Investigating the role of single point mutations in the human proteome: a computational study

In the post genomic era with the massive production of biological data the understanding of factors affecting protein stability is one of the most important and challenging tasks for highlighting the role of mutations in relation to human maladies. The problem is at the basis of what is referred to as molecular medicine with the underlying idea that pathologies can be detailed at a molecular level. To this purpose scientific efforts focus on characterising mutations that hamper protein functions and by these affect biological processes at the basis of cell physiology. New techniques have been developed with the aim of detailing single nucleotide polymorphisms (SNPs) at large in all the human chromosomes and by this information in specific databases are exponentially increasing. Eventually mutations that can be found at the DNA level, when occurring in transcribed regions may then lead to mutated proteins and this can be a serious medical problem, largely affecting the phenotype. Bioinformatics tools are urgently needed to cope with the flood of genomic data stored in database and in order to analyse the role of SNPs at the protein level. In principle several experimental and theoretical observations are suggesting that protein stability in the solvent-protein space is responsible of the correct protein functioning. Then mutations that are found disease related during DNA analysis are often assumed to perturb protein stability as well. However so far no extensive analysis at the proteome level has investigated whether this is the case. Also computationally methods have been developed to infer whether a mutation is disease related and independently whether it affects protein stability. Therefore whether the perturbation of protein stability is related to what it is routinely referred to as a disease is still a big question mark. In this work we have tried for the first time to explore the relation among mutations at the protein level and their relevance to diseases with a large-scale computational study of the data from different databases. To this aim in the first part of the thesis for each mutation type we have derived two probabilistic indices (for 141 out of 150 possible SNPs): the perturbing index (Pp), which indicates the probability that a given mutation effects protein stability considering all the “in vitro” thermodynamic data available and the disease index (Pd), which indicates the probability of a mutation to be disease related, given all the mutations that have been clinically associated so far. We find with a robust statistics that the two indexes correlate with the exception of all the mutations that are somatic cancer related. By this each mutation of the 150 can be coded by two values that allow a direct comparison with data base information. Furthermore we also implement computational methods that starting from the protein structure is suited to predict the effect of a mutation on protein stability and find that overpasses a set of other predictors performing the same task. The predictor is based on support vector machines and takes as input protein tertiary structures. We show that the predicted data well correlate with the data from the databases. All our efforts therefore add to the SNP annotation process and more importantly found the relationship among protein stability perturbation and the human variome leading to the diseasome.

Pharmaceutical residues in aquatic systems: mode of action and effects on mussel physiology

Pharmaceutical residues contaminate aquatic ecosystems as a result of their widespread human and veterinary usage. Since continuously released and not efficiently removed, certain pharmaceuticals exhibit pseudo-persistence thus generating concerns for the health of aquatic wildlife. This work aimed at assessing on mussels Mytilus galloprovincialis, under laboratory conditions, the effects of three pharmaceuticals, carbamazepine (antiepileptic), propranolol (β-blocker) and oxytetracycline (antibiotic), to evaluate if the human-based mode of action of these molecules is conserved in invertebrates. Furthermore, in the framework of the European MEECE Programme, mussels were exposed to oxytetracycline and copper at increasing temperatures, simulating variations due to climate changes. The effects of these compounds were assessed evaluating a battery of biomarkers, the expression of HSP70 proteins and changes in cAMP-related parameters. A decrease in lysosomal membrane stability, induction of oxidative stress, alterations of cAMP-dependent pathway and the induction of defense mechanisms were observed indicating the development of a stress syndrome, and a worsening in mussels health status. Data obtained in MEECE Programme confirmed that the toxicity of substances can be enhanced following changes in temperature. The alterations observed were obtained after exposure to pharmaceuticals at concentrations sometimes lower than those detected in the aquatic environment. Hence, further research is advisable regarding subtle effects of pharmaceuticals on non-target organisms. Furthermore, results obtained during a research stay in the laboratories of Cádiz University (Spain) are presented. The project aimed at measuring possible effects of polluted sediments in Algeciras Bay (Spain) and in Cádiz Bay, by assessing different physiological parameters in caged crabs Carcinus maenas and clams Ruditapes decussatus exposed in situ for 28 days. The neutral red retention assay was adapted to these species and proved to be a sensitive screening tool for the assessment of sediment quality.