Interaction between nanostructured materials and synthetic and plasmatic membranes

Understanding the interplay between nanostructured materials and cell membranes is the basis of their possible usage for therapeutics and for engineering new bio-applications. With the aim to unravel the mechanisms of interaction at the molecular scale, I have studied during my PhD the interaction between CdSe/CdS semiconductor nanorods (NRs) and polymeric micelles with model and plasmatic lipid membranes. NRs were in-house synthetized and functionalized with different amount of bis-amino polyetilenglycol (PEG) and a tertiary amine to tune their surface potential (\u3b6) between -50 mV and +10 mV. Their interaction with lipid mixtures of different composition in form of supported lipid bilayers (SLBs), lipid monolayers (LMs) and different in vitro cell lines was tested. In particular, NRs adsorption to SLBs was monitored by quartz crystal microbalance with dissipation monitoring (QCM-D) varying lipid mixtures charge and investigating the influence of gel phase domains; interactions with LMs same in composition as SLBs were measured by surface pressure-area isotherms. Results showed that tuning the mutual properties of the system regulates the interaction with NRs on the membranes and that the increase of membrane complexity inhibits it: in particular a strong interaction was registered with fluid state membranes and NRs opposite in charge when \u394\u3b6 > 70 mV, whereas the interaction was hindered in presence of gel phase domains. LMs models gave more detailed information, showing removal of lipid molecules from air-water interface or insertion of NRs between lipids according to the overall system charge. QCM-D and surface pressure-area isotherms results were in agreement. Since the polymer coating of the NRs was shown to regulate the interaction, in order to elucidate its effect I have employed also fluorescent polymeric micelles of different dimension (60 and 300 nm in diameter). I have tested the interaction of both NRs and micelles with different cell lines, namely post-natal mouse neuronal network (known to have a dynamically changing membrane potential), mouse neuroblastoma Neuro2a (that can differentiate in neuronal-like cells) and Chinese hamster ovary cells (epithelial, with a static membrane potential), using confocal microscopy both on fixed samples and in real time. Preliminary results showed adhesion of negatively charged NRs and micelles on both dynamic potential membrane cell lines. Again a threshold value was found for NRs interacting with neurons (\u3b6NR < -18 mV), similarly to what was observed with models. A neurotoxin was then introduced in the experiments, to reduce the spikes of the active cells. A satellite project is finally presented as a full paper at the end of the thesis. The project concerns the fabrication and characterization of thin anodic porous alumina (tAPA) substrates, which surface was made surface-enhanced Raman spectroscopy (SERS) -active by coating with a thin gold (Au) layer. My part in this project was related to the monitoring of the chemisorption of thiols and the formation of SLB models from lipid vesicles by using the QCM-D technique on Au substrates

High-level expression of a recombinant active microbial transglutaminase in Escherichia coli

Background: Bacterial transglutaminases are increasingly required as industrial reagents for in vitro modification of proteins in different fields such as in food processing as well as for enzymatic site-specific covalent conjugation of therapeutic proteins to polyethylene glycol to get derivatives with improved clinical performances. In this work we studied the production in Escherichia coli of a recombinant transglutaminase from Streptomyces mobaraensis (microbial transglutaminase or MTGase) as enzymatically active chimeric forms using different expression systems under the control of both lac promoter or thermoinducible phage lambda promoter. Results: Thermoinducible and constitutive expression vectors were constructed expressing Met-MTGase with chimeric LacZ 1-8 PNP 1–20 or LacZ 1–8 fusion protein under different promoters. After transformed in competent Escherichia coli K12 strains were fermented in batch and fed-bach mode in different mediums in order to select the best conditions of expression. The two most performing fusion protein systems namely short thermoinducible LacZ 1–8 Met-MTGase from NP668/1 and long constitutive LacZ 1-8 PNP 1–20 Met-MTGase from NP650/1 has been chosen to compare both efficiency of expression and biochemical qualities of the product. Proteins were extracted, purified to homogeneity and verified as a single peak obtained in RP-HPLC. The LacZ 1-8 PNP 1–20 Met-MTGase fusion protein purified from NP650/1 exhibited an activity of 15 U/mg compared to 24 U/mg for the shorter fusion protein purified from NP668/1 cell strain. Conclusions: Combining the experimental data on expression levels and specific activities of purified MTGase fusion proteins, the chimeric LacZ 1-8 Met-MTGase, which displays an enzymatic activity comparable to the wild-type enzyme, was selected as a candidate for producing microbial transglutaminase for industrial applications.Pubblicat

STARD3 and the identification of new cholesterol transport inhibitor

Although many advances in the cancer treatment have been made, the research is continuously searching new perspectives in order to provide the best possible outcome for all patients. An emerging challenge is the identification of new genes involved in cancer development and progression in order to develop novel therapeutic molecules to use alone or in combination with current therapies. In the last years, some research groups have focused their attention on a protein initially discovered to be overexpressed in breast cancer samples: the StAR-related lipid transfer domain-3 (STARD3). STARD3 is a member of a subfamily of lipid trafficking proteins characterized by a C-terminal steroidogenic acute regulatory domain (STARD), which shares a 35% of homology with the domain of StAR protein, STARD1, a transporter of cholesterol in mitochondria. They both belong to the START (steroidogenic acute regulatory protein\u2013related lipid transfer) proteins family, involved in the non-vesicular transport of lipids in membranes. The crystal structure of the START domain of STARD3 revealed a hydrophobic cavity formed by the \u3b1/\u3b2 helix grip structure of the 210 amino acids with which it binds one molecule of cholesterol at an equimolar ratio 1:1, transporting sterol from the endoplasmic reticulum (ER) to the endosomes. In human, it was demonstrated that STARD3 is overexpressed in different cancer cell lines and, in particular, in Her2 overexpressing breast cancer. In fact, STARD3 and HER2 are co-amplified and cooverexpressed in about 25% of breast cancers. The molecular mechanism by which STARD3 cooperates with others oncogene such as HER2 is still unclear. Nevertheless, STARD3 is implicated in therapy resistance of breast cancer, moreover, patients with a high level of STARD3 expression display metastasis, local recurrence and shorter overall survival. Recently, new evidences suggested a possible STARD3 overexpression also in colorectal, prostate and gastric cancers. Due to its involvement in cancer, STARD3 represents an attractive candidate as a target to cancer therapy and the identification of selective inhibitors is an undiscovered but interesting field of study. In collaboration with the University of Pisa that has developed the first pharmacophore-based virtual screening (VS) platform focuses on the identification of new inhibitors of the STARD3 mediated cholesterol transport, we carried out a study to identify a lead compound (VS1) endowed with an interesting activity, thus representing the first reported STARD3 inhibitor. The activity of the inhibitor was evaluated in breast and colorectal cancer cell lines by analyzing cell vitality and the level of focal adhesion kinase (FAK). Inhibition of STARD3 by VS1 results in a consistent reduction of cell vitality; additionally the activation of a specific STARD3 target (FAK) produced by the ligand, suggests a potent and specific activity of VS1 at cellular level

Next Generation Sequencing in rare childhood epilepsy of suspected genetic etiology

Mutations in several genes are associated with epilepsy (e.g. SCN1A, MECP2, ARX). Identifying genetic causes in epileptic syndromes is crucial to avoid a complex diagnostic work up, to provide genetic counseling, to start a tailored treatment in some cases and to avoid drugs potentially worsening seizures in others. Next Generation Sequencing (NGS) technologies allow analyzing a large number of genes in a single experiment, shortening the time to reach a definite diagnosis, and saving costs. Aim of this research was to identify gene variants underlying epilepsies with a challenging etiological classification. DNA from 81 pediatric epileptic patients was analyzed with a gene panel set up by child epileptologists, neurophysiologists and geneticists. This included 55 genes, later extended to 91, associated or not to intellectual disability, additional neurological signs, and complex malformations. In 14 patients pathogenic mutations were individuated, with an overall mutational frequency of 17,2% (14/81). 90,5% of patients had previously undergone unrevealing cytogenetic or single-gene analyses, thus our population was highly selected at the time NGS was performed. It is essential to underline that NGS must not be considered a screening examination, and that it requires a multidisciplinary approach in patients’ selection, and results interpretation

Surface-enhanced Raman scattering of self-assembled thiol monolayers and supported lipid membranes on thin anodic porous alumina

Thin anodic porous alumina (tAPA) was fabricated from a 500 nm thick aluminum (Al) layer coated on silicon wafers, through single-step anodization performed in a Teflon electrochemical cell in 0.4 M aqueous phosphoric acid at 110 V. Post-fabrication etching in the same acid allowed obtaining tAPA surfaces with ≈160 nm pore diameter and ≈80 nm corresponding wall thickness to be prepared. The tAPA surfaces were made SERS-active by coating with a thin (≈25 nm) gold (Au) layer. The as obtained tAPA–Au substrates were incubated first with different thiols, namely mercaptobenzoic acid (MbA) and aminothiol (AT), and then with phospholipid vesicles of different composition to form a supported lipid bilayer (SLB). At each step, the SERS substrate functionality was assessed, demonstrating acceptable enhancement (≥100×). The chemisorption of thiols during the first step and the formation of SLB from the vesicles during the second step, were independently monitored by using a quartz crystal microbalance with dissipation monitoring (QCM-D) technique. The SLB membranes represent a simplified model system of the living cells membranes, which makes the successful observation of SERS on these films promising in view of the use of tAPA–Au substrates as a platform for the development of surface-enhanced Raman spectroscopy (SERS) biosensors on living cells. In the future, these tAPA–Au-SLB substrates will be investigated also for drug delivery of bioactive agents from the APA pores

Anemia in Elderly Patients—The Impact of Hemoglobin Cut-Off Levels on Geriatric Domains

Background: The primary aim of this study was to evaluate the impact of anemia—according to the WHO criteria—on cognitive performances, mood, functional and nutritional status, and comorbidities in a population of subjects aged 65 years or older. The secondary aim of this study was to understand if different hemoglobin cut-off levels are associated with a variation of the mentioned domains’ impairment. Methods: We designed a cross-sectional study, including subjects aged 65 or more consecutively evaluated in an outpatient setting from July 2013 to December 2019. A sum of 1698 subjects met the inclusion criteria. They were evaluated with: MMSE and CDT (cognitive assessment), GDS (mood), BADL, IADL, PPT, and POMA (autonomies), MNA (nutritional status), and CIRS (comorbidities). Results: According to the WHO criteria, non-anemic patients reported significantly better performances than the anemics in BADL (p p = 0.0007), PPT (p = 0.0278), POMA (p = 0.0235), MNA, CIRS TOT, CIRS ICC, and CIRS ISC (p p = 0.0072), CIRS (OR: 1.08, p p = 0.0007) were significant regressors of anemia, while considering the 13 g/dL-cut-off level, age (OR: 1.04, p = 0.0001), POMA (OR: 1.03, p = 0.0172), MNA (OR = 0.95, p = 0.0036), CIRS (OR: 1.17, p p = 0.018), and gender (OR = 0.48, p p > 0.01). Conclusions: Our study showed that anemia negatively impact on geriatric people’s general status, regardless of which hemoglobin cut-off level is considered. It also highlighted that hemoglobin concentrations < 13 g/dL, regardless of gender, have an association with the impairment of the affective-functional-nutritional state as well as an increase in comorbidities; therefore, it should be pursuable to consider the elderly person “anemic” if Hb < 13 g/dL regardless of gender