382 research outputs found
Analisi strutturale e funzionale di 5 nuove mutazioni del gene CYP21A2 in pazienti affetti da deficit della 21 idrossilasi
No full textLa sindrome adrenogenitale(SAG) è un gruppo di disordini autosomici recessivi dovuti a .
deficit enzimatici nella steroidogenesi di cortisolo e aldosterone.
Il deficit da 21-idrossilasi (21 OHD) è la forma più comune, interessa circa il 90 95% dei casi.
La 21-idrossilasi è un’enzima che appartiene alla famiglia dei citocromi P-450 che catalizza la conversione del 17 idrossiprogesterone in 11 deossicortisolo, un precursore del cortisolo e la
conversione del progesterone in deossicorticosterone, un precursore dell’aldosterone.
Il fenotipo della sindrome adrenogenitale varia a seconda dell’ attività della 21-idrossilasi.
Il gene della 21-idrossilasi (CYP21A2) e` localizzato nel complesso maggiore di istocompatibilita` umano di classe 3 sul cromosoma 6.
Sullo stesso locus è presente uno pseudogene (CYP21AP),identico per il 98% al gene, che codifica per una proteina non attiva a causa di numerose mutazioni deleterie; la maggior parte delle mutazioni sono causate da ricombinazioni tra CYP21A2 e CYP21AP: solo il 5% sono nuove mutazioni del gene CYP21A2 che non originano dallo pseudogene , nel quale l’analisi funzionali dell’enzima è stato considerato utile per la correlazione genotipo fenotipo.
Quando le sequenze deleterie, normalmente presenti nello pseudogene, sono trasferite al gene attivo, quest’ultimo diventa incapace di codificare per una proteina con attività funzionale,
Generalmente, e` stata dimostrata una buona correlazione tra genenotipo-fenotipo e la forma clinica correla con l`allele mutato con attivita` enzimatica piu`alta.
Questo ha reso possibile predire un inquadramento clinico in soggetti affetti basandosi sul genotipo.
Per le nuove e rare mutazioni, in cui non erano disponibili informazioni cliniche e il fenotipo non coincideva con il genotipo,in particolar modo per i pazienti maschi e doppi eterozigoti, solo studiando le attivita` funzionali degli enzimi, ha reso possibile classificare le mutazioni in base alla severita’ della malattia.
Abbiamo trovato 5 nuove mutazioni puntiformi (L122P, M150R, Q481X ,E161X and R366H ) in 5 pazienti italiani con SAG.
Per verificare le mutazioni presenti sul gene CYP21A2 e per classificarle in base alla gravità, abbiamo costruito in vitro le mutazioni del CYP21A2 e abbiamo considerato le attività enzimatiche mutanti dopo averle fatte esprimere nelle cellule COS7.
Il modelling molecolare suggerisce un impatto maggiore sull’ analisi funzionale e strutturale della 21-idrossilasi dopo l’espressione nelle cellule COS-7 confermando una attività enzimatica ridotta Solo le mutazioni M150R e R366H influenzano in minor modo l’ attività enzimatica probabilmente associata con la forma non classica di SAG.Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorder caused by the deficiency of a steroidogenic enzyme involved in corstisol or aldosterone biosynthesis.
21-hydroxylase deficiency (21-OHD) is the most common form, accounting for 90-95% of cases.
21-hydroxylase is a cytocrome P-450 enzyme that catalyzes the conversion of 17-hydroxyprogesterone to 11-deoxycortisol, a precursor of cortisol, and the conversion of progesterone to deoxycorticosterone, a precursor of aldosterone.
The phenotype of CAH varies greatly, depending of the degree in the impairment of 21-OH activity. The 21-hydroxylase gene (CYP21A”) is a part of a complicated structure located in the human leukocyte antigen class III locus on chromosome 6p21.3. In the same locus, is present a 98% identical pseudogene (CYP21A1P), which does not encode a functionally active protein due to numerous deleterious mutations; most mutations causing 21-OH deficiency arise from recombinations between CYP21A2 and CYP21A1P. When deleterious sequences normally present in the pseudogene are transferred to the active gene, the latter becomes incapable of encoding a normal enzyme: only 5% are novel CYP21A2 mutations, in which functional analysis of mutant enzymes have been helpful to correlate genotype-phenotype.
We have found five novel point mutations (L122P, M150R, Q481X ,E161X and R366H ) in five Italian patients with CAH.
To verify the status of the point mutations in CYP21A2 gene as disease-causing mutations and to classify them according to severity, we have constructed in vitro the mutations of CYP21A2 and evaluated the activities of the mutant enzymes after expression in COS7 cells.
Molecular modeling suggests a major impact on 21-hydroxylase activity and functional analysis after expression in COS-7 cells confirms reduced enzymatic activity of the mutant enzymes. Only the M150R and R366H mutations affected the activity to a minor extent, probably associated with NC CAH
Motion Scalability for Video Coding with Flexible Spatio-Temporal Decompositions
No full textPhDThe research presented in this thesis aims to extend the scalability range of the
wavelet-based video coding systems in order to achieve fully scalable coding with a
wide range of available decoding points. Since the temporal redundancy regularly
comprises the main portion of the global video sequence redundancy, the techniques
that can be generally termed motion decorrelation techniques have a central role in
the overall compression performance. For this reason the scalable motion modelling
and coding are of utmost importance, and specifically, in this thesis possible
solutions are identified and analysed.
The main contributions of the presented research are grouped into two
interrelated and complementary topics. Firstly a flexible motion model with rateoptimised
estimation technique is introduced. The proposed motion model is based
on tree structures and allows high adaptability needed for layered motion coding. The
flexible structure for motion compensation allows for optimisation at different stages
of the adaptive spatio-temporal decomposition, which is crucial for scalable coding
that targets decoding on different resolutions. By utilising an adaptive choice of
wavelet filterbank, the model enables high compression based on efficient mode
selection. Secondly, solutions for scalable motion modelling and coding are
developed. These solutions are based on precision limiting of motion vectors and
creation of a layered motion structure that describes hierarchically coded motion.
The solution based on precision limiting relies on layered bit-plane coding of motion
vector values. The second solution builds on recently established techniques that
impose scalability on a motion structure. The new approach is based on two major
improvements: the evaluation of distortion in temporal Subbands and motion search
in temporal subbands that finds the optimal motion vectors for layered motion
structure.
Exhaustive tests on the rate-distortion performance in demanding scalable video
coding scenarios show benefits of application of both developed flexible motion
model and various solutions for scalable motion coding
Regulation of osteoblast/osteoclast cross-talk by factors potentially leading to a positive bone balance : evaluation of GH, CGRP and phenylcarboxylic acid derivatives
No full textIn this study it was evaluated the role of growth hormone (GH), calcitonin gene-related peptide (CGRP) and of new molecules of synthesis in the regulation of biomolecular mechanisms involved in the process of bone remodeling. The effect of GH and CGRP in the expression and secretion of osteoprotegerin (OPG) was evaluated in primary cultures of human osteoblast-like cells (hOB). These cells showed to express both a specific GH receptor and a CGRP receptor. GH (4.5x10-11M) induced an enhancement of OPG in a specific and receptor mediated way without the involvement of IGF-I. On the contrary CGRP (1.08x10-10M) inhibited OPG production. This effect was mediated by the CGRP induced stimulation of cAMP and involvement of phosphokinase A. Since OPG secretion was reduced, conditioned media from CGRP treated hOB, stimulated osteoclast formation from human peripheral blood mononuclear cells compared to conditioned media of untreated cells. Thus suggesting that CGRP exerts its anabolic effects on bone by modulating the fine balance of bone cell coupling. A series of novel biphenylcarboxylic acid derivatives, called ABD compounds, were tested in primary cultures of murine osteoblasts, macrophages and osteoclasts derived from mouse bone marrow. All compounds showed antiosteogenic effect mediated by inhibition of RANKL induced intracellular signalling (NFkB pathway), and by activation of p38/JNK proapoptotic pathway
Il segnale serotoninergico è importante nel controllo del rimodellamento osseo in risposta alle sollecitazioni meccaniche nel ratto
No full textThere is increasing experimental and clinical evidence that serotonin (5-HT) may regulate bone metabolism. In fact, on bone cells have been detected functional receptors for 5-HT, 5-HT transporters and the tryptophan hydroxylase, a key enzyme for the synthesis of 5-HT from tryptophan (TRP).
It is well known that depressive disorders involve 5-HT system and that low bone mass is frequent in depressed patients. Since low level of physical activity, characteristic of depressed patients, is also associated to low bone mass, it remains to be clarified the relative contribution of reduced 5-HT tone or disuse in the detrimental effect of depression on bone mass.
Therefore, the aim of this study was to investigate the effects of reduced 5-HT tone, induced by chronic TRP depletion, in the control of bone mass and bone remodelling in condition of osteopenia induced by physiological movement restraint in the rat.
For this purpose, rats received a TRP-free diet or a standard control diet for 60 days. To induce a physiological movement restraint, all rats were housed in single small size cages with a floor area of cm2 370 their motor activity was diminished. Bone mass, mineral content (BMC) and density (BMD) were measured by DXA and pQCT in the femur. Bone metabolic markers included markers of bone resorption (urinary deoxypyridinoline, DPD) and markers of bone formation (serum osteocalcin, OC).
In control diet-fed rats both femoral BMC and BMD, as measured by DXA, decreased with time due to reduced movement. TRP-free diet significantly worsened the osteopenia induced by movement restraint. The reduction of both BMC and BMD either in control diet rats or in TRP-free diet rats was higher in femoral methaphysis than in femoral diaphysis. At the end of the experimental period, pQCT analysis of femoral methaphysis showed that TRP-free diet resulted in a significant reduction of BMC on total bone, trabecular, cortical and subcortical bone in comparison with control-diet fed rats. Due to decrease in BMC and cortical thickness, the femoral strength strain index (SSI) was reduced.
These effects are due to a negative shift in the balance between bone formation and resorption with a significant reduction in bone formation as evidenced by a reduction of OC and by the impairment of the Wnt/b-catenin pathway, which enhances the sensitivity of osteoblasts/osteocytes to mechanical loading.
The present data extend our knowledge on the role of 5-HT in the regulation of bone mass. In particular, our findings demonstrate that 5-HT is involved in the control of the anabolic response of bone to mechanical loadin
-GHRELIN IS INVOLVED IN OSTEOBLAST-OSTEOCLAST INTERACTION THROUGH WNT/BETA CATENIN SIGNALLING PATHWAY
No full textAims: Considerable attention has been focussed on the effect of energy regulating peptides such as leptin and ghrelin on bone metabolism.
Ghrelin, the natural ligand of the growth hormone secretagogue receptor (GHS-R), is considered a positive regulator of bone mass. Ghrelin, in fact, acts directly on osteoblasts and stimulates osteoblast proliferation and differentiation. However, the detailed mechanisms of ghrelin’s modulation of osteoblast activity remain unclear and require further studies. The Wnt/beta-catenin signalling pathway plays a crucial role in osteoblastic cell differentiation and bone formation. When Wnt receptor binding interactions are absent, beta-catenin is phosphorilated by glycogen synthase kinase 3b (GSK-3b) leading to beta-catenin degradation in the proteasome. Following Wnt receptor activation GSK-3b is phosphorylated and inactivated, leading to the stabilization of beta-catenin and its accumulation in the cytoplasm. beta-catenin then migrates to the nucleus where it regulates transcription, and modulates the expression of target genes responsible for osteoblast proliferation and differentiation.
The aim of the present study was to investigate the involvement of the Wnt/beta-catenin signalling in the anabolic effect of ghrelin and the receptors involved in such activity.
Methods: We used primary cultures of osteoblast like cells derived from rat calvariae exposed to increasing concentrations of ghrelin (10-11-10-7M) for 60 min. beta-catenin and GSK-3b protein levels were measured by Western Blot in the whole cell lysates.
Results: Ghrelin significantly increases the phosphorylation and degradation of GSK-3b and therefore it increases beta-catenin levels and beta-catenin nuclear migration.
These effects are due to a specific interaction of ghrelin with its GHSR1a receptor since pretreatment with the GHS-R1a receptor antagonist D-LYS-GHRP6 (10-7M for 30min), prevents ghrelin-induced beta-catenin stabilization and nuclear migration. Ghrelin-dependent induction of beta-catenin increases osteoblast proliferation as shown by MTT assay.
Considering that osteoprotegerin (OPG), a target gene of the Wnt/ beta-catenin signaling, plays a key role in the regulation of cross-talk between osteoblasts and osteoclasts, we examined the effect of increasing ghrelin concentrations (10-11 - 10-8 M) on the expression of OPG mRNA measured by Real Time PCR in osteoblasts. Ghrelin (10-10 M) significantly increases OPG mRNA levels 24 h after treatment. Thus, it is conceivable that ghrelin by increasing the Wnt/beta-catenin signaling pathway not only exerts an anabolic action but it is also involved in osteoblast-osteoclast interaction.
Conclusions: In conclusion, we have shown that ghrelin stimulates osteoblast proliferation and increases OPG expression at the same time through Wnt/beta-catenin signalling pathway. Even if further in vivo studies will be required to confirm this assumption, this study suggests ghrelin as a key candidate to maintain bone homeostasis when there is imbalance between resorption and formation as occurs in osteoporosis
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