“Rational design of nanoparticles to improve anticancer drug delivery”

The need to improve current cancer therapies is a pivotal point in the drug delivery systems. A major challenge is to disclose new strategies that can combine the use of targeted nanoparticles (NPs) to the efficient delivery of chemotherapeutic agents in the vicinity of tumor masses, minimizing the side effects on healthy cells. To this purpose, a preliminary step is that of defining the optimal NP characteristics able to improve drug delivery at target tissues. However, these aspects need to be defined in suitable models that can actually mimic the main cell activities, including adhesion, migration and differentiation. These features are instead missed when cells are grown on flat plastic dishes, as it occurs in traditional two dimensional (2D) systems; thus, the 2D unnatural environment can provide inaccurate data, failing to predict the in vivo real cell response to NP treatments. In this context, the main goal of this thesis has been that of identifying the key parameters useful for a rational design of anticancer drug delivery systems. To this aim, the following issues have been addressed: - the effect of three dimensional (3D) extracellular matrix, made up of collagen type I, in controlling the diffusion and cellular uptake of NPs with variable size and surface charge; - the cytotoxic efficacy of biodegradable NPs to deliver the Doxorubicin (Dox) anticancer drug in three dimensional matrices, as a function of size; - the possibility to control “on demand” Dox release, in order to reach a more efficient tumor-specific targeting. Results indicate that, in 3D environment, size, surface charge and functionalization are all crucial NP features that can modulate their ability to diffuse through the ECM and finally reach the cells. In parallel, the same properties also influence the cytotoxic effects and the cellular responses, with smaller sizes facilitating diffusion through the collagen matrix and increasing the efficiency of NP treatments. Finally, the covalent conjugation of Dox to NPs through cleavable linkers allowed obtaining a more controlled drug release. This type of nanocarrier was made specifically responsive to matrix metalloproteinase-2 (MMP2), which is known to be over-expressed in the tumor extracellular matrix, so that the drug release from NPs, as the relative cytotoxic effect, were specifically triggered by MMP2 cleavage. Altogether, the results obtained indicate that the physical-chemical characteristics of NPs and their behaviour in a 3D environment, that better mimics in vivo growth conditions, are crucial parameters that need to be taken into consideration for a more rational design of nanocarriers finalized to drug delivery in tumor tissues

Receptor tyrosine kinase-dependent PI3K activation is an escape mechanism to vertical suppression of the EGFR/RAS/MAPK pathway in KRAS-mutated human colorectal cancer cell lines

Colorectal cancer; Epidermal growth factor receptor (EGFR); MAPK pathwayCàncer colorectal; Receptor epidèrmic de factor del creixement (EGFR); Via MAPKCáncer colorrectal; Receptor epidérmico de factor del crecimiento (EGFR); Vía MAPKBACKGROUND: Previous studies showed that the combination of an anti-Epidermal growth factor (EGFR) and a MEK-inhibitor is able to prevent the onset of resistance to anti-EGFR monoclonal antibodies in KRAS-wild type colorectal cancer (CRC), while the same combination reverts anti-EGFR primary resistance in KRAS mutated CRC cell lines. However, rapid onset of resistance is a limit to combination therapies in KRAS mutated CRC. METHODS: We generated four different KRAS mutated CRC cell lines resistant to a combination of cetuximab (an anti-EGFR antibody) and refametinib (a selective MEK-inhibitor) after continuous exposure to increasing concentration of the drugs. We characterized these resistant cell lines by evaluating the expression and activation status of a panel of receptor tyrosine kinases (RTKs) and intracellular transducers by immunoblot and qRT-PCR. Oncomine comprehensive assay and microarray analysis were carried out to investigate new acquired mutations or transcriptomic adaptation, respectively, in the resistant cell lines. Immunofluorescence assay was used to show the localization of RTKs in resistant and parental clones. RESULTS: We found that PI3K-AKT pathway activation acts as an escape mechanism in cell lines with acquired resistance to combined inhibition of EGFR and MEK. AKT pathway activation is coupled to the activation of multiple RTKs such as HER2, HER3 and IGF1R, though its pharmacological inhibition is not sufficient to revert the resistant phenotype. PI3K pathway activation is mediated by autocrine loops and by heterodimerization of multiple receptors. CONCLUSIONS: PI3K activation plays a central role in the acquired resistance to the combination of anti-EGFR and MEK-inhibitor in KRAS mutated colorectal cancer cell lines. PI3K activation is cooperatively achieved through the activation of multiple RTKs such as HER2, HER3 and IGF1R

Dyskerin Downregulation Can Induce ER Stress and Promote Autophagy via AKT-mTOR Signaling Deregulation

: Dyskerin is an evolutionarily conserved nucleolar protein implicated in a wide range of fundamental biological roles, including telomere maintenance and ribosome biogenesis. Germline mutations of DKC1, the human gene encoding dyskerin, cause the hereditary disorders known as X-linked dyskeratosis congenita (X-DC). Moreover, dyskerin is upregulated in several cancers. Due to the pleiotropic functions of dyskerin, the X-DC clinical features overlap with those of both telomeropathies and ribosomopathies. In this paper, we evaluate the telomerase-independent effects of dyskerin depletion on cellular physiology by using inducible DCK1 knockdown. This system allows the downregulation of DKC1 expression within a short timeframe. We report that, in these cellular systems, dyskerin depletion induces the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum, which in turn induces the activation of the PERK branch of the unfolded protein response. We also demonstrate that the PERK-eIF2a-ATF4-CHOP signaling pathway, activated by dyskerin downregulation, triggers a functional autophagic flux through the inhibition of the PI3K/AKT/mTOR pathway. By revealing a novel unpredicted connection between the loss of dyskerin, autophagy and UPR, our results establish a firm link between the lowering of dyskerin levels and the activation of the ER stress response, that plays a key role in the pathogenesis of several diseases

Serum miR-502: A potential biomarker in the diagnosis of concussion in a pilot study of patients with normal structural brain imaging

Establishing a diagnosis of concussion within the context of competitive sport is frequently difficult due to the heterogeneity of presentation. Over the years, many endogenous proteins, including the recent Food and Drug Administration approved for mild-to-moderate traumatic brain injury, glial fibrillary acid protein and ubiquitin carboxy-terminal hydrolase, have been studied as potential biomarkers for the diagnosis of mild traumatic brain injury. Recently, a new class of potential biomarkers, the microRNAs, has shown promise as indicators of traumatic brain injury. In this pilot study, we have analysed the ability of pre-validated serum microRNAs (mi-425-5p and miR-502) to diagnose concussion, in cases without structural pathology. Their performance has been assessed alongside a set of identified protein biomarkers for traumatic brain injury in cohort of 41 concussed athletes. Athletes with a confirmed concussion underwent blood sampling after 48 h from concussion along with magnetic resonance imaging. Serum mi-425-5p and miR-502 were analysed by quantitative reverse transcription polymerase chain reaction, and digital immunoassay was used to determine serum concentrations of ubiquitin carboxy-terminal hydrolase, glial fibrillary acid protein, neurofilament light and Tau. Results were matched with 15 healthy volunteers. No structural/haemorrhagic pathology was identified. Protein biomarkers demonstrated variability among groups reflecting previous performance in the literature. Neurofilament light was the only marker to positively correlate with symptoms reported and SCAT5 scores. Despite the sub optimal timing of sampling beyond the optimal window for many of the protein biomarkers measured, miR-502 was significantly downregulated at all time points within a week form concussion ictus, showing a diagnostic sensitivity in cases beyond 48 h and without structural pathology

Il ruolo del'ingegneria nella valorizzazione dei prodotti tipici montani

Il presente lavoro si inserisce nell'ambito del progetto "Metodi e sistemi per aumentare il valore aggiunto degli alimenti tradizionali ed a vocazione territoriale" il cui obiettivo è mettere a punto metodi, strumenti e tecnologie per il recupero e la valorizzazione di produzioni tradizionali di aree geografiche sensibili dal punto di vista socio-ambientale, quali le aree montante, al fine di promuoverne lo sviluppo. Lo studio si è articolato in 3 fasi: - definizione della metodologia; - caratterizzazione e analisi del territorio e delle realtà produttive; - ottimizzazione delle produzioni e strategie di sviluppo territoriale. Sono state individuate 5 filiere produttive costituite principalmente da micro-realtà artigianali e di ciascuna sono stati analizzati gli ambiti produttivo, tecnologico, organizzativo e gestionale. L'analisi, oltre a fotografare la situazione attuale, ha evidenziato le criticità igienico-sanitarie delle produzioni alimentati e della sicurezza e benessere degli operatori. In sintesi, i principali obiettivi raggiunti sono stati: - ottimizzazione dell'attuale produzione e messa a punto di linee produttive alternative; - individuazione dell'attuale produzione e messa a punto di linee produttive alternative; - individuazione degli elementi peculiari delle filiere al fine di proporre disciplinari di produzione; - ottimizzazione meccanico-impiantistica dell'efficienza, produttività, sicurezza e benessere degli operatori; - proposta di nuove tipologie di macchine e impianti dedicati; - sostenbilità del processo produttivo atraverso l'utilizzo di fonti energetiche alternative; - recupero e potenziamento delle infrastrutture presenti sul territorio per migliorare la logistica degli approvvigionamenti e della commercializzazione dei prodotti e l'accessibilità al territorio; - definizione di nuovi criteri progettuali delle strutture produttive e di layout innovativi; - messa a punto di sistemi di controllo al fine di garantire la qualità del prodotto. In conclusione la metodologia si è rivelata adeguata nell'analizzare in modo sistematico le filiere, individuandone i limiti. Inoltre ha permesso di suggerire tecnologie e strategie valide per la gestione del passaggio da attività amatoriale ad attività imprenditoriale

A functional connection between dyskerin and energy metabolism

The human DKC1 gene encodes dyskerin, an evolutionarily conserved nuclear protein whose overexpression represents a common trait of many types of aggressive sporadic cancers. As a crucial component of the nuclear H/ACA snoRNP complexes, dyskerin is involved in a variety of essential processes, including telomere maintenance, splicing efficiency, ribosome biogenesis, snoRNAs stabilization and stress response. Although multiple minor dyskerin splicing isoforms have been identified, their functions remain to be defined. Considering that low-abundance splice variants could contribute to the wide functional repertoire attributed to dyskerin, possibly having more specialized tasks or playing significant roles in changing cell status, we investigated in more detail the biological roles of a truncated dyskerin isoform that lacks the C-terminal nuclear localization signal and shows a prevalent cytoplasmic localization. Here we show that this dyskerin variant can boost energy metabolism and improve respiration, ultimately conferring a ROS adaptive response and a growth advantage to cells. These results reveal an unexpected involvement of DKC1 in energy metabolism, highlighting a previously underscored role in the regulation of metabolic cell homeostasis

Severity of experimental traumatic brain injury modulates changes in concentrations of cerebral free amino acids

In this study, concentrations of free amino acids (FAA) and amino group containing compounds (AGCC) following graded diffuse traumatic brain injury (mild TBI, mTBI; severe TBI, sTBI) were evaluated. After 6, 12, 24, 48 and 120 hr aspartate (Asp), glutamate (Glu), asparagine (Asn), serine (Ser), glutamine (Gln), histidine (His), glycine (Gly), threonine (Thr), citrulline (Cit), arginine (Arg), alanine (Ala), taurine (Tau), γ-aminobutyrate (GABA), tyrosine (Tyr), S-adenosylhomocysteine (SAH), l-cystathionine (l-Cystat), valine (Val), methionine (Met), tryptophane (Trp), phenylalanine (Phe), isoleucine (Ile), leucine (Leu), ornithine (Orn), lysine (Lys), plus N-acetylaspartate (NAA) were determined in whole brain extracts (n = 6 rats at each time for both TBI levels). Sham-operated animals (n = 6) were used as controls. Results demonstrated that mTBI caused modest, transient changes in NAA, Asp, GABA, Gly, Arg. Following sTBI, animals showed profound, long-lasting modifications of Glu, Gln, NAA, Asp, GABA, Ser, Gly, Ala, Arg, Citr, Tau, Met, SAH, l-Cystat, Tyr and Phe. Increase in Glu and Gln, depletion of NAA and Asp increase, suggested a link between NAA hydrolysis and excitotoxicity after sTBI. Additionally, sTBI rats showed net imbalances of the Glu-Gln/GABA cycle between neurons and astrocytes, and of the methyl-cycle (demonstrated by decrease in Met, and increase in SAH and l-Cystat), throughout the post-injury period. Besides evidencing new potential targets for novel pharmacological treatments, these results suggest that the force acting on the brain tissue at the time of the impact is the main determinant of the reactions ignited and involving amino acid metabolism

Photobiomodulation improves functional recovery after mild traumatic brain injury

Mild traumatic brain injury (mTBI) is a common consequence of head injury but there are no recognized interventions to promote recovery of the brain. We previously showed that photobiomodulation (PBM) significantly reduced the number of apoptotic cells in adult rat hippocampal organotypic slice cultures. In this study, we first optimized PBM delivery parameters for use in mTBI, conducting cadaveric studies to calibrate 660 and 810 nm lasers for transcutaneous delivery of PBM to the cortical surface. We then used an in vivo weight drop mTBI model in adult rats and delivered daily optimized doses of 660, 810 nm, or combined 660/810 nm PBM. Functional recovery was assessed using novel object recognition (NOR) and beam balance tests, whilst histology and immunohistochemistry were used to assess the mTBI neuropathology. We found that PBM at 810, 660 nm, or 810/660 nm all significantly improved both NOR and beam balance performance, with 810 nm PBM having the greatest effects. Histology demonstrated no overt structural damage in the brain after mTBI, however, immunohistochemistry using brain sections showed significantly reduced activation of both CD11b+ microglia and glial fibrillary acidic protein (GFAP)+ astrocytes at 3 days post-injury. Significantly reduced cortical localization of the apoptosis marker, cleaved caspase-3, and modest reductions in extracellular matrix deposition after PBM treatment, limited to choroid plexus and periventricular areas were also observed. Our results demonstrate that 810 nm PBM optimally improved functional outcomes after mTBI, reduced markers associated with apoptosis and astrocyte/microglial activation, and thus may be useful as a potential regenerative therapy