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Reversible Immortalization and Transfer of a Dystrophin Human Artificial Chromosome into Human Mesoangioblasts: Towards Autologous Cell Therapy of Duchenne Muscular Dystrophy
No full textHuman mesoangioblasts (MABs) are vessel–associated progenitors able to
differentiate into skeletal muscle and to reconstitute the damaged muscle fibers
when injected into a Duchenne Muscular Dystrophy (DMD) mouse model (mdx),
an incurable myopathy for which no therapy currently exists. Because of these
features, human MABs are now entered clinical experimentation based upon
allogeneic transplantation. However autologous cell therapy for DMD still faces
significant limitations, first of all the large size of the dystrophin gene that
hampers its allocation into conventional gene-delivery tools such as viral vectors.
In order to overcome these limitations, a novel approach of autologous
cell therapy to treat DMD was developed by transplanting dystrophic MABs
genetically-corrected with a Human Artificial Chromosome (HAC) containing the
entire dystrophin locus (DYS-HAC). The feasibility of this strategy was already
proved: MABs derived from mdx mouse and genetically corrected with the DYSHAC,
were able to engraft skeletal muscle and restore dystrophin positive fibers
in dystrophic mice, resulting into a significant morphological and functional
amelioration of the phenotype.
In the case of DMD MABs, an additional step of immortalization is
fundamental before DYS-HAC transferring, which requires clonal expansion and
analysis, since they undergo replicative senescence. To reversibly immortalize
normal and dystrophic human MABs, lentiviral vectors encoding floxed hTERT
IRES-HSV1-TK and Bmi-1 have been used.
Normal clones have been characterized for proliferation and proper expression
of hTERT and Bmi-1; they remained growth factor-dependent, contact-inhibited,
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non tumorigenic and myogenic in vitro. Notably, upon transplantation into
dystrophic mice, they engrafted skeletal muscle and restored dystrophin
expression. I then moved to the immortalization of DMD MABs in order to
transfer the DYS-HAC, obtaining immortalized DMD DYS-HAC MABs. These
data set the conditions for future clinical translation of this experimental strategy
for the treatment of the DMD patients.Telethon, European Research Council, Duchenne Parent Project, European Community (OptiStem and
Angioscaff), Association Française contre les Myopathies (AFM)
CureDuchenne and the Italian Ministries of Research (FIRB) and Health
On Stochastic, Irreversible Investment Problems in Continuous Time: a New Approach Based on First Order Conditions
No full textOn the top coefficients of Kazhdan-Lusztig polynomials
No full textKazhdan and Lusztig in 1979 defined, for any Coxeter group W, a family of polynomial that is know as Kazhdan-Lusztig polynomials. These polynomials are indexed by pairs of elements of W and they have fundamental
importance in several areas of mathematics as representation theory, geometry, combinatorics and topology of Schubert varieties. In this paper we want to show the combinatorial connection between special matchings and the top coefficient of Kazhdan-Lusztig polynomial. In particular we study a Conjecture, due to Brenti, in different Coxeter groups and pair of elements
Comparative genomics of V. cholerae 7th pandemic strains: analysis of Integrative Conjugative Elements, Genomic Islands and prophages
No full text
THE NEW HADRON SPECTROSCOPY
No full textIn the last ten years exotic hadron resonances have been discovered in high energy collider experiments. Indeed the internal structure of these hadrons, conventionally called XYZ, cannot be explained in terms of standard mesons or baryons, made up by a quark and an antiquark or by three quarks.
In this PhD thesis we show how the decay and, above all, the production mechanisms of the XYZ could be useful in testing some theoretical hypotheses against experimental data.
Moreover we propose to perform some indirect searches in order to discover if the experimental picture as we know it nowadays is complete or otherwise
Studio molecolare della cinetica nemaspermica
No full textLa capacità fecondante di un individuo è strettamente legata alle caratteristiche microscopiche del suo liquido seminale, ed in particolare a tre parametri fondamentali: il numero di spermatozoi, la loro morfologia e la motilità. Quest’ultimo parametro riveste un ruolo fondamentale nella riproduzione, in quanto per arrivare a fecondare la cellula uovo, gli spermatozoi devono essere in grado di risalire le vie genitali femminili.
La motilità dello spermatozoo si realizza grazie alla particolare struttura con cui è organizzato il flagello e all’energia ad essa fornita da due vie metaboliche, la fosforilazione ossidativa, che avviene nel tratto intermedio dove sono localizzati i mitocondri, e la via glicolitica che si realizza nel segmento principale nel quale sono stati individuati individuati gli enzimi glicolitici. Alterazioni nella struttura del flagello o nelle vie metaboliche che forniscono energia possono determinare ridotta o assenza di motilità, quindi infertilità.
La glicolisi è un processo altamente conservato negli eucarioti, tuttavia negli spermatozoi umani sono state individuate isoforme di alcuni enzimi glicolitici, che hanno caratteristiche strutturali e funzionali che li distinguono dalle rispettive isoforme espresse nelle cellule somatiche. Tre di essi, la gliceraldeide 3-fosfato deidrogenasi, la fosfoglicerato chinasi-2 e la lattato deidrogenasi-C, sono codificati da geni paraloghi espressi solo durante la spermatogenesi.
È stato dimostrato che topi knock-out per il gene della gliceraldeide 3-fosfato deidrogenasi spermatica sono infertili e mostrano alterazioni non solo quantitative ma anche qualitative della motilità (Miki et al. 2004).
Lo scopo di questo studio è stato quello di valutare l’espressione del gene GAPDHS che codifica per l’isoforma spermatica dell’enzima glicolitico gliceraldeide 3-fosfato deidrogenasi (GAPDH) in campioni di liquido seminale caratterizzati da normocinesia o da ipocinesia , al fine di correlare le alterazioni nella motilità nemaspermica con i livelli di mRNA
Lightwave planar circuits based on organic materials for filtering and sensing
No full textThis thesis investigates in detail optical filters based on two phenomena and their applicability in
photonics devices.
The first phenomenon is called Whispering gallery modes, discovered in 1912 from Lord Rayleigh.
The second phenomenon investigated in this thesis is the Braggs’s law, to develop optical filters
Virtual Screening for the identification of inhibitors of PLP-dependent enzymes
No full textDopa decarboxylase (DDC), a pyridoxal 5'-phosphate (PLP) enzyme responsible for the biosynthesis of dopamine and serotonin, is involved in Parkinson's disease (PD). PD is a neurodegenerative disease mainly due to a progressive loss of dopamine-producing cells in the midbrain. Co-administration of L-Dopa with peripheral DDC inhibitors (carbidopa or benserazide) is the most effective symptomatic treatment for PD. Although carbidopa and trihydroxybenzylhydrazine (the in vivo hydrolysis product of benserazide) are both powerful irreversible DDC inhibitors, they are not selective because they irreversibly bind to free PLP and PLP-enzymes, thus inducing diverse side effects. Therefore, the main goals of this study were (a) to use virtual screening to identify potential human DDC inhibitors and (b) to evaluate the reliability of our virtual-screening (VS) protocol by experimentally testing the "in vitro" activity of selected molecules. Starting from the crystal structure of the DDC-carbidopa complex, a new VS protocol, integrating pharmacophore searches and molecular docking, was developed. Analysis of 15 selected compounds, obtained by filtering the public ZINC database, yielded two molecules that bind to the active site of human DDC and behave as competitive inhibitors with K(i) values ≥10 µM. By performing in silico similarity search on the latter compounds followed by a substructure search using the core of the most active compound we identified several competitive inhibitors of human DDC with K(i) values in the low micromolar range, unable to bind free PLP, and predicted to not cross the blood-brain barrier. The most potent inhibitor with a K(i) value of 500 nM represents a new lead compound, targeting human DDC, that may be the basis for lead optimization in the development of new DDC inhibitors. To our knowledge, a similar approach has not been reported yet in the field of DDC inhibitors discovery