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Sviluppo di una piattaforma per la diagnosi prenatale non invasiva di malattie genetiche in epoca gestazionale precoce
Get PDFPrenatal diagnosis of aneuploidies and monogenic diseases is usually performed by
amniocentesis or chorionic villous sampling. However, these procedures are associated
with 0.5%-2% risk of miscarriage.
The discovery of cell free fetal DNA (cffDNA) in maternal plasma in 1997 has provided a
new source of fetal genetic material that can be safely obtained from maternal blood and
successfully processed for non invasive genetic diagnosis (NIPD).
In this study is described a new approach for non invasive prenatal diagnosis of β-
thalassemia which is based on semiconductor sequencing (Ion Torrent PGM) and fetal
haplotype inference. In particular, the approach is based on target sequencing of the
mutation site, the β°39 non sense mutation of the HBB gene, and several informative
SNPs spread in the β-globin gene cluster.
The data analysis of each cffDNA sample and the inference of the most likely inherited
haplotypes were determined by an automated pipeline which firstly constructs the parental
haplotypes, using the sequencing data from the parental DNAs and an haplotype
reference panel previously created. The pipeline, then, quantifies the allele counts
observed in each site sequenced in the corresponding cffDNA sample and finally predicts
the two haplotypes most likely inherited by the fetus using a hidden Markov Model (HMM).
The results were finally compared with the sequencing data of the fetal DNA obtained by
villocentesis.
Using these approaches we have analyzed 30 out of 37 cffDNA samples; in seven
samples, in fact, the pipeline could not proceed because of the lack of informative sites or
of other parameters useful for downstream analysis. The fetal β°39 genotype was correctly
predicted in 24/30 (80%) samples, while it was incorrectly defined in 6/30 (20%) cases.
The incorrect results obtained in these last samples was due to the erroneus inference of
the maternal haplotype. On the contrary, the paternal haplotype was correctly detected in
all 30 samples processed.
In the next future we are planning to improve the protocol by increasing the number of
potentially informative SNPs and to process a higher number of Sardinian β°39 carriers in
order to expand the haplotype reference panel. This haplotype-based approach has given
encouraging results and we think that it could be a starting point for eventual future
application of NIPD also to other monogenic disorders
Identificazione di una variante missenso nel gene RBM10 in una famiglia sarda con disabilità intellettiva X-linked
Get PDFX-linked intellectual disability (XLID) is a heterogeneous disorder, and mutations causing monogenic XLID have now been reported in over 100 genes. We report a five-generation Sardinian family in which seven affected male family members had intellectual disability and craniofacial dysmorphisms. Large-scale next generation exome resequencing of X chromosome genes detected a rare missense variant (c.995G>A, p.Arg332His) located within a highly conserved domain in the RBM10 gene at Xp11.23. Sanger sequencing confirmed the presence of the variant in affected males and in their mothers. The variant was not present in non affected male family members, has not been reported in variant databases and is disease-causing according to a web-based prediction tool. RBM10 is an RNA binding protein involved in the regulation of transcription, alternative splicing and message stabilization. RBM10 nonsense and frameshift mutations are associated with TARP syndrome characterized by Talipes equinovarus, Atrial septal defect, Robin sequence and Persistence of the left superior vena cava and pre- or postnatal lethality in affected males. RBM10 has not been reported in XLID patients until now. Genic intolerance score suggested that RBM10 may be “intolerant” of functional mutations. Although our finding suggest that RBM10 is a reasonable candidate gene for XLID, functional studies and mutations screening in other patients are needed to prove a definite causal relationship between the variant and the phenotype
VGF e stress ossidativo: alterazioni cellulari e modulazione nella Sclerosi Laterale Amiotrofica
Get PDFThe VGF gene encodes a protein precursor (617/615 AA rat/man, respectively), which is processed in vivo to various low MW peptides with different biologic activities. These include attenuation of excytotoxic injury on spinal motor neurons from G93A-SOD-1 mice, upon overexpression of the VGF precursor, or an anti-apoptotic action of the VGF peptide TLQP-21 on cerebellar granules. Recently, the C3a and gC1q have been identified as TLQP-21 receptors. Using the mouse motoneuronal cell line NSC-34 and the primary human fibroblast cell colture, we studied the expression and modulation of certain VGF peptides (VGFp) in ALS and oxidative stress and their possible neuroprotective effect. Oxidative stress was induced with Na Arsenite (SA: 0.5mM, 30/60 min) while the MTT test was used to assess cell viability. Antibodies used (for either ICC or ELISA) were specific for: the N/C-terminus of the VGF precursor, NERP-1, rat PGH, NAPPE-19 and TLQP-21, and for the C3a/gC1q TLQP receptors. Both antibody receptors were also used for ICC and western blot analysis.
In NSC-34, almost all VGFp were selectively localized in the growth cones, perinuclear region, and/or in a specific paranuclear region. Upon oxidative stress, most cells showed a rounded morphology, with loss of growth cones, while almost all VGFp were found mainly in a paranuclear region compatible with the Golgi apparatus.
In ELISA, untreated cellular extracts showed the presence of the majority VGFp with a significant reduction in TLQP and NERP-1 levels versus untreated cells (58% and 65% of reduction, respectively) after SA treatment, while the culture medium showed a marginal increase of the same peptides. Cell viability studies showed a significant protective action of TLQP-21 on stressed neurons (SA alone: 74.7% 0.08, SA and TLQP-21 together: 82.1% 0.08 of untreated controls, p0.018), with no detectable effect for NERP-1.
Results from immunocytochemistry showed the presence in NSC-34 of gC1q receptor only, confirmed also by western blot analysis.
In human fibroblasts, VGFp were selectively localized in citoplasmic and paranuclear region morfologically related to the membranous cellular system, without obvious differences between healthy and ALS subjects (with TARDBPA382T and without known mutation). In ELISA, N-terminus and NERP-1 showed a significant reduction in both groups of ALS patients (TARDBPA382T: 30% and 61% of reduction, respectively; ALS without known mutation: 37% and 62% of reduction, respectively) versus healty controls, while TLQP-21 and C-terminus showed a reduction only in certain ALS patients (TLQP-21: 37% of reduction, for ALS without known mutation; C-term: 62% of reduction, only for ALS TARDBPA382T) versus healty cells.
In conclusion, the VGFp studied are present in NSC-34, but only TLQPp are modulated and secreted after oxidative stress, with a potential neuroprotective role. In fibroblasts, VGFp showed a significant reduction in ALS cell extracts. Hence, the research in the VGF field using NSC-34 could open new perspectives for potential therapeutic tools, while the results obtained through fibroblasts could be helpful for diagnostic purposes
Mediation analysis for different types of Causal questions: Effect of Cause and Cause of Effect
Get PDFMany statistical analyses aim at a causal explanation of the data. When discussing
this topic it is important to specify the exact query we want to talk about. A
typical causal question can be categorized in two main classes: questions on the
causes of observed effects and questions on the effects of observed causes. In this
dissertation we consider both EoC and CoE causal queries from a particular perspective
that is Mediation. Mediation Analysis aims to disentangle the pathway
between exposure and outcome on a direct effect and an indirect effect arising from
the chain exposure-mediator-outcome. In the EoC framework, if the goal is to measure
the causal relation between two variables when a third is involved and plays
the role of mediator, it is essential to explicitly define several assumptions among
variables. However if any of these assumptions is not met, estimates of mediating
effects may be affected by bias. This phenomenon, known with the name of Birth
Weight paradox, has been explained as a consequence of the presence of unmeasured
confounding between the mediator and the outcome. In this thesis we discuss these
apparent paradoxical results in a real dataset. In addition we suggest useful graphical
sensitivity analysis techniques to explain the potential amount of bias capable
of producing these paradoxical results. From a CoE perspective, given empirical
evidence for the dependence of an outcome variable on an exposure variable, we
can typically only provide bounds for the “probability of causation” in the case of
an individual who has developed the outcome after being exposed. We show how
these bounds can be adapted or improved if further information becomes available.
In addition to reviewing existing work on this topic, we provide a new analysis for
the case where a mediating variable can be observed. In particular we show how
the probability of causation can be bounded in two different cases of partial and
complete mediation
Enhanced sampling methods and their application in the study of molecular permeation in gram-negative bacteria
Get PDFAntimicrobial resistance is inhibiting our ability to fight against pathogens. By selectively changing the composition and expression of influx water-filled proteins filling their outer membrane, gram- negative bacteria are able to reduce the rates at which specific polar compounds are able to permeate. A clear comprehension of the mechanism determining substrates diffusion through these pores is still missing. In this thesis, we show how biased computer simulations may offer a unique perspective in the study of molecular permeation through porins, overcoming the intrinsic limitations of both experimental techniques and standard molecular dynamics. The first test-case is Acinetobacter baumannii’s CarO. The use of substrates with varying charge and molecular weight, as well as the creation of a loop-less mutant missing the extracellular domain of the protein, allowed to determine the charge selectivity and the transition rates of polar molecules. We obtained good agreement with the results of liposome swelling assays experiments. Further, we compared the passage of two carbapenem antibiotics in a series of mutated proteins extracted from a patient undergoing long term hospital infection. We connected the mutation of few key residues to a drastic change in the internal electric field of the proteins, showing that the antibiotics follow the choreography of water molecules inside the channels. In the last section, we present a kinetic model that allows to determine for a molecule the relative probability of different conformations and the time required for the translocation through a pore. This approach allowed to connect the results of enhanced sampling MD methods with current blockages in single channel experiments.All these results together show that multiscale MD techniques can offer an exhaustive view on the mechanism of molecular diffusion through pores, helping to understand the most important charac- teristics that determine the rates of translocation of different com- pounds in gram-negative bacteria. We can use these data to com- plement experimental results and to design the next generation of antibiotics
Per un nuovo rapporto tra P.A. e cittadini nel governo dei beni comuni: "Dalla tecnologia digitale alla tecnologia istituzionale
Get PDFThis work aims to explore the theme of the commons , horizontal subsidiarity and Information and Communication technologies . Realms of a reflection on the development of a new model of Public Administration . The foundations of which are posed by a renewed social need , namely the need for citizens to take responsibility for the management of their common resources . Therefore , as a result of this reflection , it tried to trace a path through the illustration of some concrete experiences , for the definition of a " Subsidiary Paradigm " , in which public institutions work together with the citizens in the management of the commons . We have tried to demonstrate, as the common good , not being linked either to the public or to private property , represent the ideal tool for the realization of shared solutions.
The participatory management initiatives of such goods, thus become, sustainable patterns of economic and social development. This, of course, requires the involvement of citizens in all proceedings relating to the common good, through the persecution of the Principles of Subsidiarity, Transparency, Participation, democracy and equal opportunity. To the success of this model it is essential that P.A., operate with citizens through an orderly path to insights that must necessarily start from knowledge, which must be constructed, inclusive, widespread and integrated. It moves, therefore, as part of the dynamics of the Information and Public Communication, which is crucial to invest to bring the system of E-Government processes. On the Government Theme of common goods, information and communication have a key responsibility in the sense of power and ability to affect the construction of a shared relationship in the public interest realization. In this sense, they are drawn to positive and critical of ITC tools in our administrative system in order to define a strategy for Information and Communication, aimed at effective sharing of public choices. It sought to identify those useful corrective to build a subsidiary relationship , taking into account all the factors liable to the growth of a conscious citizenship of their responsibilities and their rights . As we see , then, if there is a starting point of this reflection , it is the awareness of a "disease " of our current administrative system , and just analyzing this disease , has attempted to define a possible cure , that you have chosen to locate in the introduction of a " Technology Institutional " in key subsidiary , which can effectively adapt to digital ,at the end of a real and conscious participation of citizens in decision-making and management of their common resources
Design and synthesis of photoactive molecules based on metal dithiolenes
Get PDFThis work reports on the design, synthesis and characterization of some homoleptic and heteroleptic
d8 metal (Ni, Pd, Pt) dithiolene complexes.
Chapter 1 provides the background required to properly design metal dithiolenes exhibiting desired
properties.
Chapter 2 describes the synthesis & characterization of homoleptic radical monoanionic complexes
showing multi-properties. Among these, a platinum complex prepared using a ligand containing a
quinoxaline moiety connected through a dithieno bridge to the dithiolene core, shows unusual anti-
Kasha solution proton dependent luminescence. The interaction of protons with these complexes is
described with a view to understand their photo/electrocatalytic behaviour in aqueous acidic
systems. Photocatalytic preliminary experiments on noble metal free homoleptic Ni-complex for
hydrogen production from aqueous acidic solutions are described.
Chapter 3 describes design, synthesis and characterization of chiral/achiral heteroleptic d8 metal
dithiolene complexes, suitable as donor-metal-acceptor chromophores for 2nd order NLO
applications. It describes how with the presence or absence of acid (protons) in solutions of
complexes can afford two switchable chemical forms (having different electronic properties) which
can possibly display contrast in their NLO response. It also describes, by inclusion of chirality in the
acceptor ligand, a non-centrosymmetric arrangement of molecules in crystalline medium to preserve
NLO response in the bulk. Moreover, the unusual proton dependent dual luminescent properties of
some of these complexes are described.
Lastly Chapter 4 contains conclusions, perspectives and appendices for this work
Social Media Geographic Information (SMGI): opportunities for spatial planning and governance
Get PDFThe dissertation concerns the opportunities arising from the use of social media platforms as an information resource for supporting design, analysis and decision-making in spatial planning. The widespread diffusion of Web 2.0 technologies and tools such as geobrowsers, Application Programming Interfaces (API), GPS-enabled mobile devices, and recently Location-Based Social Networks are fostering the production, collection and sharing of georeferenced information by the Internet users, namely Volunteered Geographic Information (VGI) and Social Media Geographic Information (SMGI), which are not only related to measures of the geographical component, but also to user perceptions and opinions on places, localities and daily-routine events.
The wealth of VGI and SMGI freely available through the Internet may affect current practices in regional and urban planning, offering opportunities for real-time monitoring of needs, thoughts and trends of local communities. However, several hurdles related to data accessibility and management, as well as to knowledge extraction are limiting a wider use of SMGI in practice. In the light of the above premises, the research goal is to address the different aspects required for properly using VGI and SMGI within the urban and regional planning domains.
The methodological approach is developed following two main directions. First, the approach builds on the design and development of ad-hoc tools able to deal with the issues regarding the access, management and analysis of SMGI. Second, the dissertation formalizes a novel analytical framework, called SMGI Analytics, which enables the proficient use of this information in different planning scenarios. Several case studies are discussed in order to evaluate the value of both the developed tools and the proposed framework. Then, the SMGI Analytics framework is applied on a case study concerning the municipality of Cagliari in Sardinia (Italy) investigating and characterizing a specific public space.
Finally, the dissertation proposes a critical discussion about the developed tools and instruments’ effectiveness for eliciting knowledge from SMGI. The discussion ends identifying the potentialities of obtained findings to address diverse questions related to spatial planning
New HCI techniques for better living through technology
Get PDFIn the Human Computer Interaction community, researchers work on many
projects that investigate the efficacy of new technologies for better living,
but unlike other research fields, these researchers must have an approach
that is typically multi-disciplinary.
Technology is always developing thus improving our lives in many ways
like education, health and communication. This due to the fact that it is
supposed to make life easier.
This dissertation explores three main aspects: the first is learning with
new technologies, the second is the improvement of real life by using innovative
devices while the third is the usage of mobile devices in combination
with image processing algorithms and computer graphics techniques.
We firstly describe the progress on the state of the art and related work
that have been necessary to implement such tools on commodity hardware
and deploy them in both mobile and desktop settings.
We propose the usage of different technologies in different settings, comparing
these solutions for enhancing the interaction experience by introducing
virtual/augmented reality tools for supporting this kind of activities.
We also applied well-known gamification techniques coming from different
mobile applications for demonstrating how users can be entertained and
motivated in their working out.
We describe our design and prototype of several integrated systems created
to improve the educational process, to enhance the shopping experience,
to provide new experiences for travellers and even to improve fitness
and wellness activities.
Finally, we discuss our findings and frame them in the broader context
of better living thanks to technology, drawing the lessons learnt from each
work while also proposing relative future work
Terapia genica della β Talassemia mediante editing del DNA
No full textβ (HBB) gene, resulting in absence (β0) or deficiency (β+) of β globin chain synthesis. This genetic disorder occurs most frequently in people from Mediterranean countries, such as Italy. In particular, the data indicates that about 12.6% of the Sardinian subjects are carriers of β thalassemia and these are among the highest frequencies of thalassemia genes found in a Caucasian population. In Sardinia, the disease is generally determined by a nonsense mutation at codon 39 (E39X) of exon 2 causing the interruption of β globin synthesis. Patients homozygous with E39X mutation have a severe anemia and require frequent transfusions and iron chelation. The only definitive cure today possible for β chain hemoglobinopathies is the hematopoietic stem cells transplantation, but it is limited by availability of HLA matched donors. However, in the last few years new therapeutic approaches for this genetic disease are taking place. The correction of disease-causing mutation through the technique of Genome-Editing in patient-specific stem cells and subsequent autologous transplantation would be the ideal approach for the treatment of monogenic diseases such as β thalassemia. However, due to difficulties in obtaining sufficient homologous recombination percentages for therapeutic purposes, the aim of my PhD project is to reproduce artificially the HPFH mutations identified in non-coding regions of the β globin cluster, using the system CRISPR/Cas9 associated with NHEJ pathway. In this way, we hope to restore at therapeutic levels the expression of HBG genes and consequently the synthesis of a functional HbF in order to ameliorate the phenotype of β thalassemia