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MicroRNA signature changes in human ovarian follicle in relation to reproductive aging
No full textSmall non-coding class of RNAs includes different types of molecules involved in different steps of RNA synthesis, processing, translation, as well as RNA modulation of transcription initiation, RNA degradation or protein synthesis block. Among these, microRNAs (miRNAs) represent the most studied and better characterized class of ncRNAs in terms of function and impact in human health and disease. miRNAs are short non-coding RNAs involved in the control of gene expression in different species. Specifically, miRNAs play a major role as master regulators of protein-coding genes. miRNA role in ovarian follicles has been characterized and the existence of miRNAs in human follicular fluid has been recently demonstrated. It was also reported that altered regulation of miRNA expression affects crucial pathways for follicle growth and oocyte maturation in several reproductive diseases. In addition, global miRNA analysis, on different tissues and organs and animal models has also shown that aging can influence mirna expression. Decreased female fertility with advanced maternal age has been widely documented. Although it is widely recognized that a key aspect that explains infertility in reproductive aging is the decline in oocyte quality, which also associates with higher risk of birth defects, genetic disorders and miscarriage, the molecular mechanisms underlying reproductive aging in female mammals are poorly understood. This thesis has aimed at evaluating impact of maternal age on miRNA expression profiles in human ovarian follicle and characterizing the pathways significantly affected by female ageing, identifying their regulator miRNAs. The manuscript includes three studies that for concision, they will be referred in the text, by Roman numerals, as Study I, II and III respectively. The Study I has been focused on the characterization of microRNAs in human follicular fluid (FF), the Study II on the miRNome changes in Follicular fluid exosomes from women of two different age groups; while miRNA identification in human MII oocyte and their expression profile changes in relation to reproductive aging have been shown in the Study III. Firstly, it was ascertained whether miRNAs are cargo of FF exosomes and whether they are involved in the regulation of follicle maturation. At a later stage, TaqMan Human microRNAs cards were performed to verify differently expressed miRNAs in FF respect with plasma samples, collected from 15 healthy women who underwent to intracytoplasmic sperm injections (ICSI). 37 miRNAs had significantly higher expression levels in human FF. 32 are carried by exosomes and involved in critically important pathways for follicle growth and oocyte maturation. Specifically, nine of them target and negatively regulate mRNAs expressed in the follicular microenvironment encoding inhibitors of follicle maturation and meiosis resumption. In order to reveal the contribution of miRNAs to female reproduction aging, we examined their expression changes during aging in human follicular fluid, using TLDA technology. Different miRNA distribution was found in FF exosomes from old and younger women. We detected about 50 miRNAs in FF exosomes which showed highly significant differences related to aging and were predicted to regulate ECM-receptor interaction, PI3K-Akt, p53, TGF-beta, HIF-1 and mTOR signaling pathways. Finally, we identified 57 miRNAs constantly expressed in 12 MII oocytes and 12 miRNAs displaying altered regulation in women of advanced reproductive age. By computational approach we explored the possible functions of differentially expressed miRNAs inside human germ cells, and confirmed experimentally miRNA differential expression in murine MII oocyte. Finally, a significant negative correlation miRNA-targets was found for miR-29a, miR-203 and specific mRNAs, that have been ascribed responsible for mechanisms modulating epigenetic changes underlying compromised oocyte quality caused by advanced maternal age
Neutrophil gelatinase-associated lipocalin (NGAL) and matrix metalloproteinases (MMPs) as biomarkers of bladder cancer development and progression
No full textNeutrophil gelatinase-associated lipocalin (NGAL), also called lipocalin-2, is a secreted protein belonging to the lipocalin family proteins and actively participates into the proliferation, differentiation, and development of human tissues, including tumours. It positively modulates the activity of the matrix metalloproteinases-9 (MMP-9) that are involved in the enzymatic remodelling of the extracellular matrix. MMP-9 regulates the degradation of extracellular matrix in processes such as angiogenesis, tumour growth, and metastasis. By forming the NGAL/MMP-9 complex, NGAL protects MMP-9 from proteolytic degradation, a fundamental mechanism in controlling the activity of the proteins, and enhances its enzymatic activities.
As a secreted protein, NGAL is detectable in many biologic fluids, including urine, where several neoplastic cells and other tumor microenvironmental factors can be directly released from the cancer. Our in silico analysis suggested an active role of NGAL in tumour development of several cancer types. Validation of these findings is here described in bladder cancer as a good tumor model in which investigate the role of this protein because urine is in direct contact with the primary tumor.
On these bases, the release of NGAL in both urine and serum samples from 89 bladder cancer patients was measured. Further investigations, aimed to emphasize the role of NGAL in cancer, were performed by analysing MMP-9 and NGAL/MMP-9 complex levels in the same subset of bladder cancer patients. Control experiments were performed in 119 cancer-free controls, previously enrolled in a case-control study. Urinary concentrations were standardized on creatinine level. The performance of these proteins as cancer biomarkers was evaluated through the receiver operating characteristic (ROC) analysis.
In conclusion, the present study deepens the knowledge of the molecular mechanisms sustaining NGAL expression in tumor cells and its effects on cancer metastatic behaviour. Furthermore, NGAL/MMP-9 pathway is associated with an aggressive phenotype of transitional cell carcinoma of the bladder (TCC). The elevated negative predictive value of MMP-9 and NGAL/MMP-9 complex make them candidate markers of exclusion test for TCC. These findings suggest that these proteins may be integrated in the surveillance of bladder cancer, thus improving patients complaints and diminishing their discomfort
Biofunctionalized systems for drug discovery and delivery
No full textDuring this doctoral research work new potential drug delivery vehicles for targeted treatment of cancers were developed. For this purpose, both soft and hard materials were subject of study.
Firstly, the synthesis, the characterization and the biological evaluation of monomeric â-cyclodextrins functionalized with folic acid (FA) were the focus of this research. In particular, four new conjugates (CyD-FAs), both 3- and 6-functionalized â-CyDs (â-CyD3 and â-CyD6) linked to the á- or ã-carboxylic group of the FA were synthesized, isolated and fully characterized. Furthermore, the ability of these compounds to include the anticancer drug LA-12 and to deliver it selectively to FR (+) tumor cell lines was investigated.
Since the promising results obtained with these CyD-FA conjugates as carriers for LA-12, polymeric nanoparticles based on cross-linked cyclodextrins were designed for drug delivery purposes. These systems, offer the advantages of CyD-type complexation in a synergistic way, resulting more effective than the parent CyDs. In particular, CyD-based polymers and oligomers were synthesized, functionalized with FA and tested as delivery systems towards different hydrophobic anticancer or anti-inflammatory agents. Furthermore, for selected systems, the binding constants of the formed inclusion complexes were determined as well.
Concerning hard materials, mesoporous silica nanoparticles were investigated. A new disc-shaped mesoporous material, the nanodiscs (NDs), was synthesized, isolated and fully characterized. This material was firstly used for the preparation of self-assembled monolayers (SAM), to be employed in targeted cancer cell adhesion and in-situ drug delivery. For this purpose, the NDs-monolayers were functionalized with FA as targeting moiety. Thanks to their large surface area and the possibility of high density of superficial functionalization with bioactive molecules, these systems resulted effective in binding cancer cells even upon short contact times. Moreover, exploiting the porosity of the synthesized particles, the intracellular release of small hydrophobic molecules pre-loaded in the channels of the NDs were achieved.
Secondly, preliminary biological experiments carried out to test the cellular uptake of NDs, and the drug-carrier ability were performed. Finally, in the attempt to increase the biodegradability of these interesting structures, disulfide-doped mesoporous silica nanodiscs (ss-NDs) were also prepared. Full characterization and preliminary biological assays of these hybrid materials was also performed, and their degradation in redox conditions investigated. This novel material, taking advantage of bio-redox reactions, undergoes a controlled disintegration process in presence of reducing agents (i.e. glutathione), displaying an improved drug delivery action
New alkylpiperazines as 5-HT7R ligands
No full textThe 5-HT7 receptor is the last member of the serotonin receptors family. This receptor, cloned and identified in 1993, belongs to the G protein-coupled receptor family and is positively coupled with adenylyl cyclase. Different isoforms, which differ only in the length and amino acid composition of their C-terminal tail, are generated by alternative splicing of the 5-HT7 receptor gene and are namely: 5-HT7(a), (b), (c) in rat and 5-HT7(a), (b), (d) in human. These isoforms do not show significant differences in their pharmacological profile, signal transduction or tissue distribution and the 5-HT7(a) is the most abundant in human. Since its identification, 5-HT7 receptor has been the subject of intense research efforts due to its presence in functionally relevant regions of the brain. For this reason, 5-HT7 receptor has been suggested to have a role in a wide range of physiological functions such as nociception, sleep, locomotor activity regulation, learning and memory. Also, it seems to be involved in some pathologies like anxiety, depression, epilepsy, and Fragile X syndrome. After the cloning of 5-HT7 receptor, a number of non-selective ligands, belonging to different chemical classes and showing high affinity toward this receptor, were identified; however, these compounds display multi-receptor affinity. In the last decade, there have been many efforts to discover selective agents for the 5-HT7 receptor. Examples of such molecules are characterized as long-chain arylpiperazine compounds, which are categorized as 5-HT7R ligands because they indicate high affinity and good selectivity for the receptor. Due to the high drug potential of long-chain arylpiperazines, with a number of successfully developed drugs or pharmacological tools, various structure-affinity relationships studies have been done. Furthermore, given the therapeutic potential of 5-HT7 receptor agents in central nervous system disorders, we recently worked on the development of new selective 5-HT7 receptor ligands to gain a comprehensive insight about their structure-affinity relationships and the functional properties. In this thesis, novel series of long-chain arylpiperazines were designed, synthesized, and tested to evaluate their affinity for the 5-HT7 and 5-HT1A receptors. Moreover, molecular modeling studies were performed in order to investigate these new ligands interactions with the 5-HT7 receptor
Ruolo del recettore Gabaa nelle cellule alfa pancreatiche e suo coinvolgimento nelle fasi di desensibilizzazione indotta dalla lipotossicità nel diabete di tipo II.
No full textIl GABA o acido gamma-aminobutirrico è il principale neurotrasmettitore inibitorio del sistema nervoso centrale (SNC) dei mammiferi. Circa il 20 - 50% di tutte le sinapsi utilizzano questo neurotrasmettitore. Esso è presente in quantità elevate, anche nello spazio interstiziale delle isole pancreatiche e nelle beta cellule pancreatiche dalle quali viene secreto. Il GABA oltre ad avere funzione trofica sulle beta cellule, attraverso il suo recettore (GABAA) controlla la riduzione della secrezione di glucagone insieme con lâ insulina.
Nel nostro lavoro abbiamo studiato gli effetti dell'esposizione cronica al palmitato sul signaling del GABA e sulla pathway dellâ insulina in un modello di alfa cellule pancreatiche. Le cellule alfa-TC1-C6 sono state coltivate in presenza o in assenza di palmitato (0,5 mmol / l) per 48 h. Al termine del trattamento si osservava un aumento della secrezione basale di glucagone, induzione di uno stato di insulino-resistenza (diminuzione della fosforilazione di IRS-1 e Akt), e riduzione dellâ effetto inibitorio del GABA sulla secrezione di glucagone; inoltre anche la fosforilazione del canale GABAA indotta dal GABA era notevolmente ridotta dopo l'esposizione a palmitato.
Nella seconda parte di questo lavoro abbiamo studiato la possibilità di prevenire i danni causati dell'esposizione cronica al palmitato sul nostro sistema co-trattando le nostre cellule con GLP-1 (100 nmol /l ).
Nei gruppi trattati con GLP-1, la secrezione basale di glucagone non risultava aumentata e l'effetto inibitorio del GABA sulla secrezione di glucagone non era ridotto.
Questi risultati indicano che il clone alfa-TC1 6, una linea cellulare di cellule pancreatiche alfa, coltivate per 48 ore in presenza di elevate concentrazioni di palmitato (0,5 mmol / l) sviluppano insulino resistenza attraverso la riduzione della fosforilazione di IRS-1 e Akt; questo percorso controlla la secrezione di glucagone ed è critico per la traslocazione del recettore GABAA sulle membrane cellulari e quindi per la polarizzazione della membrana delle alfa cellule. Il GLP-1 sembra in grado di prevenire tali alterazioni. Questi risultati supportano l'ipotesi che l'esposizione cronica agli acidi grassi può contribuire alla alterazione della secrezione di glucagone; e il GLP-1 mostra un effetto positivo sulla prevenzione dell'insulino-resistenza e sull'attivazione del canale GABA
Alterazioni dei Neutrofili nei Pazienti con Mieloma Multiplo
No full textNel mieloma multiplo (MM), ma non nella gammopatie monoclonali di significato incerto (MGUS), la funzione immunitaria è alterata, conseguenza di un microambiente immunologicamente ostile e di difetti cellulari, tra cui la riduzione dell immuno-sorveglianza e la disfunzione dei T-linfociti.
Recenti studi indicano che l'asse mieloide avrebbe un ruolo importante nella progressione tumorale. Dal momento che i neutrofili granulocitari su sangue periferico potrebbero riflettere il complesso sistema mieloide, abbiamo indagato alcuni aspetti fenotipici e funzionali dei neutrofili di pazienti con MM all esordio (MM-N) confrontandoli con quelli di MGUS( MGUS-N) e di soggetti sani (HS-N).
Abbiamo osservato che rispetto a MGUS-N e HS-N, MM-N all esordio hanno un'alterata espressione di alcuni markers di superficie quali CD64, CD11b, CD16 e CD62L, aumentati livelli di specie reattive dell'ossigeno (ROS), ridotta attività fagocitaria e di burst ossidativo.
MM-N hanno anche aumentati livelli di Arginasi-1 e PROK2/BV-8 e risultano avere proprietà immunosoppressive in cocolture con cellule T allogeniche, in quanto inibiscono la proliferazione e la capacità di stimolazione dei T-linfociti.
I risultati di questo studio forniscono nuove informazioni sia sul microambiente che caratterizza il MM rispetto ad MGUS e sia sui meccanismi che riguardano l aumentata suscettibilità alle infezioni di questi pazienti
Molecular mechanism of Abeta recognition and neuroprotection by the glycoconjugate beta-sheet-breaker peptide Ac-LPFFD-Th
No full textInhibition of amyloid formation may represent a promising therapeutic approach for the treatment of neurodegenerative diseases. To this regard, peptide-based inhibitors of Abeta aggregation have been widely investigated with a particular emphasis to those derived from original amyloid sequences.
The experimental work described in the present PhD Thesis aims at outlining the molecular mechanisms underlying the antifibrillogenic and neuroprotective action exhibited by a new class of trehalose conjugated pentapeptides. Trehalose (Th), a non-reducing disaccharide of alpha-(D)glucose, has been demonstrated to be effective in preventing the aggregation of several proteins. We figured out that the development of hybrid compounds may provide new molecules with improved properties that might sinergically increase the potency of their single moieties.
Since it has been demonstrated that Abeta oligomers are the toxic species, it becomes a priority to counteract the Abeta self-assembly because of its doubly dangerous effect associated with oligomers generation and removal of the neurotrophic monomeric form of Abeta1-42 peptide.
Starting from the well-known neuroprotective action of the LPFFD peptide, (C. Soto, Nat Med. 1998) we investigated whether the Ac-LPFFD-Th peptide would act as an Abeta monomer-stabilizer thus exerting a neuroprotective activity. In support of this hypothesis, time-resolved proteolysis and ESI-MS experiments, performed during my PhD experimental work, showed a direct interaction between these beta-breaker peptides and Abeta monomers.
In this work, the C-terminal trehalose conjugated Ac-LPFFD-Th derivative ability to recognize and bind low molecular weight aggregated forms of Abeta has been investigated by means of different biophysical techniques, including Th-T fluorescence, DLS, ESI-MS and NMR. Furthermore, biological assays on murine cortical primary neuronal cultures were performed in order to clarify and further characterize the mechanism of cytoprotection exhibited by the Ac-LPFFD-Th.
In this PhD thesis, we demonstrated that Ac-LPFFD-Th modifies the aggregation features of Abeta and protects neurons from Abeta oligomers toxic insult
Genomic analysis in human solid tumours: Copper Homeostasis Genes in Colorectal cancer
No full text1. The public database: Cancer Genome Atlas Network has been consulted in order to reveal the presence of somatic mutations in copper homeostasis genes (CHGs) in colorectal cancer, and such analysis, performed on 228 colorectal tumor samples, has revealed that inactivating mutations are extremely rare in CHGs.
2. The collection of whole transcriptome profiles by oligonucleotide microarrays represents the second aim of the thesis. CHGs mRNA levels have been measured in 37 colorectal carcinoma samples in comparison to matched normal colonic mucosae. The transcriptome analysis has been perfomed using the last Human Transcriptome Array (HTA 2.0, Affymetrix) which allows to analyze simultaneously 40.000 coding transcripts and 20.000 non-coding transcripts and to reveal variations of mRNA levels between in colorectal cancer samples respect to normal colonic mucosae. The gene expression analysis has showed an up-regulation of several transcripts involved in copper homeostasis pathway (such as SLC31A1, SLC31A2, COX11, SCO1, SOD1) in two different conditions. On the contrary, some genes did not show variations of expression between two tested conditions suggesting the idea that these genes, if experimental altered in their expression, could represent the good targets for specific drug treatments. The transcriptome analysis by the last generation on oligonucleotide microarrays gives a possibility to analyse gene expression levels and single exon expression levels. In the 37 human colorectal samples, the exon-level expression analysis has been revealed the presence of alternative transcripts prevalent in a condition respect to other.
3. In this part of thesis the experimental down-regulation, by short interfering, of a copper chaperone ATOX1, significantly reduced the tumor growth in a Caco2 colorectal cell line. It has been demonstrated that the copper addition increase the toxic effects of some copper binding compounds, in particular, the ionophore copper-ionophore 5-chloro-8-hydroxyquinoline (ClHQ) and the copper chelator (N,N,N'N',-tetrakis (2- pyridylmethyl)ethylenediamine (TPEN) in two human colorectal cancer cell lines (Caco-2 and HT29). Moreover, the Atox1 silencing has enhanced the toxic effects of copper-ClHQ complexes and TPEN in Caco-2 cells confirming that the inhibition of copper chaperone Atox1 could be a good strategy to attenuate the cancer cell proliferation and to increase the anticancer effects of some copper binding drugs
Per una filologia dell'opera di Pavese: edizione critica dei dialoghi con Leucò
No full textLA PRESENTE TESI DI DOTTORATO AMBISCE AD INSERIRSI TRA I PIU' RECENTI CONTRIBUTI ALLO SVILUPPO DELLA FILOLOGIA PAVESIANA, ATTRAVERSO L'EDIZIONE CRITICA DEI DIALOGHI CON LEUCO' (19
Elementi di lessicografia computazionale per un vocabolario del siciliano medievale (VSM)
No full textQuesto lavoro si prefigge di costruire l impianto lessicografico e gli strumenti informatici per un vocabolario elettronico, il Vocabolario del Siciliano Medievale (VSM) progetto già auspicato da Ruffino (1989: 337), Brincat (2011), Pagano (2011), e presentato, a grandi linee, da Pagano (2012). Dopo una rapida introduzione sulle digital humanities, vengono ripercorsi alcuni degli studi che hanno proposto classificazioni e modelli di valutazione per il vocabolario elettronico, seguiti da un analisi storico-critica delle imprese più significative nel campo della lessicografia storica. Successivamente, gli elementi fondamentali dei vocabolari elettronici sono trattati singolarmente ed esaustivamente in un capitolo dedicato.
La seconda parte si prefigge di applicare sistematicamente le teorie e le metodologie individuate al caso specifico del VSM. L avvio dei lavori è naturalmente incentrato sulla raccolta dei dati lessicografici. A partire dall analisi della rappresentatività del Corpus Artesia (Archivio Testuale del Siciliano Antico artesia.ovi.cnr.it) viene affrontato il problema del bilanciamento del sottocorpus dei testi d archivio. La ricerca ha quindi curato la codifica di oltre 60 testi per l indicizzazione in GATTO (Gestione degli Archivi Testuali del Tesoro delle Origini) e ha condotto alla realizzazione di una nuova versione della base di dati. Il lavoro è corredato dalla realizzazione dell interfaccia di consultazione e installazione per la pubblicazione su CD-ROM. La sezione si conclude con alcuni sondaggi lessicali a campione per valutare il contributo dell aggiornamento. Successivamente viene definita la microstruttura della voce, riadattando il prestigioso modello del Tesoro della Lingua Italiana delle Origini e operando una parallela riconversione della struttura logica della voce in XML/TEI. L informatizzazione della redazione è stata completata attraverso la realizzazione di un sistema on-line di redazione collaborativa, che permette l inserimento guidato delle voci con strumenti di autocompletamento e di controllo dell integrità referenziale