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Clone-specific differences and dynamics of abiotic stress effects on the Daphnia pulex proteome

Daphnia pulex unterliegt in ihrem Habitat starken Futter- und Temperatur-Schwankungen. Veränderungen der Protein-Expression innerhalb des Proteoms während akutem Hunger-, Hitze- und kombiniertem Hunger- und Hitze-Stress wurden in dieser Arbeit in zwei unterschiedlichen D. pulex-Klonen durch 2D-Gelelektrophorese und Massenspektrometrie untersucht. Viele der bestimmten Protein-Isoformen waren nach Hunger- und/oder Hitze-Stress in beiden Klonen herauf- oder herunterregeguliert, wobei die Stärke der Expression und die regulatorischen Muster variierten. Glutathion-Transferasen, V-ATPase-Untereinheiten, Zytoskelett-Proteine und Chaperone waren signifikant heraufreguliert, Proteine für Proteolyse, Kohlenhydrat-Bindung oder Metabolismus waren herunterreguliert. Die Expression aller Proteine folgte spezifischen zeitlichen Mustern. Die Verfügbarkeit von Futter zeigten Klon-spezifische Unterschiede im Überleben, dem Gesamtproteingehalt und der Protein-Expression während akutem Hitze-Stress.

Daphnia pulex is challenged by severe food and temperature changes in its habitat. To identify cellular adjustments in protein expression during acute starvation, heat-stress and the combination of both stress types; the proteomes of two D. pulex clones were studied by 2D gel electrophoresis and mass spectrometry. Several protein isoforms were up- or down-regulated in both clones during acute heat-stress and/or starvation. The expression varied in intensity and regulatory patterns between both clones. Glutathion transferases, V-ATPase subunits, cytoskeleton-proteins and chaperones were significantly up-regulated, proteins for proteolytic processes and carbohydrate binding or metabolism were down-regulated. The expression of all proteins followed specific temporal patterns. Availability of food resources revealed fundamental clone specific differences in survival, total protein content and protein expression during acute heat-stress.

Titel: Clone-specific differences and dynamics of abiotic stress effects on the Daphnia pulex proteome
Weitere Titel Clone specific differences and dynamics of abiotic stress effects on the Daphnia pulex proteome
Verfasser: Reydelet, Yann GND
Gutachter: Paul, Rüdiger J.
Organisation: FB 13: Biologie
Dokumenttyp: Dissertation/Habilitation
Medientyp: Text
Erscheinungsdatum: 2015
Publikation in MIAMI: 11.01.2016
Datum der letzten Änderung: 16.09.2016
Schlagwörter: Daphnia pulex; Proteom; 2D-Gelelektrophorese; Massenspektrometrie; Hitzestress; zelluläre Stressantwort; Überleben; Proteinexpression; Gesamtproteingehalt; Hungerstress
Daphnia pulex; 2D gel electrophoresis; starvation; heat-stress; mass spectrometry; survival; protein expression; total protein; cellular stress response; proteom
Fachgebiete: Biowissenschaften; Biologie
Sprache: Englisch
Format: PDF-Dokument
URN: urn:nbn:de:hbz:6-17229678635
Permalink: https://nbn-resolving.org/urn:nbn:de:hbz:6-17229678635
Onlinezugriff:
Inhalt:
Content I
List of Figures III
List of Tables V
List of Abbreviations VI
Abstract 1
1 Background 5
1.1 Stress – Response and Adaptation 5
1.2 Daphnia: a model organism 11
1.3 Clonal variation and local adaptation 13
1.4 Effects of changes in temperature on organisms and their coping mechanisms 17
1.5 Adaptation to the change of food availability 19
1.6 Goals of the thesis 22
2 Methods 23
2.1 Animals 23
2.2 Clone identification by allozyme characterization 23
2.3 Motility and survival assay 23
2.4 Long term swimming assay 24
2.5 Stress exposure and protein extraction 24
2.6 Evaluation of total protein amount 25
2.7 Two-dimensional gel electrophoresis 25
2.8 Analysis of differential protein expression 26
2.9 LC-MS/MS, identification and characterization of proteins 27
2.10 HSP60 quantification with Western Blots 28
3 Results 31
3.1 Clonal characterization by allozyme analysis 31
3.2 Temporal changes in motility and long-term survival under control and stress
conditions 32
3.3 Protein expression at different environmental conditions 35
3.3.1 Total protein quantities at different environmental conditions 35
3.3.2 Protein expression at control conditions (20 °C, ad libitum food supply) 36
3.3.3 Protein expression at heat stress (30 °C, ad libitum food supply) 38
3.3.4 Protein expression in two D. pulex clones at starvation stress 51
3.3.5 Protein expression at heat-and-starvation stress (30 °C, no food) 61
3.3.6 Summary of differential protein expressions 71
3.4 Validation of the protein expression changes: HSP60 74
4 Discussion 79
4.1 Major differences in stress-induced proteomic responses between the D. pulex clones G and M 81
4.2 Differences in heat-induced proteomic responses between the fed and non-fed D. pulex clones G and M 84
4.3 Differences in starvation-induced proteomic responses between the D.pulex clones G and M at 20°C and 30°C 90
4.4 Stress responses in the D.pulex clones G and M: conclusions and short outlook 95
4.5 Validation of the protein pattern by Western Blot analysis 96
4.6 Comparison of transcriptome and proteome 98
5 Supplement 99
5.1 Validation of the protein expression changes: HSP60 99
5.2 Validation of the protein pattern by Western Blot analysis 106
5.3 Regulation of gene expression 109
5.4 Regulation of gene expression via core promoters 110
5.5 Bioinformatical analysis for specific promoters and highly conserved protein
domains 115
5.6 Bioinformatic analysis of promotor regions and enhancers 118
5.7 Bioinformatic analysis of promotor and enhancer regions 129
References 135