Study of apoptotic deletion mediated by Bifidobacterium longum with construction of recombinant strains for Serpin encoding gene and phenotypes comparison in a pig cell model

The first part of the research project of the Co-Advisorship Ph.D Thesis was aimed to select the best Bifidobacterium longum strains suitable to set the basis of our study. We were looking for strains with the abilities to colonize the intestinal mucosa and with good adhesion capacities, so that we can test these strains to investigate their ability to induce apoptosis in “damaged” intestinal cells. Adhesion and apoptosis are the two process that we want to study to better understand the role of an adhesion protein that we have previously identified and that have top scores homologies with the recent serpin encoding gene identified in B. longum by Nestlè researchers. Bifidobacterium longum is a probiotic, known for its beneficial effects to the human gut and even for its immunomodulatory and antitumor activities. Recently, many studies have stressed out the intimate relation between probiotic bacteria and the GIT mucosa and their influence on human cellular homeostasis. We focused on the apoptotic deletion of cancer cells induced by B. longum. This has been valued in vitro, performing the incubation of three B.longum strains with enterocyte-like Caco- 2 cells, to evidence DNA fragmentation, a cornerstone of apoptosis. The three strains tested were defined for their adhesion properties using adhesion and autoaggregation assays. These features are considered necessary to select a probiotic strain. The three strains named B12, B18 and B2990 resulted respectively: “strong adherent”, “adherent” and “non adherent”. Then, bacteria were incubated with Caco-2 cells to investigate apoptotic deletion. Cocultures of Caco-2 cells with B. longum resulted positive in DNA fragmentation test, only when adherent strains were used (B12 and B18). These results indicate that the interaction with adherent B. longum can induce apoptotic deletion of Caco-2 cells, suggesting a role in cellular homeostasis of the gastrointestinal tract and in restoring the ecology of damaged colon tissues. These results were used to keep on researching and the strains tested were used as recipient of recombinant techniques aimed to originate new B.longum strains with enhanced capacity of apoptotic induction in “damaged” intestinal cells. To achieve this new goal it was decided to clone the serpin encoding gene of B. longum, so that we can understand its role in adhesion and apoptosis induction. Bifidobacterium longum has immunostimulant activity that in vitro can lead to apoptotic response of Caco-2 cell line. It secretes a hypothetical eukaryotic type serpin protein, which could be involved in this kind of deletion of damaged cells. We had previously characterised a protein that has homologies with the hypothetical serpin of B. longum (DD087853). In order to create Bifidobacterium serpin transformants, a B. longum cosmid library was screened with a PCR protocol using specific primers for serpin gene. After fragment extraction, the insert named S1 was sub-cloned into pRM2, an Escherichia coli - Bifidobacterium shuttle vector, to construct pRM3. Several protocols for B. longum transformation were performed and the best efficiency was obtained using MRS medium and raffinose. Finally bacterial cell supernatants were tested in a dotblot assay to detect antigens presence against anti-antitrypsin polyclonal antibody. The best signal was produced by one starin that has been renamed B. longum BLKS 7. Our research study was aimed to generate transformants able to over express serpin encoding gene, so that we can have the tools for a further study on bacterial apoptotic induction of Caco-2 cell line. After that we have originated new trasformants the next step to do was to test transformants abilities when exposed to an intestinal cell model. In fact, this part of the project was achieved in the Department of Biochemistry of the Medical Faculty of the University of Maribor, guest of the abroad supervisor of the Co-Advisorship Doctoral Thesis: Prof. Avrelija Cencic. In this study we examined the probiotic ability of some bacterial strains using intestinal cells from a 6 years old pig. The use of intestinal mammalian cells is essential to study this symbiosis and a functional cell model mimics a polarised epithelium in which enterocytes are separated by tight junctions. In this list of strains we have included the Bifidobacterium longum BKS7 transformant strain that we have previously originated; in order to compare its abilities. B. longum B12 wild type and B. longum BKS7 transformant and eight Lactobacillus strains of different sources were co-cultured with porcine small intestine epithelial cells (PSI C1) and porcine blood monocytes (PoM2) in Transwell filter inserts. The strains, including Lb. gasseri, Lb. fermentum, Lb. reuterii, Lb. plantarum and unidentified Lactobacillus from kenyan maasai milk and tanzanian coffee, were assayed for activation of cell lines, measuring nitric oxide by Griess reaction, H202 by tetramethylbenzidine reaction and O2 - by cytochrome C reduction. Cytotoxic effect by crystal violet staining and induction on metabolic activity by MTT cell proliferation assay were tested too. Transepithelial electrical resistance (TER) of polarised PSI C1 was measured during 48 hours co-culture. TER, used to observe epithelium permeability, decrease during pathogenesis and tissue becomes permeable to ion passive flow lowering epithelial barrier function. Probiotics can prevent or restore increased permeability. Lastly, dot-blot was achieved against Interleukin-6 of treated cells supernatants. The metabolic activity of PoM2 and PSI C1 increased slightly after co-culture not affecting mitochondrial functions. No strain was cytotoxic over PSI C1 and PoM2 and no cell activation was observed, as measured by the release of NO2, H202 and O2 - by PoM2 and PSI C1. During coculture TER of polarised PSI C1 was two-fold higher comparing with constant TER (~3000 ) of untreated cells. TER raise generated by bacteria maintains a low permeability of the epithelium. During treatment Interleukin-6 was detected in cell supernatants at several time points, confirming immunostimulant activity. All results were obtained using Lactobacillus paracasei Shirota e Carnobacterium divergens as controls. In conclusion we can state that both the list of putative probiotic bacteria and our new transformant strain of B. longum are not harmful when exposed to intestinal cells and could be selected as probiotics, because can strengthen epithelial barrier function and stimulate nonspecific immunity of intestinal cells on a pig cell model. Indeed, we have found out that none of the strains tested that have good adhesion abilities presents citotoxicity to the intestinal cells and that non of the strains tested can induce cell lines to produce high level of ROS, neither NO2. Moreover we have assayed even the capacity of producing certain citokynes that are correlated with immune response. The detection of Interleukin-6 was assayed in all our samples, including B.longum transformant BKS 7 strain, this result indicates that these bacteria can induce a non specific immune response in the intestinal cells. In fact, when we assayed the presence of Interferon-gamma in cells supernatant after bacterial exposure, we have no positive signals, that means that there is no activation of a specific immune response, thus confirming that these bacteria are not recognize as pathogen by the intestinal cells and are certainly not harmful for intestinal cells. The most important result is the measure of Trans Epithelial Electric Resistance that have shown how the intestinal barrier function get strengthen when cells are exposed to bacteria, due to a reduction of the epithelium permeability. We have now a new strain of B. longum that will be used for further studies above the mechanism of apoptotic induction to “damaged cells” and above the process of “restoring ecology”. This strain will be the basis to originate new transformant strains for Serpin encoding gene that must have better performance and shall be used one day even in clinical cases as in “gene therapy” for cancer treatment and prevention.

Mitochondrial Genomics: structure, inheritance and phylogenetic utility of the Mitochondrial genome in Bivalvia and Insecta

This PhD Thesis is the result of my research activity in the last three years. My main research interest was centered on the evolution of mitochondrial genome (mtDNA), and on its usefulness as a phylogeographic and phylogenetic marker at different taxonomic levels in different taxa of Metazoa. From a methodological standpoint, my main effort was dedicated to the sequencing of complete mitochondrial genomes, and the approach to whole-genome sequencing was based on the application of Long-PCR and shotgun sequences. Moreover, this research project is a part of a bigger sequencing project of mtDNAs in many different Metazoans’ taxa, and I mostly dedicated myself to sequence and analyze mtDNAs in selected taxa of bivalves and hexapods (Insecta). Sequences of bivalve mtDNAs are particularly limited, and my study contributed to extend the sampling. Moreover, I used the bivalve Musculista senhousia as model taxon to investigate the molecular mechanisms and the evolutionary significance of their aberrant mode of mitochondrial inheritance (Doubly Uniparental Inheritance, see below). In Insects, I focused my attention on the Genus Bacillus (Insecta Phasmida). A detailed phylogenetic analysis was performed in order to assess phylogenetic relationships within the genus, and to investigate the placement of Phasmida in the phylogenetic tree of Insecta. The main goal of this part of my study was to add to the taxonomic coverage of sequenced mtDNAs in basal insects, which were only partially analyzed.

"Water users must be efficient producers". Women's access to and use of land in Chókwè irrigation scheme, Mozambique

The irrigation scheme Eduardo Mondlane, situated in Chókwè District - in the Southern part of the Gaza province and within the Limpopo River Basin - is the largest in the country, covering approximately 30,000 hectares of land. Built by the Portuguese colonial administration in the 1950s to exploit the agricultural potential of the area through cash-cropping, after Independence it became one of Frelimo’s flagship projects aiming at the “socialization of the countryside” and at agricultural economic development through the creation of a state farm and of several cooperatives. The failure of Frelimo’s economic reforms, several infrastructural constraints and local farmers resistance to collective forms of production led to scheme to a state of severe degradation aggravated by the floods of the year 2000. A project of technical rehabilitation initiated after the floods is currently accompanied by a strong “efficiency” discourse from the managing institution that strongly opposes the use of irrigated land for subsistence agriculture, historically a major livelihood strategy for smallfarmers, particularly for women. In fact, the area has been characterized, since the end of the XIX century, by a stable pattern of male migration towards South African mines, that has resulted in an a steady increase of women-headed households (both de jure and de facto). The relationship between land reform, agricultural development, poverty alleviation and gender equality in Southern Africa is long debated in academic literature. Within this debate, the role of agricultural activities in irrigation schemes is particularly interesting considering that, in a drought-prone area, having access to water for irrigation means increased possibilities of improving food and livelihood security, and income levels. In the case of Chókwè, local governments institutions are endorsing the development of commercial agriculture through initiatives such as partnerships with international cooperation agencies or joint-ventures with private investors. While these business models can sometimes lead to positive outcomes in terms of poverty alleviation, it is important to recognize that decentralization and neoliberal reforms occur in the context of financial and political crisis of the State that lacks the resources to efficiently manage infrastructures such as irrigation systems. This kind of institutional and economic reforms risk accelerating processes of social and economic marginalisation, including landlessness, in particular for poor rural women that mainly use irrigated land for subsistence production. The study combines an analysis of the historical and geographical context with the study of relevant literature and original fieldwork. Fieldwork was conducted between February and June 2007 (where I mainly collected secondary data, maps and statistics and conducted preliminary visit to Chókwè) and from October 2007 to March 2008. Fieldwork methodology was qualitative and used semi-structured interviews with central and local Government officials, technical experts of the irrigation scheme, civil society organisations, international NGOs, rural extensionists, and water users from the irrigation scheme, in particular those women smallfarmers members of local farmers’ associations. Thanks to the collaboration with the Union of Farmers’ Associations of Chókwè, she has been able to participate to members’ meeting, to education and training activities addressed to women farmers members of the Union and to organize a group discussion. In Chókwè irrigation scheme, women account for the 32% of water users of the familiar sector (comprising plot-holders with less than 5 hectares of land) and for just 5% of the private sector. If one considers farmers’ associations of the familiar sector (a legacy of Frelimo’s cooperatives), women are 84% of total members. However, the security given to them by the land title that they have acquired through occupation is severely endangered by the use that they make of land, that is considered as “non efficient” by the irrigation scheme authority. Due to a reduced access to marketing possibilities and to inputs, training, information and credit women, in actual fact, risk to see their right to access land and water revoked because they are not able to sustain the increasing cost of the water fee. The myth of the “efficient producer” does not take into consideration the characteristics of inequality and gender discrimination of the neo-liberal market. Expecting small-farmers, and in particular women, to be able to compete in the globalized agricultural market seems unrealistic, and can perpetuate unequal gendered access to resources such as land and water.

Transcriptional functions of DNA Topoisomerases at a genome-wide scale and a single gene levels

The DNA topology is an important modifier of DNA functions. Torsional stress is generated when right handed DNA is either over- or underwound, producing structural deformations which drive or are driven by processes such as replication, transcription, recombination and repair. DNA topoisomerases are molecular machines that regulate the topological state of the DNA in the cell. These enzymes accomplish this task by either passing one strand of the DNA through a break in the opposing strand or by passing a region of the duplex from the same or a different molecule through a double-stranded cut generated in the DNA. Because of their ability to cut one or two strands of DNA they are also target for some of the most successful anticancer drugs used in standard combination therapies of human cancers. An effective anticancer drug is Camptothecin (CPT) that specifically targets DNA topoisomerase 1 (TOP 1). The research project of the present thesis has been focused on the role of human TOP 1 during transcription and on the transcriptional consequences associated with TOP 1 inhibition by CPT in human cell lines. Previous findings demonstrate that TOP 1 inhibition by CPT perturbs RNA polymerase (RNAP II) density at promoters and along transcribed genes suggesting an involvement of TOP 1 in RNAP II promoter proximal pausing site. Within the transcription cycle, promoter pausing is a fundamental step the importance of which has been well established as a means of coupling elongation to RNA maturation. By measuring nascent RNA transcripts bound to chromatin, we demonstrated that TOP 1 inhibition by CPT can enhance RNAP II escape from promoter proximal pausing site of the human Hypoxia Inducible Factor 1 (HIF-1) and c-MYC genes in a dose dependent manner. This effect is dependent from Cdk7/Cdk9 activities since it can be reversed by the kinases inhibitor DRB. Since CPT affects RNAP II by promoting the hyperphosphorylation of its Rpb1 subunit the findings suggest that TOP 1inhibition by CPT may increase the activity of Cdks which in turn phosphorylate the Rpb1 subunit of RNAP II enhancing its escape from pausing. Interestingly, the transcriptional consequences of CPT induced topological stress are wider than expected. CPT increased co-transcriptional splicing of exon1 and 2 and markedly affected alternative splicing at exon 11. Surprisingly despite its well-established transcription inhibitory activity, CPT can trigger the production of a novel long RNA (5’aHIF-1) antisense to the human HIF-1 mRNA and a known antisense RNA at the 3’ end of the gene, while decreasing mRNA levels. The effects require TOP 1 and are independent from CPT induced DNA damage. Thus, when the supercoiling imbalance promoted by CPT occurs at promoter, it may trigger deregulation of the RNAP II pausing, increased chromatin accessibility and activation/derepression of antisense transcripts in a Cdks dependent manner. A changed balance of antisense transcripts and mRNAs may regulate the activity of HIF-1 and contribute to the control of tumor progression After focusing our TOP 1 investigations at a single gene level, we have extended the study to the whole genome by developing the “Topo-Seq” approach which generates a map of genome-wide distribution of sites of TOP 1 activity sites in human cells. The preliminary data revealed that TOP 1 preferentially localizes at intragenic regions and in particular at 5’ and 3’ ends of genes. Surprisingly upon TOP 1 downregulation, which impairs protein expression by 80%, TOP 1 molecules are mostly localized around 3’ ends of genes, thus suggesting that its activity is essential at these regions and can be compensate at 5’ ends. The developed procedure is a pioneer tool for the detection of TOP 1 cleavage sites across the genome and can open the way to further investigations of the enzyme roles in different nuclear processes.

Social inclusion of vulnerable groups through participatory and emancipatory approaches. Implementing active citizenship and socially innovative actions in the framework of civil & human rights model of disability

The research hypothesis of the thesis is that “an open participation in the co-creation of the services and environments, makes life easier for vulnerable groups”; assuming that the participatory and emancipatory approaches are processes of possible actions and changes aimed at facilitating people’s lives. The adoption of these approaches is put forward as the common denominator of social innovative practices that supporting inclusive processes allow a shift from a medical model to a civil and human rights approach to disability. The theoretical basis of this assumption finds support in many principles of Inclusive Education and the main focus of the hypothesis of research is on participation and emancipation as approaches aimed at facing emerging and existing problems related to inclusion. The framework of reference for the research is represented by the perspectives adopted by several international documents concerning policies and interventions to promote and support the leadership and participation of vulnerable groups. In the first part an in-depth analysis of the main academic publications on the central themes of the thesis has been carried out. After investigating the framework of reference, the analysis focuses on the main tools of participatory and emancipatory approaches, which are able to connect with the concepts of active citizenship and social innovation. In the second part two case studies concerning participatory and emancipatory approaches in the areas of concern are presented and analyzed as example of the improvement of inclusion, through the involvement and participation of persons with disability. The research has been developed using a holistic and interdisciplinary approach, aimed at providing a knowledge-base that fosters a shift from a situation of passivity and care towards a new scenario based on the person’s commitment in the elaboration of his/her own project of life.

Sistemi tecnologici innovativi di involucro per il recupero del patrimonio edilizio recente. L'edilizia scolastica nel Comune di Bologna

La ricerca è volta a presentare un nuovo approccio integrato, a supporto di operatori e progettisti, per la gestione dell’intero processo progettuale di interventi di riqualificazione energetica e architettonica del patrimonio edilizio recente, mediante l’impiego di soluzioni tecnologiche innovative di involucro edilizio. Lo studio richiede necessariamente l’acquisizione di un repertorio selezionato di sistemi costruttivi di involucro, come base di partenza per l’elaborazione di soluzioni progettuali di recupero delle scuole appartenenti al secondo dopoguerra, in conglomerato cementizio armato, prevalentemente prefabbricate. Il progetto individua procedimenti costruttivi ecocompatibili per la progettazione di componenti prefabbricati di involucro “attivo”, adattabile ed efficiente, da assemblare a secco, nel rispetto dei requisiti prestazionali richiesti dalle attuali normative. La ricerca è finalizzata alla gestione dell’intero processo, supportato da sistemi di rilevazione geometrica, collegati a software di programmazione parametrica per la modellazione di superfici adattabili alla morfologia dei fabbricati oggetto di intervento. Tali strumenti informatizzati CAD-CAM sono connessi a macchine a controllo numerico CNC per la produzione industrializzata degli elementi costruttivi “su misura”. A titolo esemplificativo dell’approccio innovativo proposto, si formulano due possibili soluzioni di involucro in linea con i paradigmi della ricerca, nel rispetto dei principi di sostenibilità, intesa come modularità, rapidità di posa, reversibilità, recupero e riciclo di materiali. In particolare, le soluzioni innovative sono accomunate dall’applicazione di una tecnica basata sull’assemblaggio di elementi prefabbricati, dall’adozione di una trama esagonale per la tassellazione della nuova superficie di facciata, e dall’utilizzo del medesimo materiale termico isolante, plastico e inorganico, riciclato ed ecosostenibile, a basso impatto ambientale (AAM - Alkali Activated Materials). Le soluzioni progettuali proposte, sviluppate presso le due sedi coinvolte nella cotutela (Università di Bologna, Université Paris-Est) sono affrontate secondo un protocollo scientifico che prevede: progettazione del sistema costruttivo, analisi meccanica e termica, sperimentazione costruttiva, verifica delle tecniche di messa in opera e dei requisiti prestazionali.

Characterization of mitochondrial genomes in bivalve species with doubly uniparental inheritance of mitochondria

Many bivalve species possess two distinct mtDNA lineages, called F and M, respectively inherited maternally and paternally: this system is called doubly uniparental inheritance (DUI). The main experimental project of my PhD was the quantification of the two mtDNAs during the development of the DUI species Ruditapes philippinarum, from early embryos to sub-adults, using Real-Time qPCR. I identified the time interval in which M mtDNA is lost from female individuals, while it is retained in males (which are heteroplasmic through all of their life cycle). The results also suggested absence of mtDNA replication during early embryogenesis, a process constituting a bottleneck that highly reduces the copy number of mtDNA molecules in cells of developing larvae. In males this bottleneck may produce cells homoplasmic for M mtDNA, and could be considered as a first step of the segregation of M in the male germ line. Another finding was the characterization, in young clams approaching the first reproductive season, of a significant boost in copy number of F mtDNA in females and of M in males. Given the age of animals in which this mtDNA-specific growth was observed, the finding could probably be the outcome of the first round of gonads and gametes production. Other lines of research included the characterization of the unassigned regions in mt genomes of DUI bivalves. These regions can harbor signals involved in the control of replication and/or transcription of the mtDNA molecule, as well as additional open reading frames (ORFs) not related to oxidative phosphorylation. These features in DUI species could be associated to the maintenance of separate inheritance routes for the two mtDNAs. Additional ORFs are also found in other animal mt genomes: I summarized the presence of gene duplications as a co-author in a review focusing on animal mt genomes with unusual gene content.