Dr Mirko Pegoraro research
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Owl and Lark flies

eclosion The circadian clock provides the temporal framework for rhythmic behavioral and metabolic functions. In the modern era of industrialization, work, and social pressures, clock function is jeopardized, and can result in adverse and chronic effects on health. Understanding circadian clock function, particularly individual variation in diurnal phase preference (chronotype), and the molecular mechanisms underlying such chronotypes may lead to interventions that could abrogate clock dysfunction and improve human (and animal) health and welfare. In this paper I explored difference in expression between early rising "larks" or late rising "owls,".
  Gene Expression Associated with Early and Late Chronotypes in Drosophila melanogaster ( Pegoraro et al, 2015).

Testing the Bunning Hypothesis

Waking_Flies A major question in chronobiology focuses around the "Bunning hypothesis" which implicates the circadian clock in photoperiodic (day-length) measurement and is supported in some systems (e.g. plants) but disputed in others. Here, we used the seasonally-regulated thermotolerance of Drosophila melanogaster to test the role of various clock genes in daylength measurement. In Drosophila, freezing temperatures induce reversible chill coma, a narcosis-like state. We have corroborated previous observations that wild-type flies developing under short photoperiods (winter-like) exhibit significantly shorter chill-coma recovery times (CCRt) than flies that were raised under long (summer-like) photoperiods. Here, we show that arrhythmic mutant strains, per01, tim01 and ClkJrk, as well as variants that speed up or slow down the circadian period, disrupt the photoperiodic component of CCRt. Our results support an underlying circadian function mediating seasonal daylength measurement and indicate that clock genes are tightly involved in photo- and thermoperiodic measurements.
  Role for Circadian Clock Genes in Seasonal Timing ( Pegoraro et al, 2014).

DNA Methylation and Photoperiodic timing

Nasonia vitripennis Many organisms use the seasonal change in photoperiod to tune their seasonal response. The molecular basis of the seasonal timer is still elusive. We use the parasitic wasp Nasonia vitripennis to investigate the role of DNA methylation in the photoperiodic timing. Nasonia is a typical long day insect where short day and cold temperature induce female wasps to produce progeny that remains in diapause. The Nasonia genome has been recently sequenced reveling a mammalian like kit of DNA methylation providing the opportunity to test whether DNA methylation is involved in photoperiodic timing. We show that changes in photoperiod, induce changes in DNA methylation and that disrupting the normal level of expression of DNA methyltransferase 1 (DNMT1a) by dRNAi, as well as blocking DNA methylation pharmacologically greatly affect the photoperiodic diapause response of the wasps.
  DNA methylation in Nasonia vitripennis ( Park et al, 2011).
  DNA methylation changes induced by long and short photoperiods in Nasonia ( Pegoraro et al, 2015 ).

microRNA

how  do mirnas work MicroRNA (miRNA) is a recently discovered new class of small RNA molecules that have a significant role in regulating gene and protein expression. These small RNAs (approximately 22 nt) bind to 3' untranslated regions (3'UTRs) and induce degradation or repression of translation of their mRNA targets. Hundreds of miRNAs have been identified in various organisms and have been shown to play a significant role in development and normal cell functioning. Recently, a few studies have suggested that miRNAs may be an important regulators of circadian rhythmicity, providing a new dimension (posttranscriptional) of our understanding of biological clocks. Corrently, I am investigating the role of miRNA both in the circadian and photoperiodic systems of Drosophila melanogaster. Under the supervision of Dr Eran Tauber, I am studing mirna functions with a global approach (microarray) and a more specific approach (real time pcr and in situ hybridization).
  Review on mirna (Pegoraro and Tauber 2008).

Molecular clock and timeless

drosophila molecular clock The circadian clock may be evolved in response of early needs to anticipate enviromental changes. Even today is possible to see the effect of natural selection on circadian clock genes like period and timeless in Drosophila melanogaster. A natural length polimorphism in the timeless gene affects the incidence of diapause, probably enhancing fly's adaptation to seasonal changes.
  tim paper (Sandrelli et al. 2007).
  tim paper (Tauber et al. 2007).

Cryptochrome

drosophila molecular clock Genetic variations in circadian clock genes may serve as molecular adaptations, allowing populations to adapt to local environments. Here, we carried out a survey of genetic variation in Drosophila cryptochrome (cry), the fly's dedicated circadian photoreceptor. An initial screen of 10 European cry alleles revealed substantial variation, including seven nonsynonymous changes. The SNP frequency spectra and the excessive linkage disequilibrium in this locus suggested that this variation is maintained by natural selection. We focused on a non-conservative SNP involving a leucine - histidine replacement (L232H) and found that this polymorphism is common, with both alleles at intermediate frequencies across 27 populations surveyed in Europe, irrespective of latitude. Remarkably, we were able to reproduce this natural observation in the laboratory using replicate population cages where the minor allele frequency was initially set to 10%. Within 20 generations, the two allelic variants converged to approximately equal frequencies. Further experiments using congenic strains, showed that this SNP has a phenotypic impact, with variants showing significantly different eclosion profiles. At the long term, these phase differences in eclosion may contribute to genetic differentiation among individuals, and shape the evolution of wild populations.
  CRYL232H paper (Pegoraro et al. 2014).

Photoperiodism "Diapause"

drosophila molecular clock Diapause is a protective response to unfavorable environments that results in a suspension of insect development and is most often associated with the onset of winter. The ls-tim mutation in the Drosophila melanogaster clock gene timeless has spread in Europe over the past 10,000 years, possibly because it enhances diapause. Studing distribution of alleles of genes involved in diapause response like timeless and cryptochrome is possible to elucidate the evolution of this facinating process. The diapause response of populations distributed along Europe can be extremely important in order to better understand climate changes. In fact in a global warming enviroment we'll aspect a southernization of the diapause response with a selection of "southern alleles".
  Review on diapause (Emerson et al. 2009).

per and Bombyx mori

bombyx mori I have been involved in the process of knocking down the gene period in the lepidopteran Bombyx mori. The period post-translational silencing induced a disruption of egg-hatching rhythm, as well as a reduction in egg-to-adult developmental time, without altering silk production parameters. These could be a practical way to shortening the rearing time without reducing silk production.
  B.mori paper (Sandrelli et al. 2007).

Silencing Surf1

silencing Surf1 Mutations in Surf1, a human gene involved in the assembly of cytochrome c oxidase (COX), cause Leigh syndrome, the most common infantile mitochondrial encephalopathy, characterized by a specific COX deficiency. We report the generation and characterization of functional knockdown (KD) lines for Surf1 in Drosophila melanogaster. The results reported in Zordan et al. 2006 indicate important functions for SURF1 specifically related to COX activity and suggest a crucial role of mitochondrial energy pathways in organogenesis and CNS development and function.
  Silencing Surf1 (Zordan et al. 2006).
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© 2009 Dr Mirko Pegoraro
mp248@le.ac.uk
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