Proteine disaccoppianti e termogenesi adattativa in Drosophila

Uncoupling proteins (UCPs) are anion carriers located in the inner mitochondrial membrane. They dissipate the proton gradient generated by mitochondrial respiratory chain during electron transport. UCPs allow protons to flow back into the matrix, dissipating the proton gradient as heat, without leading to ATP generation (Skulachev, 1996). In mammals, five members of the UCP protein family have been identified. UCP1 is the only bona fide uncoupling protein is present in brown adipose tissue where mediates mitochondrial thermogenesis (Nicholls and Locke; 1984). The only UCPs characterized in Drosophila melanogaster UCP4A, UCP4B, UCP4C, and UCP5 (Hanák and Ježek, 2001). Previous findings, by using KD models of Ucp4 paralogs, have shown that metabolism during larval stages is fuelled by glycolysis and is uncoupled from ATP synthesis in relation to the expression of Ucp4C. The main aim of my PhD project was to investigate the molecular mechanisms of larval thermogenesis in D. melanogaster – I focused on the characterization of Ucp4C, for which I generated a KO model. Ucp4C KO egg-to-adult survival at 14°C was significantly decreased compared to controls. However, unlike Ucp4C KD flies, which showed 100% mortality when developing at low temperature (Da-Ré et al., 2014), about 20% of Ucp4C KO flies were able to reach the adult stage. This suggests that KD mortality was not simply due to reduced expression of Ucp4C but might also be a result of synergic off-target effects. In addition, I investigated the expression of the other Ucp paralogs, showing that their levels are modified in the Ucp4C KO compared to those of the wild type w[1118] – that suggests compensatory effects. Moreover, I studied the temporal expression of Ucp4C and Ucp4B in larvae, and detected a significant oscillation of Ucp4B mRNA over the 24 hours, but not of Ucp4C. Respiration profiles were obtained by measuring mitochondrial oxygen consumption rate (OCR) in isolated mitochondria. I confirmed the coupled state of isolated larval mitochondria from both w[1118] and Ucp4C KO. Next, isolated mitochondria of both genotypes were treated with palmitate (UCPs activator). The results showed similar effects - slight increase in respiration in both Ucp4C KO and control larvae that might relate to compensatory effects in Ucp4C KO flies and suggests the potential existence of different uncoupling mechanisms that might be triggered when the classic Ucp4C-dependent mechanism is abolished. I used thermography to investigate larval heat generation. Temperature difference between surrounding medium and larvae was lower in Ucp4C KO than in w[1118]. In addition, I tested by monitoring w[1118] and Ucp4C KO adult flies’ survival at 14°C, if flies lacking Ucp4C KO are affected by exposure to cold. This was higher in females compared to males in both genotypes. My project also encompasses work on Drosophila suzukii, a pest species which, over recent years, has become a serious economical danger for agriculture. Currently utilised pest control methods are not very effective, thus new strategies are needed. First, I tested if temperature influences Ucp4C expression also in D. suzukii. Ucp4C levels in larvae were modulated by temperature (14°C, 18°C, 23°C), with the highest values at the lowest temperatures. I also measured longevity of adult flies exposed to constant temperature of 23, 18 or 15°C. As expected, flies grown at 23°C had the shortest survival period while flies reared at 15°C survived for the longest time. I also designed an approach to interfere the Ucp4C gene in D. suzukii by delivering dsRNA with the food. This was obtained by soaking the larvae in a solution containing dsRNA. This treatment resulted in a significant decrease of Ucp4C expression compared to untreated control larvae, suggesting that it might serve as a basis for the development of strategies applicable in the field.