In this study, employing an integrated approach that involves transcriptomics, metabolomics, and other techniques, we aimed to understand the mechanistic roles of the cryoprotective system underlying extreme freeze tolerance in the larvae of the drosophilid fly, Chymomyza costata. Metabolomics unveiled the complex composition of the seasonally accumulated larval innate mixture of putative cryoprotectants. This mixture was predominantly composed of proline and trehalose, supplemented by minor components (glutamine, asparagine, betaine, sarcosine, glycerophospho-choline, and ethanolamine). We identified food ingestion as a significant source for the direct assimilation of amino compounds, while glycogen and phospholipids served as the primary internal sources for the biosynthesis of other cryoprotectants. The utilization of MALDI-MSI to observe cryoprotectants revealed distinct behaviors of proline and trehalose during ecologically relevant, gradual inoculative extracellular freezing of larvae. Trehalose exhibited accumulation in partially dehydrated hemolymph, inducing a transition to the amorphous glass phase. On the other hand, proline migrated to the boundary between extracellular ice and dehydrated hemolymph and tissues, forming a layer of dense viscoelastic liquid. Finally, through a combination of in vivo and in vitro assays, we found that cell membranes are likely targets of freezing injury, while their integrity is sustained by accumulated small cryoprotective molecules and proteins in cold-acclimated C. costata larvae. Contrary to our expectations, our assays did not provide support for the hypothesis that proteins (soluble enzymes) require in vivo stabilization through the accumulation of cryoprotectants.
Anotace v angličtině
In this study, employing an integrated approach that involves transcriptomics, metabolomics, and other techniques, we aimed to understand the mechanistic roles of the cryoprotective system underlying extreme freeze tolerance in the larvae of the drosophilid fly, Chymomyza costata. Metabolomics unveiled the complex composition of the seasonally accumulated larval innate mixture of putative cryoprotectants. This mixture was predominantly composed of proline and trehalose, supplemented by minor components (glutamine, asparagine, betaine, sarcosine, glycerophospho-choline, and ethanolamine). We identified food ingestion as a significant source for the direct assimilation of amino compounds, while glycogen and phospholipids served as the primary internal sources for the biosynthesis of other cryoprotectants. The utilization of MALDI-MSI to observe cryoprotectants revealed distinct behaviors of proline and trehalose during ecologically relevant, gradual inoculative extracellular freezing of larvae. Trehalose exhibited accumulation in partially dehydrated hemolymph, inducing a transition to the amorphous glass phase. On the other hand, proline migrated to the boundary between extracellular ice and dehydrated hemolymph and tissues, forming a layer of dense viscoelastic liquid. Finally, through a combination of in vivo and in vitro assays, we found that cell membranes are likely targets of freezing injury, while their integrity is sustained by accumulated small cryoprotective molecules and proteins in cold-acclimated C. costata larvae. Contrary to our expectations, our assays did not provide support for the hypothesis that proteins (soluble enzymes) require in vivo stabilization through the accumulation of cryoprotectants.
Klíčová slova
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Klíčová slova v angličtině
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Rozsah průvodní práce
132
Jazyk
AN
Anotace
In this study, employing an integrated approach that involves transcriptomics, metabolomics, and other techniques, we aimed to understand the mechanistic roles of the cryoprotective system underlying extreme freeze tolerance in the larvae of the drosophilid fly, Chymomyza costata. Metabolomics unveiled the complex composition of the seasonally accumulated larval innate mixture of putative cryoprotectants. This mixture was predominantly composed of proline and trehalose, supplemented by minor components (glutamine, asparagine, betaine, sarcosine, glycerophospho-choline, and ethanolamine). We identified food ingestion as a significant source for the direct assimilation of amino compounds, while glycogen and phospholipids served as the primary internal sources for the biosynthesis of other cryoprotectants. The utilization of MALDI-MSI to observe cryoprotectants revealed distinct behaviors of proline and trehalose during ecologically relevant, gradual inoculative extracellular freezing of larvae. Trehalose exhibited accumulation in partially dehydrated hemolymph, inducing a transition to the amorphous glass phase. On the other hand, proline migrated to the boundary between extracellular ice and dehydrated hemolymph and tissues, forming a layer of dense viscoelastic liquid. Finally, through a combination of in vivo and in vitro assays, we found that cell membranes are likely targets of freezing injury, while their integrity is sustained by accumulated small cryoprotective molecules and proteins in cold-acclimated C. costata larvae. Contrary to our expectations, our assays did not provide support for the hypothesis that proteins (soluble enzymes) require in vivo stabilization through the accumulation of cryoprotectants.
Anotace v angličtině
In this study, employing an integrated approach that involves transcriptomics, metabolomics, and other techniques, we aimed to understand the mechanistic roles of the cryoprotective system underlying extreme freeze tolerance in the larvae of the drosophilid fly, Chymomyza costata. Metabolomics unveiled the complex composition of the seasonally accumulated larval innate mixture of putative cryoprotectants. This mixture was predominantly composed of proline and trehalose, supplemented by minor components (glutamine, asparagine, betaine, sarcosine, glycerophospho-choline, and ethanolamine). We identified food ingestion as a significant source for the direct assimilation of amino compounds, while glycogen and phospholipids served as the primary internal sources for the biosynthesis of other cryoprotectants. The utilization of MALDI-MSI to observe cryoprotectants revealed distinct behaviors of proline and trehalose during ecologically relevant, gradual inoculative extracellular freezing of larvae. Trehalose exhibited accumulation in partially dehydrated hemolymph, inducing a transition to the amorphous glass phase. On the other hand, proline migrated to the boundary between extracellular ice and dehydrated hemolymph and tissues, forming a layer of dense viscoelastic liquid. Finally, through a combination of in vivo and in vitro assays, we found that cell membranes are likely targets of freezing injury, while their integrity is sustained by accumulated small cryoprotective molecules and proteins in cold-acclimated C. costata larvae. Contrary to our expectations, our assays did not provide support for the hypothesis that proteins (soluble enzymes) require in vivo stabilization through the accumulation of cryoprotectants.
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Klíčová slova v angličtině
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Zásady pro vypracování
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Zásady pro vypracování
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Seznam doporučené literatury
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ilustrace, grafy, tabulky
Převzato z knihovny
Ne
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Hodnocení vedoucího
Záznam průběhu obhajoby
Průběh obhajoby 15:30 - 17:30
1. předseda komise přivítal přítomné, představil uchazeče a členy komise. 2. školitel představil uchazeče a zhodnotil jeho doktorské studium. 3. uchazeč přednesl obsah své disertační práce. 4. oponentka Jantina Toxopeus přednesla své hodnocení a položila otázky uchazeči: fáze diapauzního vývoje hmyzu obecně a C. costata speciálně, vliv rychlosti chlazení na kryoprotektivní funkci prolinu a trehalózy, toxicita vysokých koncentrací kryoprotektantů. 5. oponentka Jacqueline Lebenzon přednesla své hodnocení a položila otázky uchazeči: metabolické role prolinu v hmyzím organismu,přezimování C. costata v přírodních podmínkách, vliv dietárního prolinu na fitness dospělců C. costata, mitochondriální stabilita během chladového stresu. 6. Uchazeč uspokojivě zodpověděl všechny dotazy oponentek. 7. Členové komise položili dotazy uchazeči a ten na ně odpovídal: Nedvěd: design experimentu na preferenci různých larválních diet C. costata, Kodrík: metodika barvení hmyzích tkání pro mikroskopické analýzy, Doležal T.: metabolismus glykogenu, kolagenu, trehalózy a prolinu, role LsP1 a Lsp2, Vácha: intra vs. extra-celulární led. 8. dotazy z pléna: Štětina: vliv prolinu na mitochondriální membrány. 9. předseda komise ukončil veřejnou část obhajoby a zahájil interní jednání členů komise za přítomnosti obou oponentek a školitele. 10. předseda a členové komise (7) a oponentky (2) hlasovali: 9 x prospěl, 0 x neprospěl. 11. předseda komise veřejně vyhlásil výsledek hlasování: uchazeč prospěl (všemi hlasy), komise doporučuje udělení titulu Ph.D. Robertu Grgacovi
členové komise: Vladimír Košťál (školitel),Dalibor Kodrík,Martin Vácha,Robert Hanus,Oldrich Nedved,Tomas Dolezal,Ivo Sauman,Petr Dolezal,Jantina Toxopeus,Jacqueline Lebenzon,