De novo synthesis | EPFL Graph Search

In chemistry, de novo synthesis () refers to the synthesis of complex molecules from simple molecules such as sugars or amino acids, as opposed to recycling after partial degradation. For example, nucleotides are not needed in the diet as they can be constructed from small precursor molecules such as formate and aspartate. Methionine, on the other hand, is needed in the diet because while it can be degraded to and then regenerated from homocysteine, it cannot be synthesized de novo. Nucleotide Nucleotide#Synthesis De novo pathways of nucleotides do not use free bases: adenine (abbreviated as A), guanine (G), cytosine (C), thymine (T), or uracil (U). The purine ring is built up one atom or a few atoms at a time and attached to ribose throughout the process. Pyrimidine ring is synthesized as orotate and attached to ribose phosphate and later converted to common pyrimidine nucleotides. Cholesterol Cholesterol#Biosynthesis Cholesterol is an essential structural

Investigation of physiological roles of AMPK-activated protein kinase y3

Philipp Jurijvic Rhein

Clinical trials have shown that direct activators of an evolutionary-conserved metabolic sensor, AMP-activated protein kinase (AMPK), are beneficial in preventing/treating a range of metabolic disorders, including type 2 diabetes. These activators, including 991/MK-8722, bind at regulatory site, termed the allosteric drug and metabolite (ADaM-site) at the interface between the catalytic alpha subunit and regulatory beta subunit. In addition, another approach for direct activation of AMPK is through mimicking its natural ligands, AMP/ADP, by binding to the nucleotide site in the y3 subunit, e.g. 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). Interestingly, their ability to influence glucose homeostasis is reported to be through activation of AMPK complexes found within the skeletal muscle. The AMPKy3 is unique as an AMPK subunit isoform since it is selectively expressed in glycolytic skeletal muscle fibres. To gain more insight into the y3 isoform-specific regulation of glucose metabolism, we used a y3 knock-out (y3-/-) mouse model to investigate the effects of the AICAR and 991/MK-8722. We confirm that y3 isoform expression and activity is high glycolytic muscles, including extensor digitorum longus (EDL), whereas there is very little detected in oxidative soleus (SOL) muscle. We show that AICAR and 991/MK-8722 stimulated glucose uptake in both EDL and SOL muscles. Interestingly, only the ability of AICAR to induce glucose uptake was blunted in EDL muscle taken from y3-/- mice. Consistent with this, whole-body glucose clearance was also impaired in y3-/- mice in response to an AICAR, whilst MK-8722 lowered blood glucose similarly between WT and y3-/- mice. Despite the presence of y1-containing complexes, which are the most predominant complexes in both EDL and SOL muscles, this suggests that different AMPKy isoforms play different roles in glucose homeostasis in response to AICAR. We further looked at de novo glycogen synthesis and glucose utilisation in skeletal muscle. Despite similar basal glucose uptake between muscles from WT and y3-/-, y3-deficiency results in lower basal glycogen content only in glycolytic muscles. We show that this is not due to an inability to synthesise glycogen de novo, since MK-8722 was able to increase glycogen levels in EDL. Instead, we show that the decrease in basal glycogen in y3-/- EDL, may be linked lower expression of UDP-glucose pyrophosphorylase 2 (UGP2). This suggests that AMPKy3 may play a role in steering the molecular fate of glucose inside the cell. Taken together, whilst we show that AMPKy3 is dispensable for ex vivo glucose transport and in vivo glucose homeostasis in response to ADaM-site AMPK activators, nucleotide-mediated activation of y3 by AICAR is impaired. Further research is required to understand the nucleotide regulation of different y-containing AMPK complexes. This differential activation mechanism could be exploited to specifically target AMPKy3 complexes in the muscle, and potentially avoid deleterious effects of activation of AMPK complexes in other tissues.

EPFL2021

A Single Metabolite which Modulates Lipid Metabolism Alters Hematopoietic Stem/Progenitor Cell Behavior and Promotes Lymphoid Reconstitution

Sonja Giger, Marlen Knobloch, Matthias Lütolf, Mergim Ramosaj

Fatty acid beta-oxidation (FAO), the breakdown of lipids, is a metabolic pathway used by various stem cells. FAO levels are generally high during quiescence and downregulated with proliferation. The endogenous metabolite malonyl-CoA modulates lipid metabolism as a reversible FAO inhibitor and as a substrate for de novo lipogenesis. Here we assessed whether malonyl-CoA can be exploited to steer the behavior of hematopoietic stem/progenitor cells (HSPCs), quiescent stem cells of clinical relevance. Treatment of mouse HSPCs in vitro with malonyl-CoA increases HSPC numbers compared with nontreated controls and ameliorates blood reconstitution capacity when transplanted in vivo, mainly through enhanced lymphoid reconstitution. Similarly, human HSPC numbers also increase upon malonyl-CoA treatment in vitro. These data corroborate that lipid metabolism can be targeted to direct cell fate and stem cell proliferation. Physiological modulation of metabolic pathways, rather than genetic or pharmacological inhibition, provides unique perspectives for stem cell manipulations in health and disease.

2020

Engineering next generation organoids through guided stem cell self-organisation

Mikhail Nikolaev

One of the long-standing goals in the field of tissue engineering is the fabrication of de novo tissues or even organs to provide better tissue models for basic research and drug discovery, and to alleviate a shortage of donor organs in the future. Research in recent decades to understand tissue biology has led to the discovery of stem cell niches and the development of innovative protocols for the generation of three-dimensional in vivo-like tissues called organoids. Organoids represent multiple histological and functional aspects of real organs and offer unprecedented perspectives for modelling tissue development, regeneration, and disease in vitro. However, relying so far almost exclusively on spontaneous self-organisation of stem cells, organoids develop uncontrollably into small structures of highly variable size and architecture. Moreover, the closed, cystic architecture of most epithelial organoids limits their lifespan and makes experimental manipulation difficult. In this thesis, three innovative engineering technologies were developed that offer the possibility of steering in vitro stem cell patterning and organoid formation into more controllable and physiologically relevant miniature tissues.	The aim of the first project was to study how predefined geometric constraints, as they exist in vivo, affect organoid development. This required the development of a novel fabrication process of the topographically micro-patterned hydrogel surfaces. This approach allowed the generation of large arrays of the identical organoids, having pre-defined shape and size to study cellular mechanisms involved in the proper, stereotypical cell type pattering of intestinal organoids.		The second project focused on replicating tissue dynamics and aspects of organ physiology by developing a novel three-dimensional organ-on-chip approach. Using stem cells derived from patient biopsies, this technology enables to production of miniature versions of patientâs organs that can be used in personalized diagnostics and medical treatment assays.	Bioprinting has long been considered as one of the most promising technologies for fabricating organs in vitro. However, despite encouraging advances made in recent years, the degree of multicellular spatial complexity and function of organoids remains unmatched by any existing bioprinting technology. Therefore, the third project was aimed to develop a new bioprinting strategy that preserves local stem cell self-organization potential and extends organoids growth to the macroscopic scale.	Overall, this thesis introduces several cutting-edge technologies that offer the possibility to control in vitro organoid development and should therefore become efficient research tools to facilitate the translation of organoid technology towards pharmaceutical and clinical applications. Hydrogel micro-pattering is an efficient technology that can be widely used to explore the mechanisms by which tissue geometry can regulate morphogenesis of different organs and can be readily applied for high-throughput screening in pharmacological assays. The microscope-based bioprinter can be set-up in any laboratory to guide stem cell self-organization and tissue morphogenesis from millimetre to centimetre scales. The microfluidic organoid-on-chip concept should enable the creation of various miniature organs with complex three-dimensional spatial organisation and multicellular complexity for basic research and drug discovery.

EPFL2021

Engineering next generation organoids through guided stem cell self-organisation

Mikhail Nikolaev

One of the long-standing goals in the field of tissue engineering is the fabrication of de novo tissues or even organs to provide better tissue models for basic research and drug discovery, and to alleviate a shortage of donor organs in the future. Research in recent decades to understand tissue biology has led to the discovery of stem cell niches and the development of innovative protocols for the generation of three-dimensional in vivo-like tissues called organoids. Organoids represent multiple histological and functional aspects of real organs and offer unprecedented perspectives for modelling tissue development, regeneration, and disease in vitro. However, relying so far almost exclusively on spontaneous self-organisation of stem cells, organoids develop uncontrollably into small structures of highly variable size and architecture. Moreover, the closed, cystic architecture of most epithelial organoids limits their lifespan and makes experimental manipulation difficult. In this thesis, three innovative engineering technologies were developed that offer the possibility of steering in vitro stem cell patterning and organoid formation into more controllable and physiologically relevant miniature tissues.	The aim of the first project was to study how predefined geometric constraints, as they exist in vivo, affect organoid development. This required the development of a novel fabrication process of the topographically micro-patterned hydrogel surfaces. This approach allowed the generation of large arrays of the identical organoids, having pre-defined shape and size to study cellular mechanisms involved in the proper, stereotypical cell type pattering of intestinal organoids.		The second project focused on replicating tissue dynamics and aspects of organ physiology by developing a novel three-dimensional organ-on-chip approach. Using stem cells derived from patient biopsies, this technology enables to production of miniature versions of patientâs organs that can be used in personalized diagnostics and medical treatment assays.	Bioprinting has long been considered as one of the most promising technologies for fabricating organs in vitro. However, despite encouraging advances made in recent years, the degree of multicellular spatial complexity and function of organoids remains unmatched by any existing bioprinting technology. Therefore, the third project was aimed to develop a new bioprinting strategy that preserves local stem cell self-organization potential and extends organoids growth to the macroscopic scale.	Overall, this thesis introduces several cutting-edge technologies that offer the possibility to control in vitro organoid development and should therefore become efficient research tools to facilitate the translation of organoid technology towards pharmaceutical and clinical applications. Hydrogel micro-pattering is an efficient technology that can be widely used to explore the mechanisms by which tissue geometry can regulate morphogenesis of different organs and can be readily applied for high-throughput screening in pharmacological assays. The microscope-based bioprinter can be set-up in any laboratory to guide stem cell self-organization and tissue morphogenesis from millimetre to centimetre scales. The microfluidic organoid-on-chip concept should enable the creation of various miniature organs with complex three-dimensional spatial organisation and multicellular complexity for basic research and drug discovery.

EPFL2021

Investigation of physiological roles of AMPK-activated protein kinase y3

Philipp Jurijvic Rhein

EPFL2021