ADAPTATIVE EVOLUTION OF THE HEAT SHOCK RESPONSE IN ANTARCTIC CILIATES\ud

During my PhD, the general objective of my work was to\ud contribute to a better knowledge of the molecular basis of Heat Shock Response in eukaryotic microorganisms. In particular, I was interested in analyzing the plasticity of hsp70 gene expression in marine species of ciliates inhabiting the cold and thermally stable coastal sea water of Antarctica. Useful organism models for analysis of adaptation of gene expression were represented by two Antarctic species of Euplotes, E. focardii andE. nobilii, which showed up marked differences in their capacity\ud to activate hsp70 gene expression in response to thermal insults.\ud The specific aims of my PhD projects were:\ud 1. To perform a comparative analysis of hsp70 gene\ud structures (at level of coding and non-coding, regulatory\ud regions) between the two Antarctic species of Euplotes, E.\ud focardii and E. nobilii, with the ultimate goal to identify the causes of the unresponsiveness to thermal stress in E.\ud focardi. (Chapter 1)\ud 2. To perform a comparative analysis of hsp70 gene\ud structures (at level of coding and non coding, regulatory\ud regions) and a preliminary examination of the DNA-binding\ud activity of Heat Shock Factors by means of Electrophoretic\ud Mobility Shift Assay approaches in E. nobili and E. raikovi a species inhabiting temperate waters and thus, adapted to\ud a fluctuating thermal environment. The possibility to\ud compare the transcriptional machinery of closely related\ud species adapted to different thermal regimes, might\ud provide valuable clues for unraveling the molecular\ud mechanisms of transcription in the cold. (Chapter 2)\u

UV Radiation and Visible Light Induce hsp70 Gene Expression in the Antarctic Psychrophilic Ciliate Euplotes focardii

The psychrophilic ciliate Euplotes focardii inhabits the shallow marine coastal sediments of Antarctica, where, over millions of years of evolution, it has reached a strict molecular adaptation to such a constant-temperature environment (about −2 °C). This long evolution at sub-zero temperatures has made E. focardii unable to respond to heat stress with the activation of its heat shock protein (hsp) 70 genes. These genes can, however, be expressed in response to other stresses, like the oxidative one, thus indicating that the molecular adaptation has exclusively altered the heat stress signaling pathways, while it has preserved hsp70 gene activation in response to other environmental stressors. Since radiative stress has proved to be affine to oxidative stress in several organisms, we investigated the capability of UV radiation to induce hsp70 transcription. E. focardii cell cultures were exposed to several different irradiation regimes, ranging from visible only to a mixture of visible, UV-A and UV-B. The irradiation values of each spectral band have been set to be comparable with those recorded in a typical Antarctic spring. Using Northern blot analysis, we measured the expression level of hsp70 immediately after irradiation (0-h-labeled samples), 1 h, and 2 h from the end of the irradiation. Surprisingly, our results showed that besides UV radiation, the visible light was also able to induce hsp70 expression in E. focardii. Moreover, spectrophotometric measurements have revealed no detectable endogenous pigments in E. focardii, making it difficult to propose a possible explanation for the visible light induction of its hsp70 genes. Further research is needed to conclusively clarify this point. Antonietta La Terza and Roberto Marangoni are joint last authors

Rule based modeling of gene regulation and biosynthesis of tryptophan in E. coli

The genetic regulation of the Trp operon in the bacterium E. coli relies on a sophisticated control mechanism. It tightly couples the advance of transcribing RNA polymerase to the efficiency of the contemporaneous translation of the nascent transcript by a ribosome. The concurrent control of this process involves interdependencies between multiple molecular actors. Within process algebra based modeling languages focused on pairwise interaction, its representation required sophisticated coding tricks. In this work, we abstract the mechanism of transcriptional attenuation within a novel rule base modeling language. It allows non-trivial concurrent control by representing molecules as parametrized terms

Adaptive evolution of the heat-shock response in the Antarctic psychrophilic ciliate, Euplotes focardii: hints from a comparative determination of the hsp70 gene structure

The Antarctic psychrophilic ciliate Euplotes focardii manifests a dramatic reduction in the activation of its hsp70 gene in response to a heat-shock, while oxidative and chemical stresses activate the transcription of this gene to appreciable extents. To investigate the genetic causes of this eccentric behaviour of E. focardii in the hsp70 gene transcription activation, we carried out a comparative structural analysis of this gene between E. focardii and another Antarctic Euplotes, E. nobilii, which manifests a psychrotrophic behaviour and an inducible thermal response. No substantial difference was detected in the organization of the hsp70 5’ promoter region, both species bearing canonical regulatory cis-acting elements deputed to bind transcriptional trans-activating factors. Adenine-rich elements favouring mRNA degradation were instead detected in the hsp70 3’ regulatory region of E. nobilii, but not in that of E. focardii. These observations lend further support to the hypothesis that the causes of the Euplotes focardii unresponsiveness to thermal stress resides in some structural, or functional modifications of transcriptional trans-activating factors