Molecular genetics of inherited cystic kidney diseases: new diagnostic approaches

Background. Hhereditary cystic kidney diseases are a heterogeneous spectrum of disorders leading to renal failure. Clinical features and family history can help to distinguish the recessive from dominant diseases but the differential diagnosis is difficult due the phenotypic overlap. The molecular diagnosis is often the only way to characterize the different forms. A conventional molecular screening is suitable for small genes but is expensive and time-consuming for large size genes. Next Generation Sequencing (NGS) technologies enables massively parallel sequencing of nucleic acid fragments. Purpose. The first purpose was to validate a diagnostic algorithm useful to drive the genetic screening. The second aim was to validate a NGS protocol of PKHD1 gene. Methods. DNAs from 50 patients were submitted to conventional screening of NPHP1, NPHP5, UMOD, REN and HNF1B genes. 5 patients with known mutations in PKHD1 were submitted to NGS to validate the new method and a not genotyped proband with his parents were analyzed for a diagnostic application. Results. The conventional molecular screening detected 8 mutations: 1) the novel p.E48K of REN in a patient with cystic nephropathy, hyperuricemia, hyperkalemia and anemia; 2) p.R489X of NPHP5 in a patient with Senior Loken Syndrome; 3) pR295C of HNF1B in a patient with renal failure and diabetes.; 4) the NPHP1 deletion in 3 patients with medullar cysts; 5) the HNF1B deletion in a patient with medullar cysts and renal hypoplasia and in a diabetic patient with liver disease. The NGS of PKHD1 detected all known mutations and two additional variants during the validation. The diagnostic NGS analysis identified the patient’s compound heterozygosity with a maternal frameshift mutation and a paternal missense mutation besides a not transmitted paternal missense mutation. Conclusions. The results confirm the validity of our diagnostic algorithm and suggest the possibility to introduce this NGS protocol to clinical practice.

Nature and relevance of inherited blueprints on business models choices and innovation: an assessment

One important metaphor, referred to biological theories, used to investigate on organizational and business strategy issues is the metaphor about heredity; an area requiring further investigation is the extent to which the characteristics of blueprints inherited from the parent, helps in explaining subsequent development of the spawned ventures. In order to shed a light on the tension between inherited patterns and the new trajectory that may characterize spawned ventures’ development we propose a model aimed at investigating which blueprints elements might exert an effect on business model design choices and to which extent their persistence (or abandonment) determines subsequent business model innovation. Under the assumption that academic and corporate institutions transmit different genes to their spin-offs, we hence expect to have heterogeneity in elements that affect business model design choices and its subsequent evolution. This is the reason why we carry on a twofold analysis in the biotech (meta)industry: under a multiple-case research design, business model and especially its fundamental design elements and themes scholars individuated to decompose the construct, have been thoroughly analysed. Our purpose is to isolate the dimensions of business model that may have been the object of legacy and the ones along which an experimentation and learning process is more likely to happen, bearing in mind that differences between academic and corporate might not be that evident as expected, especially considering that business model innovation may occur.

The genetic history of Italians: new-insights from uniparentally-inherited markers

Genetic differences among human groups can be ascribed both to the broad-scale extents of pre-historical and historical migrations and to the fine-scale impacts of socio-cultural and geographic heterogeneity. In this thesis, the genetic information provided by uniparental markers were exploited to address different aspects of the Italian population history, by combining macro- and micro-geographic investigations at different spatial and temporal scales. To firstly assess the overall Italian variability, Y-chromosome and mtDNA markers were deeply typed in ~900 individuals from continental Italy, Sicily and Sardinia. Sex-biased patterns and contrasting demographic histories were observed for males and females. Differential European and Mediterranean contributions were invoked to explain the paternal genetic sub-structure observed in peninsular Italy, compared to the homogeneous maternal genetic landscape. If Neolithic showed to be one principal determinant of the detected paternal structure, local insights into specific Italian regional contexts highlighted the importance of Post-Neolithic contributions. Among them, migrations from the Balkans (particularly Greece) during late Metal Ages, played a relevant role in the cultural and genetic transitions occurred in Sicily and Southern Italy. On a finer geographic and temporal perspective, the more recent layers of Italian genetic history and some aspects of the gene-culture interaction were assessed by exploring the genetic variability within two “marginal populations”: Arbereshe of Southern Italy and Partecipanza in Northern Italy. The Arbereshe are Albanian-speaking communities settled in Sicily and Calabria since the end of Middle Ages. Despite sharing common genetic and cultural backgrounds, these groups revealed diverging micro-evolutionary histories, implying different founding events and different patterns of cultural isolation and local admixture. Partecipanza is an idiosyncratic institution of Medieval origin aimed at sharing and devolving collective lands. This case-study exemplified that socio-economic stratification within the same population may induce sex-biased genetic structuring and the maintenance of otherwise hidden historical genetic traces.

Urine proteome in animals of veterinary interest: species comparison and new biomarkers of nephropathy

Urine is considered an ideal source of biomarkers, however in veterinary medicine a complete study on the urine proteome is still lacking. The present work aimed to apply proteomic techniques to the separation of the urine proteome in dogs, cats, horses, cows and some non-conventional species. High resolution electrophoresis (HRE) was also validated for the quantification of albuminuria in dogs and cats. In healthy cats, applying SDS-PAGE and 2DE coupled to mass spectrometry (MS), was produced a reference map of the urine proteome. Moreover, 13 differentially represented urine proteins were linked with CKD, suggesting uromodulin, cauxin, CFAD, Apo-H, RBP and CYSM as candidate biomarkers to be investigated further. In dogs, applying SDS-PAGE coupled to MS, was highlighted a specific pattern in healthy animals showing important differences in patients affected by leishmaniasis. In particular, uromodulin could be a putative biomarker of tubular damage while arginine esterase and low MW proteins needs to be investigated further. In cows, applying SDS-PAGE, were highlighted different patterns between heifers and cows showing some interesting changes during pregnancy. In particular, putative alpha-fetoprotein and b-PAP needs to be further investigated. In horses, applying SDS-PAGE, was produced a reference profile characterized by 13±4 protein bands and the most represented one was the putative uromodulin. Proteinuric horses showed the decrease of the putative uromodulin band and the appearance of 2 to 4 protein bands at higher MW and a greater variability in the range of MW between 49 and 17 kDa. In felids and giraffes was quantified proteinuria reporting the first data for UTP and UPC. Moreover, by means of SDS-PAGE, were highlighted species-specific electrophoretic patterns in big felids and giraffes.

Acquired and inherited cardiomyopathies: evaluation of cardiotoxic effects in preclinical models

Cardiomyopathies are a heterogeneous group of myocardial disorders defined by structural and functional alterations of the heart. These cardiac diseases can have both non-genetic and genetic origin. Nevertheless, a different etiology can trigger the same phenotype, as in the case of anthracycline-induced cardiotoxicity and desmin-related cardiomyopathy (DRM). Therefore, the aim of this study was to investigate the cellular mechanisms driving the development of these cardiotoxic conditions in in vitro models. Doxorubicin (DOX) is a commonly used antineoplastic drug for the treatment of a wide range of tumors. Besides, its clinical use is restricted because of dose-dependent cardiotoxicity. Our findings provided evidence that phospholipase C Beta 2 (PLCβ2) may have a critical role in DOX-induced cardiotoxicity in undifferentiated and differentiated H9c2 cell line. Interestingly, the results obtained revealed that cardiomyocytes are less sensitive to DOX, following the evaluation of cellular mechanisms such as: oxidative stress, apoptosis and cell proliferation. Nonetheless, the treatment induced a significant upregulation of PLCβ2 associated to morphological changes in both models, demonstrating the implication in a hypertrophic response. On the other hand, a hereditary DRM was associated to a missense mutation of aB crystallin (CRYAB), a chaperone protein involved in the regulation of the intermediate filament network. Since research has only been conducted on transgenic (TG) mice and neonatal rat cardiomyocytes, this study aimed at investigating cellular mechanisms triggered by CRYABR120G mutation in a hiPSC-derived DRM model. Our model confirmed the impairment of the cytoskeletal organization resulting in the formation of desmin and CRYAB aggregates and myofibril misalignment. Moreover, the missense mutation confirmed a hypertrophic cardiomyopathy phenotype, a feature of DRM patients, on cardiac engineered tissues. Lastly, these data obtained suggest that further research on PLCβ2 and CRYAB are needed to comprehend the molecular mechanisms behind the development of these 2 cardiac diseases.