| 1. |
- Grüttner, Jana, 1992-
(författare)
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The interplay between Giardia intestinalis and host intestinal epithelial cells : A tale of immune activation and suppression
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Giardia intestinalis is a non-invasive, protozoan parasite causing the diarrheal disease giardiasis in a variety of mammals, including humans. The parasite has two main life cycle stages; disease-causing trophozoites and infectious cysts. Trophozoites colonize the host’s upper small intestine by attaching to intestinal epithelial cells (IECs). G. intestinalis infections can cause a broad spectrum of clinical outcomes, ranging from acute and chronic infections to asymptomatic carriage. In most cases, the infection causes only a low degree of mucosal inflammation. The molecular basis of how the parasite causes such diverse clinical outcomes is poorly defined. Here, we used in vitro infection models to investigate host-parasite interactions between G. intestinalis and IECs. In paper I, we assessed transcriptional changes of Caco-2 cells during infections with different life cycle stages of the parasite by RNA-sequencing (RNA-seq). Infections with trophozoites, cysts or encysting cells showed that each life cycle stage of the parasite induced the expression of both a common set of host genes and unique, life cycle stage specific responses. The IECs showed large expression changes in immune signalling, transcriptional regulation, apoptosis, metabolism and oxidative stress. We could also observe that the parasites’ contact with IECs inhibited its encystation processes. In paper II, we mapped global protein changes of Caco-2 cells during G. intestinalis trophozoite infections using quantitative proteomics. We could confirm expression changes in oxidative stress, immune response, signalling, metabolism and apoptosis on protein level. We further investigated the interplay between IECs and G. intestinalis using small intestinal organoids, to more accurately mimic intact primary host tissue. The infection dynamics of trophozoites on intestinal organoid-derived monolayers were assessed using live-cell microscopy (paper III). We investigated swimming behaviour of trophozoites on the intestinal epithelial surface. Trophozoites swan in repetitive circling and intermittent attachment patterns during preattachment swimming, possibly to explore the epithelium surface and to find a suitable permanent attachment site. We further assessed early immune responses of IECs during trophozoite infections using intestinal organoid-derived monolayers (paper IV). Transcriptional expression of immune response genes was dependent on the trophozoite inoculate. Trophozoites that were preconditioned to maximize the trophozoites’ fitness triggered only a negligible inflammatory transcriptional response, while lysed and “non-fit” trophozoites induced a strong inflammatory response. “Fit” trophozoites could even suppress the inflammatory response triggered by lysed trophozoites, demonstrating the immune modulating capacity of the parasite. In conclusion, this thesis has increased our understanding of the interplay between G. intestinalis and IECs.
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| 2. |
- Gunja, Sethu Madhava Rao, 1986-
(författare)
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PARN - A Tale of A de-Tailor : Functional importance of poly(A) degradation in developmental and telomere biology disorders
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Poly(A)-specific ribonuclease (PARN) is a eukaryotic 3’-5’exoribonuclease that removes poly(A) tails of many coding and non-coding RNAs. In this thesis, we have studied the physiological role of PARN. We have found that genetic lesions in the human PARN gene are associated with a spectrum of human developmental disorders, including telomere biology disorders (TBDs). TBDs encompass a spectrum of developmental disorders associated with telomere dysfunction and include idiopathic pulmonary fibrosis (IPF), aplastic anaemic (AA), dyskeratosis congenita (DC) and Hoyeraal-Hreidarsson syndrome (HHS). Patients with mono-allelic mutations in PARN suffer from developmental and neurological disorders, whereas bi-allelic mutations are associated with severe disorders, e.g., DC or HHS.Transcriptome analysis revealed that PARN deficient patients were affected in a number of cellular pathways. The most affected were the ribosome/translation, cell-cell adhesion, cell cycle and cell signaling pathways. We also found that PARN deficient patients were defective in the biogenesis of a large number of non-coding RNAs (ncRNAs), including snoRNAs, scaRNAs, miRNAs and rRNAs. Deficiency in snoRNA and rRNA biogenesis correlated with blood disorders and/or bone marrow failure. PARN deficient patients also displayed defects in the maturation of the telomerase RNA component that correlated with telomere shortening.To further understand the physiological role of PARN in TBDs over generations and throughout the life span of an organism, we have established a parn loss-of-function zebrafish model, which recapitulates TBD phenotypes in human patients. In keeping with the human patients, homozygous parn deficient fish exhibited aberrant snoRNA profile, perturbed telomerase RNA maturation and short telomeres. In addition, we found that the zygotic parn mutant fish exhibited a spectrum of developmental defects from early embryogenesis to adult stage. The whole array of disease phenotypes observed in PARN deficient human patients and the parn loss-of-function zebrafish model indicate that PARN has essential roles in regulating growth and development throughout the life of an organism.
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| 3. |
- Pottmeier, Philipp, 1987-
(författare)
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Genetic Sex Differences in Early Human Neuronal Development : An Investigation in Embryo Tissue and Embryonic Stem Cells
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Sex differences in the human body affect many different organs and tissues, some of them have an effect on the human brain and its development. In the developing nervous system, sex differences can bias the number or functionality of neurons, glial cells or synapses. As a result, neural networks might develop with a sex-specific bias. A number of neurodevelopmental diseases, such as Tourette-Syndrome or Attention-Deficit/Hyperactivity Disorder, show sex differences in symptoms, onset and prevalence. It seems likely that sex differences in brain development contribute to differences in neurological disease susceptibility between males and females. In my work, I am investigating sex differences in gene expression during neuronal development in human embryo brain tissue, embryonic stem cells and neural stem cells. Of particular interest for sex differences are the genes of the sex chromosomes, since a large number of X-linked genes and even some Y-linked genes are implicated in neurodevelopment.In our first study, we found that Y chromosome genes are highly expressed in fetal brain tissues and 5 X/Y homologous genes have an increased gene dosage in male samples. We suggest 6 novel long non-coding RNAs that were expressed in previously unannotated regions of the Y chromosome in male fetal brain tissue. In our second study, we identified an increased rate of proliferation in male neural stem cells but similar neuronal differentiation trajectories in cells of both sexes. An increased expression of DCX and DLG4 suggests a faster differentiation of male neural stem cells, but sex differences disappeared after 14 days. Male cells overexpressed MASH1 and RELN, markers for Cajal-Retzius neurons, and the two demethylases KDM5D and UTY. Female cells overexpressed RMST a long non-coding RNA critical for neurogenesis. In the third study, sex-biased gene expression was investigated in human embryonic stem cells during 37 days of neuronal differentiation. Male and female cell lines showed sex-biased expression of genes involved in neurodevelopment, suggesting a sex difference in differentiation trajectory. We propose 13 sex-biased candidate genes that could strongly affect neuronal development. In addition, we confirmed the gene dosage compensation of X/Y homologs escaping XCI through the Y-homolog and identified a significant expression of the Y-homologs TXLNGY and UTY after 37 days of neuronal differentiation. We have also measured a significant increase of the Y-linked genes PCDH11Y, UTY and USP9Y during differentiation. The fourth study was an investigation of sex differences in H3 methylation and acetylation marks in embryonic stem cells. We found that H3K4me3, a transcription activation mark, was enriched at promotor sites of major pluripotency genes and related pathways, in female cell lines.In conclusion, we confirm the importance of Y chromosome genes for neuronal development and show that sex differences in gene expression exist during neuronal differentiation.
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| 4. |
- Younes, Sara
(författare)
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Uncovering biomarkers and molecular heterogeneity of complex diseases : Utilizing the power of Data Science
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Uncovering causal drivers of complex diseases is yet a difficult challenge. Unlike single-gene disorders complex diseases are heterogeneous and are caused by a combination of genetic, environmental, and lifestyle factors which complicates the identification of patient subgroups and the disease causal drivers. In order to study the dimensions of complex diseases analyzing different omics data is a necessity.The main goal of this thesis is to provide computational approaches for analyzing omics data of two complex diseases; mainly, Acute Myeloid Leukaemia (AML) and Systemic Lupus Erythematosus (SLE). Additionally, we aim at providing a method that would deal with integration issues that usually arise when combining complex diseases omics (specifically metabolomics) data from multiple data sources. AML is a cancer of the myeloid blood cells that is known for its heterogeneity. Patients usually respond to treatment and achieve a complete remission state. However, a majority of patients relapse or develop treatment resistance. In paper I, we focus on investigating recurrent genomic alterations in adult and pediatric relapsed and primary resistant AML that may explain disease progression. In paper II, we characterize changes in the transcriptome of AML over the course of the disease, incorporating machine learning analysis.SLE is a heterogeneous autoimmune disease characterized by unpredictable periods of flares. The flares are presented as different SLE disease activities (DA). Studies on the combinatorial effects of genes towards the manifestation of SLE DAs in patients’ subgroups have been limited. In paper III, we analyze gene expression data of pediatric SLE using interpretable machine learning. The aim was to study the co-predictive transcriptomic factors driving disease progression, discover the disease subtypes, and explore the relationship between transcriptomics factors and the phenotypes associated with the discovered subtypes.Recently, Metabolomics has been a crucial dimension in major multi-omics complex disease studies. Small-compound databases contain a large amount of information for metabolites. However, the existing redundancy of information in the databases leads to major standardization issues. In paper IV, we aim at resolving the inconsistencies that exist when linking and combining metabolomics data from several databases by introducing the new R package MetaFetcheR.
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| 5. |
- Behra, Phani Rama Krishna
(författare)
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Comparative genomics of the genus Mycobacterium : Genome evolution, phylogeny and diversity
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The genus Mycobacterium includes more than 190 species, and many cause severe diseases such as tuberculosis and leprosy. According to the "World Health Organization", in year 2019 alone, 10 million people developed TB, and 1.4 million died. TB had been in decline in developed countries, but made its reappearance as an opportunistic pathogen targeting immuno-compromised AIDS victims. Also, non-tuberculosis mycobacteria (NTM) infections have emerged as a major infectious agent in recent times. NTM occupy diverse ecological niches and can be isolated from soil, tap water, and groundwater. This thesis has investigated the Mycobacterium species from a genomic perspective, focusing on the biology of virulence factors, mobile genetic elements, tRNAs, and non-coding RNAs and their evolutionary distribution and possible relationship with phenotypic diversity. As part of this study, we have sequenced 153 mycobacterial genomes, including type strains, environmental samples, isolates from hospital patients, infected fish, and outbreak samples in an animal facility at Uppsala University. We have provided a phylogenetic tree based on 387 (and 56) core genes covering most species (244 genomes) constituting the Mycobacterium genus. The core gene phylogeny resulted in 33 clades. Subsequently, we have covered different clade groups, such as, M. marinum, M. mucogenicum, M. chelonae and M. chlorophenolicum and investigated the NTM clade-specific genome diversity and evolution. Our examination of non-coding genes showed that the total number of tRNA genes per species varies between 42 and 90. Among the species with more than 50 tRNAs, additional tRNA genes are likely acquired through horizontal gene transfer (HGT), as supported by the presence of closely linked HNH endonuclease gene and GOLLD RNA. We have explored the presence of selenocysteine utility and the gene for selenoprotein "formate dehydrogenase" among 244 mycobacterial genomes. For the M. chlorophenolicum clade, we have explored genes with a role in the bioremediation process. Comparative genomics of M. marinum and M. chelonae clade groups suggest new clusters or subspecies. Mutational hotspots are relatively higher in M. marinum compared to that in M. tuberculosis and M. salmoniphilum. Relatively higher number of hotspots in M. marinum is likely related to its ability to occupy different ecological niches. Finally, the thesis uncovered IS elements, phage sequences, plasmids, tRNA, and ncRNA contributing to mycobacterial evolution.
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| 6. |
- Cantoni, Federico
(författare)
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Fabrication advances of microvasculature models on-chip
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Despite the technological advances of the last decades, drug development remains a lengthy and costly process with uncertainties still associated with the poor predictive power of the in vitro and animal models. To address this limitation, microphysiological systems have been introduced in an attempt to increase the biological relevance of in vitro devices. One of the current challenges in MPS is the integration of a vasculature network to sustain 3D cultures to closely mimic human physiology. This thesis proposed a new strategy to recreate a more representative vasculature system directly on-chip. As a first step, the 2-photon polymerization was investigated as a 3D printing technique to recreate structures with cell-relevant feature size and resolution. Subsequently, the 2-photon polymerization 3D printing was combined with micromolding to recreate a multi-hydrogel vasculature network integrated on-chip for cell culture. The synergy of the two methods ensured the generation of a high-fidelity multi-hydrogel scaffold for cell co-culture. To preserve the delicate cell culture while still ensuring the sample manipulation for monitoring and analysis, a customized microphysiological system carrier with an integrated heating and perfusion system was also developed. Finally, the possibility of tuning the properties of the 3D-printed hydrogel by controlling the printing parameters was investigated to guide glioblastoma cells to a vascularized compartment. Overall, the thesis not only demonstrated the fabrication versatility of 2 photon polymerization for a vasculature model directly on-chip but also showed the benefits in integrating microphysiological systems on a carrier.
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| 7. |
- Cediel-Ulloa, Andrea, 1989-
(författare)
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Novel Endpoints To Unravel Developmental Neurotoxicity : From DNA methylation responses to methylmercury to the in vitro identification of endocrine disruptors
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The developing brain is especially sensitive to environmental stressors due to its dependence on the precise spatiotemporal regulation of multiple signals, and the long time period required for its formation. Some chemicals can interfere with molecular and cellular processes driving brain development, including epigenetic processes such as DNA methylation. Hence, identification of DNA methylation changes induced by chemical exposure may serve as early molecular markers for developmental neurotoxicity (DNT). Chemicals known as endocrine disruptors (EDCs) can produce adverse effects due to their capability to alter the endocrine system. Since brain development is highly dependent on endocrine signals, the potential adverse effects of EDCs on brain development needs to be addressed. Detection of DNT in the regulatory context has been based on in vivo testing, however, the financial costs and time intensive characteristics of these methods have resulted in a limited assessment of the DNT hazard of chemicals. In addition, in order to regulate EDCs, it is paramount to demonstrate that their adverse effects are a product of disruption of endocrine signals. Yet, at the moment, there are no approved methods which address both an endocrine mode of action and adverse neurodevelopmental outcomes. This doctoral thesis had two main aims: Firstly, to identify epigenetic changes, at the level of DNA methylation, underlying DNT induced by exposure to methylmercury (MeHg); and secondly, to develop new approach methods (NAMs) for the detection of DNT induced by endocrine disruption. Epigenetic effects were studied both in epidemiological data and experimentally in vitro. Associations between prenatal MeHg exposure and DNA methylation of GRIN2B and NR3C1 were found in children. In vitro validation of DNA methylation changes found in epigenome-wide association studies of populations exposed to MeHg, uncovered the potential involvement of the Mediator Complex Subunit 31 (MED31) in MeHg DNT. To contribute to the endocrine disruption (ED)-induced DNT field, the applicability of an in vitro model composed of murine neural progenitor cells (the C17.2 cell-line) was evaluated. We found that C17.2 neural differentiation and morphology were sensitive to retinoic acid (RAR), retinoic X (RXR), peroxisome proliferator-activated β/δ (PPARβ/δ), and glucocorticoid (GR) agonism. Furthermore, two out of 25 tested EDCs decreased neurite outgrowth and branching in the C17.2 system. These effects were recovered by co-exposure of the chemicals with antagonists of RAR, RXR, or PPARβ/δ, indicating that their DNT effect is mediated by hormonal disruption. Altogether, this thesis contributed to the development of new methodologies and endpoints for the assessment of DNT induced by MeHg and EDCs.
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| 8. |
- Ghandour, Salim
(författare)
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Novel Materials and Implant Designs for the Treatment of Degenerative Disc Disease
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The overarching objective of this thesis was to develop intervertebral implants for degenerated discs through design, fabrication, and mechanical validation. The research had four primary aims, each addressing different facets of implant development.The first aim was to design a structurally optimized fusion intervertebral cage capable of accommodating weak bioactive materials. Topology optimization was employed to design cages using titanium and calcium phosphate. The cage’s integrity was verified using finite element simulations, fabricated using additive manufacturing, and validated using ASTM F2077. Imaging techniques were utilized to assess the quality of the produced cages. These evaluations confirmed the mechanical reliability of the produced cage, able to incorporate around 75% in volume of a bioactive calcium phosphate material, protected by the titanium.The second aim was to develop a clinically relevant degeneration model for the biomechanical evaluation of percutaneous cement discoplasty (PCD). To this end, voids were generated in ovine functional spinal units (FSUs) using papain. The results were compared with clinical data where parameters such as void volume percentage, disc height, and morphology of the induced injury were assessed. FSUs were treated with PCD using bone cement, and mechanically evaluated under healthy, injured, and treated conditions to determine if PCD could stabilize the spinal segment. The void induced showed similar parameters compared to the clinical data. Further, the stability of the spine was significantly reduced after degeneration and restored after treatment, highlighting the effectiveness of the degeneration method and PCD treatment.The third aim was to evaluate the suitability of novel bone cements for their use in PCD. This study examined the tensile and fatigue properties of a low-modulus cement (VSLA) primarily intended for vertebroplasty. The formulation was tested in tensile and fatigue. VSLA showed a significant decrease in tensile and fatigue properties when compared to commercial cements. This study set a baseline for future low-modulus cements that may be tested for use in PCD.The fourth aim was to evaluate an alternative cement due to the low viscosity of VSLA, which may not be suitable for discoplasty. This study assessed the fatigue and long-term properties of a high-viscosity low-modulus cement (hv-LA-PMMA). The hv-LA-PMMA showed a significant reduction of mechanical and fatigue properties when compared to its commercial base. Notably, the fatigue properties were similar to those of the annulus fibrosus in the disc. Additionally, its high viscosity renders it a promising alternative to the bone cements currently utilized in PCD.In conclusion, this thesis successfully addressed the design, fabrication, and mechanical validation of two types of intervertebral implants for degenerated discs. The research outcomes contribute with valuable insights into the design of fusion cages, a degeneration model to evaluate PCD, and the assessment of low-modulus cements for use in PCD.
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| 9. |
- Krakovka, Sascha
(författare)
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Deradicalising Giardiasis Treatment
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Giardia intestinalis is a unicellular parasite causing the disease giardiasis. This disease is mostly prevalent in low resource settings, mandating low-cost treatment options. Treatment is mainly based on antiparasitics from the classes of 5-nitroimidazoles and benzimidazoles, which both target parasite DNA and other cellular macromolecules based on radicals generated from the drugs. In recent years resistance to those classes and cross-resistance in between them has become a problem, hence alternative antigiardials are needed.In this thesis we enhanced our understanding of the crucial differentiation process of encystation, which produce the environmentally stable and infective form of the parasite, the cyst (Paper I). Resistance development has been slow in G. intestinalis until now and it has been shown that resistance to the main treatment option, metronidazole, can be lost after en- and excystation so an enhanced understanding of this process can help us to identify the cause of this loss of drug tolerance. In the second study (Paper II) we followed this up by analysing two metronidazole resistant lines and one revertant on their ability to produce infective cysts, while cross checking with growth rates, metronidazole resistance level, transcriptomics and proteomics. We found the resistant cells lines to have deeply disturbed cellular pathways with the main resistance mechanisms being a reduction of uptake, a reduction of activation rate and an upregulation of oxidative stress responses. Cyst production and growth rates were highly reduced giving those lines a clear disadvantage when no drug pressure is applied. Most changes, phenotypically and expression wise, were reset in the revertant. As next step we evaluated alternative antigiardials and their targets. In Paper III we characterised the giardial thymidine kinase, on which this parasite depends completely to supply thymidine for DNA synthesis. We identified a nucleoside analogue, azidothymidine, that is targeting this enzyme and efficiently inhibits growth and encystation of trophozoites both in vivo and in vitro. Azidothymidine is currently used in HIV treatment, has a good safety profile and is comparatively cheap, which makes it a good candidate for treatment of giardiasis. In conclusion, this study has focussed on several aspects of nitroimidazole resistance in G. intestinalis throughout the life cycle as well as repurposing of antibiotics from other drug classes that could be used to fill our arsenal of antigiardials with new alternatives.
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| 10. |
- Lin, Weifeng
(författare)
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Development of advanced chemical biology tools for microbiome metabolism : Chemoselective probes for enhanced metabolomics analysis
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The human microbiome has a profound impact on host physiology by generating highly reactive compounds that can contribute to the development of diseases. These microbial metabolites have a substantial potential that can serve as valuable indicators or biomarkers for different health conditions. Nevertheless, elucidating the microbiota composition and function remains challenging due to its remarkable diversity and complex. Furthermore, conducting a comprehensive analysis of the entire metabolome in a single analytical measurement is difficult. Researchers often employ derivatization techniques in analytical chemistry, which involve modifying the chemical structure of molecules to enhance their detectability, ionization properties and stability during analysis. However, derivatization carries the risk of introducing artifacts or chemical alterations that may compromise the accuracy of analytical results. Consequently, more advanced techniques are urgently required to improve the precision of derivatization-based metabolomics.In response to this challenge, we have developed chemoselective probes immobilized onto magnetic beads to capture metabolites within biological samples. This innovative method improves the mass spectrometric sensitivity by up to a factor of one million, due to the efficient removal of sample matrix background through magnetic separation and improved ionization properties of the metabolites via derivatization. Our approach, termed quant-SCHEMA, has demonstrated the qualitative detection of metabolites containing carbonyl and amine groups with exceptional sensitivity and reproducibility. Additionally, we have successfully applied this method with improved probe design to quantitatively analyse carbonyl-containing metabolites, leading to the discovery of four valuable nutritional biomarkers. Furthermore, we have developed a precise quantification method for short-chain fatty acids (SCFAs) based on this chemoselective probe. The successful implementation of our chemoselective probes highlights the importance of chemical biology tools in advancing metabolomics, which we have termed chemical metabolomics,This comprehensive mass spectrometric analysis expands the horizons of metabolomics-driven biomarker discovery. We envision that our innovative chemical biology tool will find widespread utility in metabolomics analysis, providing valuable insights into microbial interactions with the human host and the development of diseases.
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