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Fourier transform infrared microspectroscopy for the analysis of the biochemical composition of C. elegans worms
Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM).ORCID-id: 0000-0002-5534-2425
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM).
2016 (Engelska)Ingår i: Worm, E-ISSN 2162-4054, Vol. 5, nr 1, artikel-id e1132978Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Changes in intermediary metabolism have profound effects on many aspects of C. elegans biology including growth, development and behavior. However, many traditional biochemical techniques for analyzing chemical composition require relatively large amounts of starting material precluding the analysis of mutants that cannot be grown in large amounts as homozygotes. Here we describe a technique for detecting changes in the chemical compositions of C. elegans worms by Fourier transform infrared microspectroscopy. We demonstrate that the technique can be used to detect changes in the relative levels of carbohydrates, proteins and lipids in one and the same worm. We suggest that Fourier transform infrared microspectroscopy represents a useful addition to the arsenal of techniques for metabolic studies of C. elegans worms.

Ort, förlag, år, upplaga, sidor
Taylor & Francis, 2016. Vol. 5, nr 1, artikel-id e1132978
Nyckelord [en]
C. elegans, carbohydrate, composition, Fourier transform infrared, lipid, microspectroscopy, protein
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
URN: urn:nbn:se:umu:diva-111049DOI: 10.1080/21624054.2015.1132978PubMedID: 27073735OAI: oai:DiVA.org:umu-111049DiVA, id: diva2:866396
Anmärkning

Originally included in thesis in manuscript form, with the title "Fourier transform infrared microspectroscopy for the analysis of the biochemical composition of C. elegans".

Tillgänglig från: 2015-11-02 Skapad: 2015-11-02 Senast uppdaterad: 2018-06-07Bibliografiskt granskad
Ingår i avhandling
1. Regulation of energy balance in Caenorhabditis elegans 
Öppna denna publikation i ny flik eller fönster >>Regulation of energy balance in Caenorhabditis elegans 
2015 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Alternativ titel[sv]
Reglering av energibalans i Caenorhabditis elegans
Abstract [en]

Obesity is a medical condition in which excess body fat has been accumulated. It is most commonly caused by imbalance between energy intake and energy expenditure (lack of physical activity and lower metabolic rate, etc.). The control of energy metabolism involves multiple tissues and signalling pathways and there is a great need for further understanding of these different interactions.

In this study, I use Caenorhabditis elegans to study these complex pathways at the level of a whole organism. The downstream target of mTOR, p70 S6 kinase (S6K), has been implicated in the phosphorylation of multiple substrates and the regulation of growth and metabolism. In this study the worm homolog of S6K, rsks-1, found to be important for fat metabolism. Previous work in our lab found that RSKS-1::GFP is expressed at high levels in a set of sensory neurons and upregulated in ASJ, ASE and BAG sensory neurons in starved worms or mutants with low insulin activity. In this study, I found that the upregulation of rsks-1 expression was affected by serotonin, but not by the other neurotransmitters. Combined with the result that rsks-1 is required for the expression of TGFβ and insulin in ASI, rsks-1 may control dietary sensing by affecting insulin and TGFβ signalling within nervous system. Quantification of fat accumulation by TLC/GC revealed that in comparison to wild type worms, rsks-1 mutants have more than two-fold higher levels of triglycerides. This was confirmed by FT-IR microspectroscopy analysis. rsks-1 mutants also contain disproportionately high levels of C16:1n9 and C18:1n9 lipids compared with wild type worms. Genetic analysis has shown that rsks-1 acts either downstream of, or in parallel to the insulin and TGFβ pathways to affect fat levels. My studies showed that rsks-1 affects fat metabolism by influencing mRNA levels of genes encoding proteins in the β-oxidation pathway. Combined with defects in dietary sensing, fatty acid absorption, fertility and mitochondria function, the loss of rsks-1 activity induced much more energy storage than wild type by making a profound metabolic shift. These results are consistent with the metabolomics data analysis. Tissue specific RNAi showed that rsks-1 was required in many different tissues to regulate fat metabolism. Taken together, it can be concluded that RSKS-1 activity is needed for co-ordination of metabolic states in C. elegans. In order to understand more about the physiology behind fat accumulation, I analysed a mutant, aex-5, that has significantly lowered lipid levels. I found that this defect is associated with a significant reduction in the rate at which dietary fatty acids are taken up from the intestinal lumen. The aex-5 gene, which encodes a Kex2/subtilisin-family, Ca2+-sensitive proprotein convertase, is required for a discrete step in an ultraradian rhythmic phenomenon called the defecation motor program (DMP). Combined with other results, we conclude that aex-5 and other defecation genes may affect fat uptake by promoting the correct distribution of acidity within the intestinal lumen.

This dissertation also described how to use Fourier transform infrared (FT-IR) microspectroscopy to detect lipids, proteins and carbohydrates directly in single worm. In conclusion, in this thesis I have uncovered several components that play roles in dietary sensing, fatty acid synthesis, adiposity regulation and fatty acid absorption in C. elegans.   

Ort, förlag, år, upplaga, sidor
Umea: Research Media, 2015. s. 70
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 1762
Nyckelord
C.elegans, fat metabolism, S6 Kinase, FT-IR
Nationell ämneskategori
Biologiska vetenskaper
Forskningsämne
molekylär medicin (genetik och patologi)
Identifikatorer
urn:nbn:se:umu:diva-110688 (URN)978-91-7601-380-9 (ISBN)
Disputation
2015-11-24, Betula, NUS 6A–L - Biomedicinhuset, Major groove, Umeå, 09:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2015-11-03 Skapad: 2015-10-26 Senast uppdaterad: 2018-06-07Bibliografiskt granskad

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Sheng, MingGorzsás, AndrásTuck, Simon

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