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I solventi clorurati sono composti organici di sintesi appartenenti alla classe degli idrocarburi alifatici clorurati, largamente impiegati in diversi settori industriali e, come conseguenza, ampiamente diffusi nell’ambiente come inquinanti e particolarmente pericolosi anche per la salute umana. Tra i metodi di bonifica di siti contaminati da solventi clorurati, quelli basati sul biorisanamento sfruttano uno dei peculiari metabolismi batterici coinvolti nella biodegradazione di tali composti: la declorurazione riduttiva anaerobia in cui il composto clorurato è utilizzato come accettore terminale di elettroni in un vero e proprio processo di respirazione anaerobia, detta dealorespirazione; il cometabolismo aerobio in cui il composto clorurato viene fortuitamente ossidato ad opera di ossigenasi batteriche normalmente utilizzate per l’uptake del substrato di crescita preferenziale; l’ossidazione aerobia diretta in cui il composto clorurato funge da donatore di elettroni e substrato di crescita primario per il microrganismo. Il presente lavoro di tesi di dottorato si è occupato della caratterizzazione microbiologica di un’acqua contaminata da solventi clorurati, prevalentemente da 1,2-dicloroetano (1,2-DCA), e della valutazione del suo potenziale di biodegradazione, con l’obiettivo di sfruttare consorzi microbici autoctoni alla matrice contaminata per la definizione di un eventuale intervento di biorisanamento in situ. Le comunità microbiche autoctone dell’acqua contaminata sono state dapprima caratterizzate in termini di struttura, diversità e composizione, mediante ARISA (Automated Ribosomal Intergenic Spacer Analysis) e 16S rRNA metabarcoding, evidenziando una grande variabilità: campioni di acqua raccolti da diversi punti di prelievo e in diversi periodi sono risultati infatti piuttosto eterogenei, sia da un punto di vista chimico-fisico che da un punto di vista microbiologico. Abbastanza limitata è risultata la presenza di specie batteriche note in grado di degradare solventi clorurati. La biodegradazione di 1,2-DCA è stata successivamente valutata mediante analisi gascromatografiche in spazio di testa effettuate in microcosmo. Allo scopo di indagare tutti i metabolismi biodegradativi noti nei confronti di 1,2-DCA, sono stati allestiti e opportunamente biostimolati microcosmi in aerobiosi e anaerobiosi. I microcosmi sono stati realizzati all’interno di vials con acqua contaminata, un opportuno terreno di coltura e una quota aggiunta di 1,2-DCA. Il monitoraggio della concentrazione di 1,2-DCA nel tempo ha mostrato elevati abbattimenti nei microcosmi anaerobi biostimolati in presenza/assenza di lattato e nei microcosmi biostimolati in presenza di O2. In entrambi, la caratterizzazione della comunità microbica ha evidenziato la presenza di batteri in grado di svolgere declorurazione riduttiva di solventi clorurati e dealogenazione idrolitica di 1,2-DCA. Nel DNA metagenomico dei suddetti microcosmi è stata anche rilevata la presenza di sequenze dei geni per una dealogenasi riduttiva specifica per 1,2-DCA (dcaA) e per la aloalcano dealogenasi (dhlA). Un’efficiente biodegradazione di 1,2-DCA è stata evidenziata anche in una simulazione di sistema di Barriera Reattiva Permeabile in colonna biostimolato con poli-β-idrossibutirrato (PHB), quale substrato fermentabile e donatore di elettroni a lento rilascio per i processi di declorurazione riduttiva. Il sistema in colonna è stato alimentato con l’acqua contaminata, inoculata con un consorzio batterico precedentemente arricchito su PHB. Infine, è stata avviata una sperimentazione che vede l’utilizzo di carrier biopolimerici biodegradabili come innovativa tecnologia di immobilizzazione di batteri biodegradatori direttamente reclutati dall’acqua contaminata, allo scopo di ottenere una maggiore efficienza di biodegradazione di solventi clorurati, a basso costo e in maniera ecocompatibile. Supporti in acido polilattico hanno dato preliminari evidenze sia di assorbimento del contaminante 1,2-DCA, sia di adesione microbica quando immersi nell’acqua contaminata in un sistema a scala di microcosmo.

The binomial “autophagy-cancer” is intricate and methylomic studies can help to understand it by changing point of view from a gene level to an -omic one. Recently, autophagy-modulating properties of several phytochemicals have attracted attention in anticancer research. We evaluated whether Indicaxanthin (IND), the peculiar known beneficial phytochemical of prickly pear, seasonally available in the southern Italy, could induce autophagy in Caco2 cells, and whether it results from an epigenomic modification and/or a direct molecular interaction. IND increased autophagy in Caco-2 cells; the methylomic signature, obtained by Reduced Representation Bisulfite Sequencing (15 million of clusters) reported that 14 main genes of autophagy, showed a different methylation consistent with the induction of this phenomenon. Among these: MTOR, ATG13, BECN1, TFEB, ATG3, WIPI2, TECPR1, SNAP29, VPS11, VPS16. By traditional approaches we confirmed the demethylation of BECN1 gene and the increase of Beclin1 levels. By in-silico molecular modelling, we displayed a possible interference of IND, by competitive mechanisms, in the Beclin1-Bcl2 interaction. Methylomic signature and molecular modelling has been helpful to understand autophagy IND-induced in intestinal epithelial tumour cells. Our results suggest that the pro-autophagic action promoted by this phytochemical involves both epigenomic modulation and post-translation mechanisms by direct interaction with key targets of autophagy pathway.

Gliomas are the prevalent forms of brain cancer and derive from glial cells. Among them, astrocytomas are the most frequent. Astrocytes are fundamental for most brain functions, as they contribute to neuronal metabolism and neurotransmission. When they acquire cancer properties, their functions are altered, and, in addition, they start invading the brain parenchyma. Thus, a better knowledge of transformed astrocyte molecular properties is essential. With this aim, we previously developed rat astrocyte clones with increasing cancer properties. In this study, we used proteomic analysis to compare the most transformed clone (A-FC6) with normal primary astrocytes. We found that 154 proteins are downregulated and 101 upregulated in the clone. Moreover, 46 proteins are only expressed in the clone and 82 only in the normal cells. Notably, only 11 upregulated/unique proteins are encoded in the duplicated q arm of isochromosome 8 (i(8q)), which cytogenetically characterizes the clone. Since both normal and transformed brain cells release extracellular vesicles (EVs), which might induce epigenetic modifications in the neighboring cells, we also compared EVs released from transformed and normal astrocytes. Interestingly, we found that the clone releases EVs containing proteins, such as matrix metalloproteinase 3 (MMP3), that can modify the extracellular matrix, thus allowing invasion.

The production of polyhydroxyalkanoates (PHA) using industrial wastewaters as feedstocks is a current and challenging topic. This study investigated the production of biopolymers by a mixed microbial culture under different OLRs equal to 1 kgCOD m-3d-1 (Period 1), 2 kgCOD m-3d-1 (Period 2) and 3 kgCOD m-3d-1 (Period 3). The maximum PHA content was achieved in Period 2 (0.38 gPHA gTSS-1), whereas lower values were obtained in Period 1 (0.13 gPHA gTSS-1) and Period 3 (0.26 gPHA gTSS-1). Overall, the maximum PHA productivity resulted equal to 0.08 gPHA L-1h-1 (P2), 0.05 gPHA L-1h-1 (P1) and 0.04 gPHA L-1h-1 (P3), respectively. The molecular weight of the PHA increased from Period 1 (250 kDa) to Period 2 (417 KDa) and Period 3 (463 KDa), although resulting in a slight decrease of crystallinity degree. Microbial community analysis, revealed a reduction in bacterial diversity and a progressive shift of the microbial community with the increasing OLR. Alpha-diversity indexes based on Operational Taxonomic Units (OTUs) at 99% identity revealed higher species richness (Taxa (S) 280) and diversity (Shannon (H) 4,06) in Period 1, whereas Period 3 was characterized by reduced richness and diversity and higher dominance (Taxa (S) 133, Shannon (H) 2,40). Based on the results obtained, it was pointed out that the OLR variation determined significant effects on the process performances, as well as on the productivity and quality of the biopolymers. This means that OLR is a key control parameter to maximize the PHA production and control the physical-chemical characteristics of the polymers.

Gliomas are complex and heterogeneous tumors that originate from the glial cells of the brain. The malignant cells undergo deep modifications of their metabolism, and acquire the capacity to invade the brain parenchyma and to induce epigenetic modifications in the other brain cell types. In spite of the efforts made to define the pathology at the molecular level, and to set novel approaches to reach the infiltrating cells, gliomas are still fatal. In order to gain a better knowledge of the cellular events that accompany astrocyte transformation, we developed three increasingly transformed astrocyte cell lines, starting from primary rat cortical astrocytes, and analyzed them at the cytogenetic and epigenetic level. In parallel, we also studied the expression of the differentiation-related H1.0 linker histone variant to evaluate its possible modification in relation with transformation. We found that the most modified astrocytes (A-FC6) have epigenetic and chromosomal alterations typical of cancer, and that the other two clones (A-GS1 and A-VV5) have intermediate properties. Surprisingly, the differentiation-specific somatic histone H1.0 steadily increases from the normal astrocytes to the most transformed ones. As a whole, our results suggest that these three cell lines, together with the starting primary cells, constitute a potential model for studying glioma development.

We examined the effects of the ferrocene-based histone deacetylase-3 inhibitor Pojamide (N1-(2-aminophenyl)-N8-ferrocenyloctanediamide) and its two derivatives N1-(2-aminophenyl)-N6-ferrocenyladipamide and N1-(2-aminophenyl)-N8-ferroceniumoctanediamide tetrafluoroborate on triple-negative MDA-MB-231 breast cancer cells. Viability/growth assays indicated that only the first two compounds at 70 &mu, M concentration caused an approximate halving of cell number after 24 h of exposure, whereas the tetrafluoroborate derivative exerted no effect on cell survival nor proliferation. Flow cytometric and protein blot analyses were performed on cells exposed to both Pojamide and the ferrocenyladipamide derivative to evaluate cell cycle distribution, apoptosis/autophagy modulation, and mitochondrial metabolic state in order to assess the cellular basis of the cytotoxic effect. The data obtained show that the cytotoxic effect of the two deacetylase inhibitors may be ascribed to the onset of non-apoptotic cell death conceivably linked to a down-regulation of autophagic processes and an impairment of mitochondrial function with an increase in intracellular reactive oxygen species. Our work expands the list of autophagy-regulating drugs and also provides a further example of the role played by the inhibition of autophagy in breast cancer cell death. Moreover, the compounds studied may represent attractive and promising targets for subsequent molecular modeling for anti-neoplastic agents in malignant breast cancer.

The carbazole compounds PK9320 (1-(9-ethyl-7-(furan-2-yl)-9H-carbazol-3-yl)-N-methylmethanamine) and PK9323 (1-(9-ethyl-7-(thiazol-4-yl)-9H-carbazol-3-yl)-N-methylmethanamine), second-generation analogues of PK083 (1-(9-ethyl-9H-carbazol-3-yl)-N-methylmethanamine), restore p53 signaling in Y220C p53-mutated cancer cells by binding to a mutation-induced surface crevice and acting as molecular chaperones. In the present paper, these three molecules have been tested for mutant p53-independent genotoxic and epigenomic effects on wild-type p53 MCF-7 breast adenocarcinoma cells, employing a combination of Western blot for phospho-γH2AX histone, Comet assay and methylation-sensitive arbitrarily primed PCR to analyze their intrinsic DNA damage-inducing and DNA methylation-changing abilities. We demonstrate that small modifications in the substitution patterns of carbazoles can have profound effects on their intrinsic genotoxic and epigenetic properties, with PK9320 and PK9323 being eligible candidates as “anticancer compounds” and “anticancer epi-compounds” and PK083 a “damage-corrective” compound on human breast adenocarcinoma cells. Such different properties may be exploited for their use as anticancer agents and chemical probes.

Human parathyroid hormone-related protein (PTHrP) is a polyhormone that undergoes proteolytic cleavage producing smaller peptides which exert diversified biological effects. PTHrP signalization is prominently involved in breast development and physiology, but the studies have been focused onto either N-terminal species or full-length protein introduced by gene transfer techniques. Our present work investigates for the first time the effect of the mid-region PTHrP secretory form, that is, the fragment [38-94], on HB2 non-tumoral breast epithelial cells. We examined viability/proliferation of cells grown in PTHrP-containing media supplemented with different serum concentration and on different substrates, extending our investigation to check whether (a) by analogy with MDA-MB231 cells, also HB2 cell chromatin possesses genome-wide binding sites for mid-region PTHrP, and (b) the peptide is endowed with modulating properties toward the expression of proliferation/differentiation signatures by HB2 cells. Our results indicate that mid-region PTHrP acts as a cell growth/differentiation stimulator in routine and "nutrient stress" culture conditions, accordingly reprogramming gene expression, and is able to bind to cytogenetic preparations from HB2 cells. This supports the concept that the physiological mechanisms involving PTHrP during breast development may include mature processed forms of the protein different from the N-terminal fragment. © 2018 BioFactors, 45(2):279-288, 2019.