introduction of choline and the various clinical outcomes associated with choline supplementation during pregnancy. Chapter 2 presents findings of altered whole-body glucose tolerance in choline supplemented (CS) male offspring prenatally challenged by a maternal HF diet (HF+CS). Here we demonstrate benefits of choline on blood glucose control and metabolic regulation of the visceral adipose tissue. Chapter 3 presents an investigation into the global and site-specific methylation in fetal and postnatal liver and brain in response to maternal HF and CS. Here we demonstrate an increase in DNA methylation and decrease in gene expression of Srebp1, a gene that mediates de novo lipogenesis, by maternal CS in the HF condition during the fetal period. However, this epigenetic change was not maintained in adulthood. Global DNA methylation of liver and adipose demonstrates increased methylation during the prenatal period yet decreased methylation during postnatal HF feeding of maternal CS offspring, suggesting that methylation marks established by maternal CS cannot be maintained during postnatal HF feeding. Chapter 4 explores the lipidomic changes in the fetal and postnatal period in response to maternal HF and CS. We found increases in ether-linked phospholipids (plasmalogens) which serve as antioxidants for cellular membrane lipids in HFCS male offspring, suggesting a potential role of maternal CS in redox regulation and membrane integrity. Chapter 5 explores the metabolic outcomes of mouse offspring exposed to prenatal HFCS and 16-weeks of postnatal western diet (WD) challenge. Results suggest that prenatal HFCS has mixed effects on offspring phenotypes and MAFLD biomarkers, and the effects were sexually dimorphic. Further, systems approaches were used to assess the transcriptomic and lipidomic changes related to maternal HFCS after the 16-week WD challenge in offspring. Chapter 6 presents the rationale for a systems approach in interpreting the findings presented, discusses limitations in the study and future directions for research in preventing MASH (steatohepatitis) pathogenesis

Impaired Mitochondrial Bioenergetics under Pathological Conditions

Mitochondria are the powerhouses of cells; however, mitochondrial dysfunction causes energy depletion and cell death in a variety of diseases. Altered oxidative phosphorylation and ion homeostasis are associated with ROS production resulting from the disassembly of respiratory supercomplexes and the disruption of electron transfer chains. In pathological conditions, the dysregulation of mitochondrial homeostasis promotes Ca2+ overload in the matrix and ROS accumulation, which induces the mitochondrial permeability transition pore formation responsible for mitochondrial morphological changes linked to membrane dynamics, and ultimately, cell death. Finally, studies on the impaired mitochondrial bioenergetics in pathology could provide molecular tools to counteract diseases associated with mitochondrial dysfunction