STRUCTURAL CHARACTERIZATION OF POLYMERIC MATRICES FOR BIOMEDICAL APPLICATIONS

2013/2014L’obiettivo finale di questa tesi di dottorato è la determinazione di alcune importanti micro e nano caratteristiche strutturali di matrici polimeriche destinate ad applicazioni biomedicali. In particolare, la nostra attenzione si è focalizzata sull’applicazione della Risonanza Magnetica Nucleare a basso campo (LF-NMR), una metodica non distruttiva utilizzata, in particolar modo, nel campo alimentare per la caratterizzazione di sistemi porosi e non. La prima parte di questo lavoro è stata dedicata allo studio di un sistema omogeneo costituito da alginato e pluronico F127. In particolare, grazie alla combinazione della risonanza magnetica ad alto e basso campo, della reologia e del microscopio elettronico a trasmissione (TEM) è stato possibile capire le caratteristiche strutturali di queste matrici, utilizzate al fine di prevenire la restenosi coronarica. La seconda parte del lavoro è stata dedicata allo studio di sistemi porosi, principalmente utilizzati come scaffold per la medicina rigenerativa e l’ingegneria tissutale. Infatti, la risonanza magnetica nucleare a basso campo è in grado di fornire informazioni sulla dimensione media dei pori, un fattore chiave per la crescita cellulare. Infatti, affinchè le cellule possano crescere all’interno di una matrice polimerica, i pori devono avere delle opportune dimensioni (intorno ai 100 m in diametro). Al fine di verificare la robustezza e affidabilità di questa tecnica, sono stati considerati diversi sistemi: a) green coffee seads, b) gomme stirene/butadiene, c) gel di acido acrilico e cellulosa batterica. Una volta che l'affidabilità del metodo NMR a basso campo è stato definitivamente dimostrato, l'attenzione si è spostata sui sistemi, dal punto di vista biomedico, più interessanti. In particolare, sono state considerati due differenti tipologie di scaffold: a) alginato / idrossiapatite scaffold e b) Poly Left Lactide (PLLA) scaffold. Le prove effettuate sugli scaffold che hanno dato esito positivo sono l’ulteriore conferma della validità della tecnica. Lo studio della proliferazione cellulare all’interno della struttura sembra fattibile ed estremamente interessante, in quanto per la natura non distruttiva dell’analisi sarà probabilmente possibile seguire passo passo la crescita delle cellule nello stesso campione di scaffold a tempi crescenti. Pertanto a conclusione di questo lavoro si può ragionevolmente asserire che l’NMR è uno strumento molto affidabile e che le tecniche da noi riportate sono valide sia per i risultati ottenuti (coerenti a quelli ottenuti con altre tecniche analitiche), sia per la facilità di applicazione a molteplici materiali. Si auspica pertanto che la diffusione nel mondo scientifico e industriale della macchina NMR negli anni a venire sia celere e capillare.XXVII Ciclo198

Investigating alcohol consumption during pregnancy for the prevention of Fetal Alcohol Spectrum Disorders (FASD)

The term FASD (Fetal Alcohol Spectrum Disorders) is used to describe the entire spectrum of pathologies and disorders caused by alcohol exposure in uterus. Alcohol assumed in pregnancy passes directly through the placental barrier causing a broad range of symptoms whose severity can greatly vary in degree. The alcohol teratogenic effect may result in physical damage and specific facial anomalies, growth delays, neurological defects along with intellectual disabilities and behavioral problems. Children affected show difficulties in verbal learning, memory, visual-spatial abilities, attention, logic and math abilities, information processing, executive functions as well as in many other domains and in general coping with daily life. Total abstention from alcohol during pregnancy is strongly recommended, as a safe threshold of consumption has not been established yet. Hence, the early identification of alcohol consumption in pregnancy is crucial. Specific methodologies to overcome difficulties related to the identification of alcohol behavior in pregnant women are needed and intervention protocols should be implemented to prevent damage in offsprings. This paper gives an overview on this pathology, from clinical delineation to epidemiology and risk factors with a special focus to promote alcohol-free pregnanc

Changing isotopic food webs of two economically important fish in mediterranean coastal lakes with different trophic status

Transitional waters are highly productive ecosystems, providing essential goods and services to the biosphere and human population. Human influence in coastal areas exposes these ecosystems to continuous internal and external disturbance. Nitrogen-loads can affect the composition of the resident community and the trophic relationships between and within species, including fish. Based on carbon (δ13C) and nitrogen (δ15N) stable isotope analyses of individuals, we explored the feeding behaviour of two ecologically and economically important omnivorous fish, the eel Anguilla anguilla and the seabream Diplodus annularis, in three neighbouring lakes characterised by different trophic conditions. We found that A. anguilla showed greater generalism in the eutrophic lake due to the increased contribution of basal resources and invertebrates to its diet. By contrast, the diet of D. annularis, which was mainly based on invertebrate species, became more specialised, focusing especially on polychaetes. Our results suggest that changes in macroinvertebrate and fish community composition, coupled with anthropogenic pressure, affect the trophic strategies of high trophic level consumers such as A. anguilla and D. annularis. Detailed food web descriptions based on the feeding choices of isotopic trophospecies (here Isotopic Trophic Units, ITUs) enable identification of the prey taxa crucial for the persistence of omnivorous fish stocks, thus providing useful information for their management and habitat conservation

Lovastatin induces apoptosis of k-ras-transformed thyroid cells via inhibition of ras farnesylation and by modulating redox state.

Transformation of thyroid cells with either K-ras or H-ras viral oncogenes produces cell types with different phenotype and different response to the inhibition of the prenylation pathway by 3-hydroxy-3-methylglutaryl-CoA reductase or farnesyltransferase inhibitors. These inhibitors induce apoptosis in K-ras-transformed FRTL-5 cells (FRTL-5-K-Ras) whereas cell cycle arrest is induced in H-ras-transformed FRTL-5 (FRTL-5-H-Ras). In FRTL-5-K-Ras cells, the product of K-ras gene is implicated in the scavenging of reactive oxygen species (ROS) through the activation of extracellular-signal-regulated kinase (ERK)1/2 kinases. We observed that lovastatin blocked ras activation through inhibition of farnesylation and induced apoptosis, increasing ROS levels through inhibition of ERK1/2 signaling and Mn-SOD expression. Lovastatin-induced apoptosis was due to intracellular ROS increase since both, the antioxidant compound pyrrolidinedithiocarbamate or the SOD-mimetic compound, antagonized apoptosis. Moreover, both p38 mitogen-activated protein kinase and nuclear factor kappaB pathways, activated as a consequence of high ROS levels, are involved in the apoptotic effect, indicating that cell death induced by lovastatin was dependent on oxidative stress. Lovastatin antitumor efficacy in K-ras-dependent thyroid tumors was further confirmed in vivo, proposing a new therapeutic strategy for those tumor diseases that are sustained by an inappropriate K-ras expression

Identification of two regions in the p140Cap adaptor protein that retain the ability to suppress tumor cell properties

p140Cap is an adaptor protein that negatively controls tumor cell properties, by inhibiting in vivo tumor growth and metastasis formation. Our previous data demonstrated that p140Cap interferes with tumor growth and impairs invasive properties of cancer cells inactivating signaling pathways, such as the tyrosine kinase Src or E-cadherin/EGFR cross-talk. In breast cancer p140Cap expression inversely correlates with tumor malignancy. p140Cap is composed of several conserved domains that mediate association with specific partners. Here we focus our attention on two domains of p140Cap, the TER (Tyrosine Enriched Region) which includes several tyrosine residues, and the CT (Carboxy Terminal) which contains a proline rich sequence, involved in binding to SH2 and SH3 domains, respectively. By generating stable cell lines expressing these two proteins, we demonstrate that both TER and CT domains maintain the ability to associate the C-terminal Src kinase (Csk) and Src, to inhibit Src activation and Focal adhesion kinase (Fak) phosphorylation, and to impair in vitro and in vivo tumor cell features. In particular expression of TER and CT proteins in cancer cells inhibits in vitro and in vivo growth and directional migration at a similar extent of the full length p140Cap protein. Moreover, by selective point mutations and deletion we show that the ability of the modules to act as negative regulators of cell migration and proliferation mainly resides on the two tyrosines (Y) inserted in the EPLYA and EGLYA sequences in the TER module and in the second proline-rich stretch contained in the CT protein. Gene signature of cells expressing p140Cap, TER or CT lead to the identification of a common pattern of 105 down-regulated and 128 up-regulated genes, suggesting that the three proteins can act through shared pathways. Overall, this work highlights that the TER and CT regions of p140Cap can efficiently suppress tumor cell properties, opening the perspective that short, defined p140Cap regions can have therapeutic effects

Effect of different soil treatments on production and chemical composition of essential oils extracted from Foeniculum vulgare Mill., Origanum vulgare L. and Thymus vulgaris L

The aim of the present study was to investigate how essential oil production and associated chemical composition and related biological activity could be influenced by different cultivation treatments and distillation method. Foeniculum vulgare Mill. (fennel), Origanum vulgare L. (oregano), and Thymus vulgaris L. (thyme) were cultivated in absence of any fertilizer (control) and in presence of three different fertilizers: a chemical one with augmented of mineral phosphorus and potassium, a second added with hydrolysed organic substance and mineral phosphorus and potassium (organic-mineral) and a third one treated with high content of organic nitrogen of protein origin (organic). The plants were subjected to steam distillation using two modalities: recycled and continuous to obtain 32 essential oil samples. Chemical composition analysis was performed by gas chromatography-mass spectrometry; in vitro antimicrobial activity was evaluated by broth microdilution method. In general, the recycled distillation method appeared to have a slightly higher yield than the continuous method. The "mineral" and "organic-mineral" treatments resulted in the higher yield compared to the "organic" or "control" treatments, and this was particularly evident in the recycled method. The "control" plants had a lower yield of essential oils. Anethole (13.9-59.5%) and estragole (13.4-52.2%) were the main constituents of fennel oils, p-cymene and its derivatives carvacrol and thymol were the main constituents of oregano and thyme samples. The antimicrobial activity of thyme oils on Staphylococcus aureus ranged from 0.31 to 0.16% (v/v); a lower effect of oregano samples and no activity of fennel samples were observed. The essential oils failed to inhibit the growth of Pseudomonas aeruginosa strains

Multi-Gene Next-Generation Sequencing Panel for Analysis of BRCA1/BRCA2 and Homologous Recombination Repair Genes Alterations Metastatic Castration-Resistant Prostate Cancer

: Despite significant therapeutic advances, metastatic CRPC (mCRPC) remains a lethal disease. Mutations in homologous recombination repair (HRR) genes are frequent in mCRPC, and tumors harboring these mutations are known to be sensitive to PARP inhibitors. The aim of this study was to verify the technical effectiveness of this panel in the analysis of mCRPC, the frequency and type of mutations in the BRCA1/BRCA2 genes, as well as in the homologous recombination repair (HRR) genes. A total of 50 mCRPC cases were analyzed using a multi-gene next-generation sequencing panel evaluating a total of 1360 amplicons in 24 HRR genes. Of the 50 cases, 23 specimens (46.0%) had an mCRPC harboring a pathogenic variant or a variant of uncertain significance (VUS), whereas in 27 mCRPCs (54.0%), no mutations were detected (wild-type tumors). BRCA2 was the most commonly mutated gene (14.0% of samples), followed by ATM (12.0%), and BRCA1 (6.0%). In conclusion, we have set up an NGS multi-gene panel that is capable of analyzing BRCA1/BRCA2 and HRR alterations in mCRPC. Moreover, our clinical algorithm is currently being used in clinical practice for the management of patients with mCRPC

A two-tiered mechanism of EGFR inhibition by RALT/MIG6 via kinase suppression and receptor degradation

The EGFR kinase inhibitor RALT/MIG6 also functions as an endocytic adaptor to promote receptor internalization by scaffolding AP-2 and intersectins

AIRO Breast Cancer Group Best Clinical Practice 2022 Update

Introduction: Breast cancer is the most common tumor in women and represents the leading cause of cancer death. Radiation therapy plays a key-role in the treatment of all breast cancer stages. Therefore, the adoption of evidence-based treatments is warranted, to ensure equity of access and standardization of care in clinical practice.Method: This national document on the highest evidence-based available data was developed and endorsed by the Italian Association of Radiation and Clinical Oncology (AIRO) Breast Cancer Group.We analyzed literature data regarding breast radiation therapy, using the SIGN (Scottish Intercollegiate Guidelines Network) methodology (www.sign.ac.uk). Updated findings from the literature were examined, including the highest levels of evidence (meta-analyses, randomized trials, and international guidelines) with a significant impact on clinical practice. The document deals with the role of radiation therapy in the treatment of primary breast cancer, local relapse, and metastatic disease, with focus on diagnosis, staging, local and systemic therapies, and follow up. Information is given on indications, techniques, total doses, and fractionations.Results: An extensive literature review from 2013 to 2021 was performed. The work was organized according to a general index of different topics and most chapters included individual questions and, when possible, synoptic and summary tables. Indications for radiation therapy in breast cancer were examined and integrated with other oncological treatments. A total of 50 questions were analyzed and answered.Four large areas of interest were investigated: (1) general strategy (multidisciplinary approach, contraindications, preliminary assessments, staging and management of patients with electronic devices); (2) systemic therapy (primary, adjuvant, in metastatic setting); (3) clinical aspects (invasive, non-invasive and micro-invasive carcinoma; particular situations such as young and elderly patients, breast cancer in males and cancer during pregnancy; follow up with possible acute and late toxicities; loco-regional relapse and metastatic disease); (4) technical aspects (radiation after conservative surgery or mastectomy, indications for boost, lymph node radiotherapy and partial breast irradiation).Appendixes about tumor bed boost and breast and lymph nodes contouring were implemented, including a dedicated web application. The scientific work was reviewed and validated by an expert group of breast cancer key-opinion leaders.Conclusions: Optimal breast cancer management requires a multidisciplinary approach sharing therapeutic strategies with the other involved specialists and the patient, within a coordinated and dedicated clinical path. In recent years, the high-level quality radiation therapy has shown a significant impact on local control and survival of breast cancer patients. Therefore, it is necessary to offer and guarantee accurate treatments according to the best standards of evidence-based medicine