This thesis exemplifies the application of metabolomics as a tool for the scientific exploration of molecules aimed at unravelling molecular mechanisms involved in cellular processes shaping... Show moreThis thesis exemplifies the application of metabolomics as a tool for the scientific exploration of molecules aimed at unravelling molecular mechanisms involved in cellular processes shaping biological phenotypes. Such approaches are already enabling us to identify molecular targets tailored to clinical applications serving as diagnostic markers as well as the identification of novel patho-physiologies and disease relevant mechanisms. The thesis is divided into five research chapters (chapters 2-6) in which different analytical technologies were used to explore metabolic alterations, i.e., liquid chromatography mass spectrometry (LC-MS), differential mobility spectroscopy (DMS) and nuclear magnetic resonance spectroscopy (NMR). To explore the applicability of metabolomics analysis as an important tool in patho-physiological research, I applied these different technologies in diverse experimental frameworks, as for example different in vitro and in vivo models, intervention studies, and the combination of multi-omics data. Show less
The investigation into the bidirectional communication between the gut and its host is a rapidly growing area of research, attracting considerable attention for its potential to provide crucial... Show moreThe investigation into the bidirectional communication between the gut and its host is a rapidly growing area of research, attracting considerable attention for its potential to provide crucial insights into health and disease. Recognizing the metabolic flexibility of the gut microbiota and its ability to respond to environmental shifts, this thesis goes beyond underestimated metabolites such as SCFAs. The hypothesis underlying this research is that the composition of the gut microbiota intertwines with broader metabolic processes in the host. Therefore, this thesis, aims to explore the alterations observed in metabolites linked to immune signalling pathways in both fecal and blood specimens. The primary objective is to unravel the correlation between these alterations and variations in the composition of gut microbiota, particularly within the context of cognitive impairment. In addition, since metabolomics methods are often optimized for blood analysis rather than fecal samples, developing of a sample preparation technique for comprehensively extracting metabolites from the complex fecal matrix, as well as analysing global metabolites, comprise significant focus of this thesis. Show less
Unlike the commonly held view, saliva is not a homogenous biofluid, but rather a complex mix of serous and mucus type secretions. These individual secretions present with characteristic molecular... Show moreUnlike the commonly held view, saliva is not a homogenous biofluid, but rather a complex mix of serous and mucus type secretions. These individual secretions present with characteristic molecular compositions, functions, and rhythm. The thesis displays empirical proof of the biochemical diversity of subtypes of saliva at both spatial and temporal dimensions. Furthermore, the implications and prospects of such distinctive biochemistry are discussed. Show less
Prognostic information is needed to balance benefits and risks of cancer treatment in older patients. Metabolomics-based scores were previously developed to predict 5- and 10-year mortality ... Show morePrognostic information is needed to balance benefits and risks of cancer treatment in older patients. Metabolomics-based scores were previously developed to predict 5- and 10-year mortality (MetaboHealth) and biological age (MetaboAge). This study aims to investigate the association of MetaboHealth and MetaboAge with 1-year mortality in older patients with solid tumors, and to study their predictive value for mortality in addition to established clinical predictors. This prospective cohort study included patients aged ≥ 70 years with a solid malignant tumor, who underwent blood sampling and a geriatric assessment before treatment initiation. The outcome was all-cause 1-year mortality. Of the 192 patients, the median age was 77 years. With each SD increase of MetaboHealth, patients had a 2.32 times increased risk of mortality (HR 2.32, 95% CI 1.59–3.39). With each year increase in MetaboAge, there was a 4% increased risk of mortality (HR 1.04, 1.01–1.07). MetaboHealth and MetaboAge showed an AUC of 0.66 (0.56–0.75) and 0.60 (0.51–0.68) for mortality prediction accuracy, respectively. The AUC of a predictive model containing age, primary tumor site, distant metastasis, comorbidity, and malnutrition was 0.76 (0.68–0.83). Addition of MetaboHealth increased AUC to 0.80 (0.74–0.87) (p = 0.09) and AUC did not change with MetaboAge (0.76 (0.69–0.83) (p = 0.89)). Higher MetaboHealth and MetaboAge scores were associated with 1-year mortality. The addition of MetaboHealth to established clinical predictors only marginally improved mortality prediction in this cohort with various types of tumors. MetaboHealth may potentially improve identification of older patients vulnerable for adverse events, but numbers were too small for definitive conclusions. The TENT study is retrospectively registered at the Netherlands Trial Register (NTR), trial number NL8107. Date of registration: 22–10-2019. Show less
The circadian system has evolved to benefit the fitness of the organism. A properly functioning clock improves overall performance and promotes health. By gaining more knowledge about how the... Show moreThe circadian system has evolved to benefit the fitness of the organism. A properly functioning clock improves overall performance and promotes health. By gaining more knowledge about how the system works and responds to changes, therapies can be developed to promote the functioning of the circadian system. In this thesis, the response of the circadian system to changes in daylength (e.g. long summer days and short winter days was investigated. In addition the functioning of the circadian system with aging was investigated. This is relevant since aging is known to be accompanied by a weakening of the circadian system in humans, which has been associated with deterioration of a number of age-related conditions such as arteriosclerosis, type 2 diabetes and neurodegenerative diseases such as Parkinson's and Alzheimer's disease. In addition, a large proportion of the elderly will experience fragmentation of sleep, meaning that people have difficulty sleeping at night, while during the day they are very sleepy. Promoting the circadian rhythm with relatively simple interventions, such as correctly timed exposure to (day) light, physical activity and food intake can support the circadian system and promote general health. Show less
Unsaturated fatty acids (UFA) are crucial for T-cell effector functions, as they can affect the growth, differentiation, survival, and function of T cells. Nonetheless, the mechanisms by which UFA... Show moreUnsaturated fatty acids (UFA) are crucial for T-cell effector functions, as they can affect the growth, differentiation, survival, and function of T cells. Nonetheless, the mechanisms by which UFA affects T-cell behavior are ill-defined. Therefore, we analyzed the processing of oleic acid, a prominent UFA abundantly present in blood, adipocytes, and the fat pads surrounding lymph nodes, in CD4+ T cells. We found that exogenous oleic acid increases proliferation and enhances the calcium flux response upon CD3/CD28 activation. By using a variety of techniques, we found that the incorporation of oleic acid into membrane lipids, rather than regulation of cellular metabolism or TCR expression, is essential for its effects on CD4+ T cells. These results provide novel insights into the mechanism through which exogenous oleic acid enhances CD4+ T-cell function. Show less
The ideas underlying this thesis are that lipidomics may improve the diagnosis of liver metabolic diseases, and can provide further insights into the underlying pathophysiology of these diseases.... Show moreThe ideas underlying this thesis are that lipidomics may improve the diagnosis of liver metabolic diseases, and can provide further insights into the underlying pathophysiology of these diseases. However to achieve this, the measurement of lipids should be further improved by enabling comprehensive coverage, accurate identification and quantitation. Therefore, in this thesis, new analytical methodologies using LC-MS for the accurate detection and quantitation of lipids are developed. These methods are then applied to MCADD samples to identify lipid biomarkers, which are further correlated with gene expression analysis to understand the underlying biochemical pathways. Show less
Sluiskes, M.; Goeman, J.; Beekman, M.; Slagboom, E.; Akker, E. van den; Putter, H.; Rodríguez-Girondo, M. 2024
Aging is a multifaceted and intricate physiological process characterized by a gradual decline in functional capacity, leading to increased susceptibility to diseases and mortality. While... Show moreAging is a multifaceted and intricate physiological process characterized by a gradual decline in functional capacity, leading to increased susceptibility to diseases and mortality. While chronological age serves as a strong risk factor for age-related health conditions, considerable heterogeneity exists in the aging trajectories of individuals, suggesting that biological age may provide a more nuanced understanding of the aging process. However, the concept of biological age lacks a clear operationalization, leading to the development of various biological age predictors without a solid statistical foundation. This paper addresses these limitations by proposing a comprehensive operationalization of biological age, introducing the “AccelerAge” framework for predicting biological age, and introducing previously underutilized evaluation measures for assessing the performance of biological age predictors. The AccelerAge framework, based on Accelerated Failure Time (AFT) models, directly models the effect of candidate predictors of aging on an individual’s survival time, aligning with the prevalent metaphor of aging as a clock. We compare predictors based on the AccelerAge framework to a predictor based on the GrimAge predictor, which is considered one of the best-performing biological age predictors, using simulated data as well as data from the UK Biobank and the Leiden Longevity Study. Our approach seeks to establish a robust statistical foundation for biological age clocks, enabling a more accurate and interpretable assessment of an individual’s aging status. Show less
The outbreaks of AIDS and COVID-19 showed clearly how infectious viruses can influence people’s lives. Investigating the changes in the host metabolism may provide a paradigm shift to consider... Show moreThe outbreaks of AIDS and COVID-19 showed clearly how infectious viruses can influence people’s lives. Investigating the changes in the host metabolism may provide a paradigm shift to consider immune-metabolic interactions as therapeutic targets. The aim of this thesis is to examine the interplay between the immune system and metabolism during viral infections, such as HIV and coronavirus. These investigations will utilize metabolomic and lipidomic mass spectrometry techniques to gain a comprehensive understanding of the metabolic changes that occur during viral infections. To enhance the coverage of the lipidome, a new method will be developed. Show less
With ageing populations, the prevalence of age-related disorders such as dementia is on the rise. As there is currently no curable treatment for dementia, the vascular component of dementia is... Show moreWith ageing populations, the prevalence of age-related disorders such as dementia is on the rise. As there is currently no curable treatment for dementia, the vascular component of dementia is increasingly recognised as a key modifiable cause. This thesis aims to investigate biological pathways between risk factors of cardiometabolic disease and cognitive function, in a population of older adults at increased risk of cardiovascular disease (CVD). We hypothesise that changes in physiological functioning caused by (sub)clinical CVD are possible mediators within the pathway leading to cognitive dysfunction. In the first part of this thesis, we studied electrocardiogram-based intervals and serum cardiac biomarkers (such as troponin) in relation to cognitive function. In the second part of this thesis, we studied the interplay of body mass index and serum leptin, loss of body weight and body weight variability, as well as metabolomics-based health scores in relation to cognitive function. We found that various cardiometabolic risk factors are associated with worse cognitive function. The results of this thesis strongly suggest that subclinical changes in cardiometabolic health may exist before cognitive dysfunction becomes apparent. Treating these cardiometabolic risk factors may be of benefit to future cognitive health. Show less
Per- and polyfluoroalkyl substances (PFAS) are widely used and persistent chemicals, leading to ubiquitous exposure. Although high PFAS levels have been associated with an adverse cardiovascular... Show morePer- and polyfluoroalkyl substances (PFAS) are widely used and persistent chemicals, leading to ubiquitous exposure. Although high PFAS levels have been associated with an adverse cardiovascular risk profile, the distribution of levels and relations with cardio-metabolic risk markers in the general population have not been fully characterized. We assessed the association between blood levels of perfluorooctaneic acid (PFOA), perfluorooctane sulfonic acid (PFOS), and perfluorohexanesulfonic acid (PFHxS) and a range of lipoproteins and metabolites as well as clinical lipid measurements. We used data from participants of the Netherlands Epidemiology of Obesity study (NEO) (n = 584) and the Rhineland Study (n = 1962), jointly spanning an age range of 30 to 89 years. PFAS were measured with the Metabolon HD4 platform, and lipoprotein and metabolite profiles were measured using Nightingale's nuclear magnetic resonance-spectroscopy platform, and mainly comprised lipoprotein markers. Using linear regression analyses, we quantified age-, sex-, and education-adjusted associations of PFOA, PFOS, and PFHxS with clinical lipid measurements and 224 lipoproteins and metabolites. Higher levels of PFAS, particularly PFOS and PFHxS, were associated with higher concentrations of total lipid, cholesterol and phospholipid content in most HDL, IDL, LDL, and VLDL subclasses. The effect sizes were age-dependent for the majority of the associations, with the deleterious effects of PFAS being generally stronger in people below compared to those above median age. Our observation that in the general population even low PFAS concentrations are associated with an unfavorable lipid profile, calls for further critical regulation of PFAS substances. Show less
The progressive loss and degeneration of dopaminergic neurons is a major pathological hallmark of Parkinson's disease (PD). The onset and progression of PD can be triggered by multiple risk factors... Show moreThe progressive loss and degeneration of dopaminergic neurons is a major pathological hallmark of Parkinson's disease (PD). The onset and progression of PD can be triggered by multiple risk factors, for instance, genetic mutation, environmental exposure, and aging. Each factor may cause common or unique metabolic disturbances, ultimately converging into a complex metabolic disorder with diverse clinical phenotypes. Through metabolome analysis, the full picture of the metabolic landscape depicting a biological system can be revealed. Metabolites function as key elements and direct read-outs of a system’s functional status. Alterations in metabolite concentrations are informative for inferring and understanding underlying metabolic activity. In addition, stable isotope labeling techniques coupled with metabolomics can bring us an extra-dynamic vision of the metabolic landscape. Changes in labeling patterns of metabolites help identify alterations with metabolic fluxes through pathways. This thesis aimed to develop a comprehensive analytical strategy for characterizing the metabolic activity related to PD neurodegeneration, especially focused on the improvement in metabolome coverage and data quality, facilitating the use of stable isotope labeling in in-depth metabolism investigations, and developing a computational workflow for metabolic flux analysis. These methods were applied to investigate metabolic dysregulation of dopaminergic neurons due to genetic and environmental factors. Show less
De uitkomsten beschreven in dit proefschrift dragen bij aan de bestaande overtuiging dat een verfijndere classificatie voor depressie, op basis van symptoomprofielen en hun mogelijke biologische... Show moreDe uitkomsten beschreven in dit proefschrift dragen bij aan de bestaande overtuiging dat een verfijndere classificatie voor depressie, op basis van symptoomprofielen en hun mogelijke biologische onderbouwing, overwogen dient te worden. Inmiddels wordt adipositas in de dagelijkse praktijk op meer dan alleen het BMI beoordeeld, namelijk ook de tailleomtrek en het lipidenprofiel. Echter, dergelijke aandacht bestaat nog niet voor de heterogeniteit van depressie. Een grotere bewustwording van de verschillende manifestaties van depressie-symptomatologie, die het gevolg kunnen zijn van uiteenlopende pathofysiologische mechanismen, is van essentieel belang. Wanneer een patiënt met depressie een atypisch energie-gerelateerd symptoomprofiel heeft, kan het nuttig zijn om diens metabole biomarkers te controleren om mogelijke ontwikkeling van cardiometabole ziekten te voorkomen. In de klinische praktijk moeten wij ons bij de behandeling van patiënten met depressie ook meer bewust worden van de correlatie tussen symptoomprofielen van depressie en afzonderlijke biologische en klinische manifestaties. Het is cruciaal om goed te kijken naar de symptomen die bij elke patiënt tot uiting komen. De resultaten van dit proefschrift tonen aan dat patiënten met een depressie die atypische energie-gerelateerde depressieve symptomen vertonen, genetisch en klinisch kwetsbaar zijn voor aan insulineresistentie gerelateerde ziekten (namelijk adipositas, metabole ontregelingen en diabetes mellitus type 2). Een gepersonaliseerde aanpak kan behulpzaam zijn in preventie van deze chronische en complexe ziekten. Hierbij dient er rekening gehouden worden met de heterogeniteit van depressie en de associatie tussen atypische energie-gerelateerde symptomen van depressie en deze ziekten. Show less
BackgroundB vitamins such as folate (B9), B6, and B12 are key in one carbon metabolism, which generates methyl donors for DNA methylation. Several studies have linked differential methylation to... Show moreBackgroundB vitamins such as folate (B9), B6, and B12 are key in one carbon metabolism, which generates methyl donors for DNA methylation. Several studies have linked differential methylation to self-reported intakes of folate and B12, but these estimates can be imprecise, while metabolomic biomarkers can offer an objective assessment of dietary intakes. We explored blood metabolomic biomarkers of folate and vitamins B6 and B12, to carry out epigenome-wide analyses across up to three European cohorts. Associations between self-reported habitual daily B vitamin intakes and 756 metabolites (Metabolon Inc.) were assessed in serum samples from 1064 UK participants from the TwinsUK cohort. The identified B vitamin metabolomic biomarkers were then used in epigenome-wide association tests with fasting blood DNA methylation levels at 430,768 sites from the Infinium HumanMethylation450 BeadChip in blood samples from 2182 European participants from the TwinsUK and KORA cohorts. Candidate signals were explored for metabolite associations with gene expression levels in a subset of the TwinsUK sample (n = 297). Metabolomic biomarker epigenetic associations were also compared with epigenetic associations of self-reported habitual B vitamin intakes in samples from 2294 European participants.ResultsEighteen metabolites were associated with B vitamin intakes after correction for multiple testing (Bonferroni-adj. p < 0.05), of which 7 metabolites were available in both cohorts and tested for epigenome-wide association. Three metabolites — pipecolate (metabolomic biomarker of B6 and folate intakes), pyridoxate (marker of B6 and folate) and docosahexaenoate (DHA, marker of B6) — were associated with 10, 3 and 1 differentially methylated positions (DMPs), respectively. The strongest association was observed between DHA and DMP cg03440556 in the SCD gene (effect = 0.093 ± 0.016, p = 4.07E−09). Pyridoxate, a catabolic product of vitamin B6, was inversely associated with CpG methylation near the SLC1A5 gene promoter region (cg02711608 and cg22304262) and with SLC7A11 (cg06690548), but not with corresponding changes in gene expression levels. The self-reported intake of folate and vitamin B6 had consistent but non-significant associations with the epigenetic signals.ConclusionMetabolomic biomarkers are a valuable approach to investigate the effects of dietary B vitamin intake on the human epigenome. Show less
Viral hemorrhagic fever (VHF) is a group of acute diseases caused by highly infectious viruses including Ebola, Lassa, Dengue viruses. Its high mortality rate poses high risk to public health,... Show moreViral hemorrhagic fever (VHF) is a group of acute diseases caused by highly infectious viruses including Ebola, Lassa, Dengue viruses. Its high mortality rate poses high risk to public health, however, studies on VHF have been hampered due to the non-availability of proper models and incomplete knowledge on its mechanism. In order to fill this gap, this thesis presented new bioanalytical, lab-on-chip and single-cell assays to investigate changes in vascular biology and macrophage immunometabolism induced by VHF viruses. Firstly, an organ chip was developed to mimic the hemorrhagic shock syndrome caused by VHF viruses in vitro and test experimental drug candidates. In addition, acoustic force spectroscopy was applied to investigate the effect of Dengue on the cellular viscoelastic properties of endothelial cells at single-cell level. Then, metabolic profiling of endothelial cells and macrophages upon Ebola viral protein exposure was performed on bulk-level. Finally, the immunometabolism of human macrophages upon polarization was investigated by live single-cell metabolomics, setting the stage for future host-pathogen studies at single-cell level. Overall, this thesis will facilitate the understanding of VHF viruses and the development of treatment strategies. More importantly, the technologies developed here expectedly open up opportunities to combat the viruses that threaten global society. Show less
Paulino Fernandez, H.; Choi, Y.H.; Vrieling, K.; Bresser, M. de; Sewalt, B.A.; Tonolo, F. 2023
Cannabis sativa L. is a plant with a wide range of potential medicinal applications. In recent years, polyploidy has gained attention as a potential strategy for rapidly improving C. sativa, which,... Show moreCannabis sativa L. is a plant with a wide range of potential medicinal applications. In recent years, polyploidy has gained attention as a potential strategy for rapidly improving C. sativa, which, unlike other modern crops, has not yet benefitted from this established biotechnological application. Currently, no reports on high THCA and CBDA drug-type polyploid cultivars have been published. Moreover, it still needs to be clarified if different cultivars react similarly to polyploidization. For these reasons, we set out to evaluate and compare the phenotype and chemotype of three high Δ9-tetrahydrocannabinolic acid (THCA) and one high cannabidiolic acid (CBDA) drug-type cultivars in their diploid, triploid and tetraploid state through agronomic and metabolomic approaches. Our observations on plant morphology revealed a significant increase in plant height and leaf size with increasing ploidy levels in a cultivar-dependent manner. In contrast, cannabinoids were negatively affected by polyploidization, with the concentration of total cannabinoids, THCA, CBDA and cannabigerolic acid (CBGA) decreasing significantly in higher ploidy levels across all four cultivars. Headspace analysis of volatiles revealed that total volatile content decreased in triploids. On the other hand, tetraploids reacted differently depending on the cultivars. Two THCA dominant cultivars showed an increase in concentrations, while in the other two cultivars, concentrations decreased. Additionally, several rare compounds not present in diploids appeared in higher ploidy levels. Moreover, in one high THCA cultivar, a couple of elite tetraploid genotypes for cannabinoid and volatile production were identified, highlighting the role of cultivar and genotypic variability as an important factor in Cannabis sativa L. polyploids. Overall, our observations on plant morphology align with the giga phenotype observed in polyploids of other plant species. The decrease in cannabinoids and volatiles production in triploids have relevant implications regarding their commercial use. On the other hand, this study found that tetraploidization is a suitable approach to improve Cannabis sativa L. medicinal potential, although the response is cultivar and genotype-dependent. This work lays the ground for further improving, evaluating and harnessing Cannabis sativa L. chemical diversity by the breeding, biotechnological and pharmaceutical sectors. Show less
Metabolomics is a powerful tool that can provide a comprehensive insight into the complexity of human biology and the pathophysiology of diseases. By analyzing the metabolome, which refers to the... Show moreMetabolomics is a powerful tool that can provide a comprehensive insight into the complexity of human biology and the pathophysiology of diseases. By analyzing the metabolome, which refers to the complete set of small (endogenous) molecules (with a mass <1500 Da) present within a given biological system, a direct functional read-out of an organism's physiological status can be obtained. In particular, metabolomics has emerged as a promising approach for neuroscience research, as it helps to shed light on the molecular mechanisms underlying neurodegenerative and neuropsychiatric diseases. One of the main challenges in (brain) metabolomics studies is the reliable and sensitive analysis of volume-limited biological samples. Therefore, in this thesis, innovative microscale analytical workflows based on capillary electrophoresis (CE) coupled to mass spectrometry (MS) are developed in order to generate metabolic profiles in volume-restricted samples. The ultimate aim of this thesis is to showcase that the technical reproducibility, detection sensitivity, and metabolic coverage of CE-MS are sufficient to allow use for different material-limited matrices of interest for neuroscience research. Show less
Lipid signaling is an essential biological event/process in a plethora of pathophysiological conditions. The underlying idea of this thesis is that many of the roles and the complex interplay of... Show moreLipid signaling is an essential biological event/process in a plethora of pathophysiological conditions. The underlying idea of this thesis is that many of the roles and the complex interplay of the individual signaling lipids in inflammatory processes and related conditions in health and disease is not well known, and therefore has to be studied integrally as a complex network. In order to study this complex interplay, an improved broad analytical method is necessary to analyze a wide range of different signaling lipid classes such as oxylipins, (nitro) free fatty acids, endocannabinoids, bile acids and different subclasses of lysophospholipids. Therefore, the aim of this thesis is to develop a better method to study signaling lipids, and to apply it to study the role of these molecules in several relevant biological questions for a better understanding of inflammation related pathophysiology including autoimmune diseases, neurodegeneration and regulatory effect of exercise training. Show less
