Molecular interactions: metal ions and protein chaperones in the urease system from Helicobacter pylori

Although nickel is a toxic metal for living organisms in its soluble form, its importance in many biological processes recently emerged. In this view, the investigation of the nickel-dependent enzymes urease and [NiFe]-hydrogenase, especially the mechanism of nickel insertion into their active sites, represent two intriguing case studies to understand other analogous systems and therefore to lead to a comprehension of the nickel trafficking inside the cell. Moreover, these two enzymes have been demonstrated to ensure survival and colonization of the human pathogen H. pylori, the only known microorganism able to proliferate in the gastric niche. The right nickel delivering into the urease active site requires the presence of at least four accessory proteins, UreD, UreE, UreF and UreG. Similarly, analogous process is principally mediated by HypA and HypB proteins in the [NiFe]-hydrogenase system. Indeed, HpHypA and HpHypB also have been proposed to act in the activation of the urease enzyme from H. pylori, probably mobilizing nickel ions from HpHypA to the HpUreE-HpUreG complex. A complete comprehension of the interaction mechanism between the accessory proteins and the crosstalk between urease and hydrogenase accessory systems requires the determination of the role of each protein chaperone that strictly depends on their structural and biochemical properties. The availability of HpUreE, HpUreG and HpHypA proteins in a pure form is a pre-requisite to perform all the subsequent protein characterizations, thus their purification was the first aim of this work. Subsequently, the structural and biochemical properties of HpUreE were investigated using multi-angle and quasi-elastic light scattering, as well as NMR and circular dichroism spectroscopy. The thermodynamic parameters of Ni2+ and Zn2+ binding to HpUreE were principally established using isothermal titration calorimetry and the importance of key histidine residues in the process of binding metal ions was studied using site-directed mutagenesis. The molecular details of the HpUreE-HpUreG and HpUreE-HpHypA protein-protein assemblies were also elucidated. The interaction between HpUreE and HpUreG was investigated using ITC and NMR spectroscopy, and the influence of Ni2+ and Zn2+ metal ions on the stabilization of this association was established using native gel electrophoresis, light scattering and thermal denaturation scanning followed by CD spectroscopy. Preliminary HpUreE-HpHypA interaction studies were conducted using ITC. Finally, the possible structural architectures of the two protein-protein assemblies were rationalized using homology modeling and docking computational approaches. All the obtained data were interpreted in order to achieve a more exhaustive picture of the urease activation process, and the correlation with the accessory system of the hydrogenase enzyme, considering the specific role and activity of the involved protein players. A possible function for Zn2+ in the chaperone network involved in Ni2+ trafficking and urease activation is also envisaged.

An evolutionary approach to the design of experiments for combinatorial optimization with an application to enzyme engineering

In a large number of problems the high dimensionality of the search space, the vast number of variables and the economical constrains limit the ability of classical techniques to reach the optimum of a function, known or unknown. In this thesis we investigate the possibility to combine approaches from advanced statistics and optimization algorithms in such a way to better explore the combinatorial search space and to increase the performance of the approaches. To this purpose we propose two methods: (i) Model Based Ant Colony Design and (ii) Naïve Bayes Ant Colony Optimization. We test the performance of the two proposed solutions on a simulation study and we apply the novel techniques on an appplication in the field of Enzyme Engineering and Design.

Dynamic stabilization of Rayleigh-Taylor instability of ablation fronts in inertial confinement fusion

One of the most important problems in inertial confinement fusion is how to find a way to mitigate the onset of the Rayleigh-Taylor instability which arises in the ablation front during the compression. In this thesis it is studied in detail the possibility of using for such a purpose the well-known mechanism of dynamic stabilization, already applied to other dynamical systems such as the inverted pendulum. In this context, a periodic acceleration superposed to the background gravity generates a vertical vibration of the ablation front itself. The effects of different driving modulations (Dirac deltas and square waves) are analyzed from a theoretical point of view, with a focus on stabilization of ion beam driven ablation fronts, and a comparison is made, in order to look for optimization.

Land use change to perennial energy crops in Northern Italy: Effects on soil organic carbon sequestration and distribution, soil enzyme activities and microbial communities.

Studies on soil organic carbon (SOC) sequestration in perennial energy crops are available for North-Central Europe, while there is insufficient information for Southern Europe. This research was conducted in the Po Valley, a Mediterranean-temperate zone characterised by low SOC levels, due to intensive management. The aim was to assess the factors influencing SOC sequestration and its distribution through depth and within soil fractions, after a 9-year old conversion from two annual systems to Miscanthus (Miscanthus × giganteus) and giant reed (Arundo donax). The 13C natural abundance was used to evaluate the amount of SOC in annual and perennial species, and determine the percentage of carbon derived from perennial crops. SOC was significantly higher under perennial species, especially in the topsoil (0-0.15 m). After 9 years, the amount of C derived from Miscanthus was 18.7 Mg ha-1, mostly stored at 0-0.15 m, whereas the amount of C derived from giant reed was 34.7 Mg ha-1, evenly distributed through layers. Physical soil fractionation was combined with 13C abundance analysis. C derived from perennial crops was mainly found in macroaggregates. Under giant reed, more newly derived-carbon was stored in microaggregates and mineral fraction than under Miscanthus. A molecular approach based on denaturing gradient gel electrophoresis (DGGE) allowed to evaluate changes on microbial community, after the introduction of perennial crops. Functional aspects were investigated by determining relevant soil enzymes (β-glucosidase, urease, alkaline phosphatase). Perennial crops positively stimulated these enzymes, especially in the topsoil. DGGE profiles revealed that community richness was higher in perennial crops; Shannon index of diversity was influenced only by depth. In conclusion, Miscanthus and giant reed represent a sustainable choice for the recovery of soils exhausted by intensive management, also in Mediterranean conditions and this is relevant mainly because this geographical area is notoriously characterised by a rapid turnover of SOC.

Natural compounds Camptothecin and Triptolide: highly specific enzyme inhibitors and tools to dissect transcriptional functions

With this work I elucidated new and unexpected mechanisms of two strong and highly specific transcription inhibitors: Triptolide and Campthotecin. Triptolide (TPL) is a diterpene epoxide derived from the Chinese plant Trypterigium Wilfoordii Hook F. TPL inhibits the ATPase activity of XPB, a subunit of the general transcription factor TFIIH. In this thesis I found that degradation of Rbp1 (the largest subunit of RNA Polymerase II) caused by TPL treatments, is preceded by an hyperphosphorylation event at serine 5 of the carboxy-terminal domain (CTD) of Rbp1. This event is concomitant with a block of RNA Polymerase II at promoters of active genes. The enzyme responsible for Ser5 hyperphosphorylation event is CDK7. Notably, CDK7 downregulation rescued both Ser5 hyperphosphorylation and Rbp1 degradation triggered by TPL. Camptothecin (CPT), derived from the plant Camptotheca acuminata, specifically inhibits topoisomerase 1 (Top1). We first found that CPT induced antisense transcription at divergent CpG islands promoter. Interestingly, by immunofluorescence experiments, CPT was found to induce a burst of R loop structures (DNA/RNA hybrids) at nucleoli and mitochondria. We then decided to investigate the role of Top1 in R loop homeostasis through a short interfering RNA approach (RNAi). Using DNA/RNA immunoprecipitation techniques coupled to NGS I found that Top1 depletion induces an increase of R loops at a genome-wide level. We found that such increase occurs on the entire gene body. At a subset of loci R loops resulted particularly stressed after Top1 depletion: some of these genes showed the formation of new R loops structures, whereas other loci showed a reduction of R loops. Interestingly we found that new peaks usually appear at tandem or divergent genes in the entire gene body, while losses of R loop peaks seems to be a feature specific of 3’ end regions of convergent genes.