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.

Caratterizzazione ed isolamento di intermedi delle reazioni di sostituzione elettrofila e nucleofila in serie aromatica

1,3,5–Tris(N,N-dialkylamino)benzene derivatives are strongly activated neutral carbon nucleophiles able to stress some reactivity aspects toward more or less activated electrophilic substrates. These very interesting electron-rich benzenes have been firstly synthesized in 1967 and extensively studied. Their supernucleophilic character permits to perform reactions in particularly mild conditions, and make them suitable for mechanistic investigations. In many reactions they permit to isolate –complexes in electrophilic aromatic reactions. The possibility to form moderately stable Wheland intermediates depends both, on the activation of the reagents and on the experimental conditions which makes slow the proton elimination in the re-aromatization process. In presence of a carbon super electrophile reagent as 4,6-dinitrobenzofuroxan or 4,6-dinitrotetrazolepiridine, 1,3,5–tris(N,N-dialkylamino)benzene derivatives afford C–C coupling products which are “double σ complexes”, Wheland–like on the 1,3,5-tris(N,N-dialkylamino)benzene moiety, and Meisenheimer–like on the electrophile moiety. We named these complexes as Wheland–Meisenheimer (W-M) complexes. These complexes are moderately stable at low temperature and they were characterized by NMR spectroscopy methods. Others nucleophile reagents as 2-aminothiazole derivatives give a Wheland-Meisenheimer complex with 4,6-dinitrobenzofuroxan.

ATG based combination therapy - a novel clinically relevant approach to promote regulation and induce long-term allograft survival

Regulatory T cells (Treg) actively regulate alloimmune responses and promote transplantation tolerance. Polyclonal anti-thymocyte globulin (ATG), a widely used induction therapy in clinical organ transplantation, depletes peripheral T cells. However, resistance to tolerance induction is seen with certain T cell depleting strategies and is attributed to alterations in the balance of naïve, memory and regulatory T cells. Here we report a novel reagent, murine ATG (mATG), depletes T cells but preferentially spares CD25+ natural Tregs which limit skewing of T cell repertoire toward T-effector-memory (Tem) phenotype among the recovering T cells. T-cell depletion with mATG combined with CTLA4Ig and Sirolimus synergize to prolong graft survival by tipping the Treg/Tem balance further in favor of Tregs by preserving Tregs, facilitating generation of new Tregs by a conversion mechanism and limiting Tem expansion in response to alloantigen and homeostatic proliferation. These results provide the rationale for translating such novel combination therapies to promote tolerance in primate and human organ transplantation.

Pyrolysis-Gas Chromatography-Mass Spectometry and Chemometric Analysis for the Characterization of Complex Matrices

The present PhD thesis was focused on the development and application of chemical methodology (Py-GC-MS) and data-processing method by multivariate data analysis (chemometrics). The chromatographic and mass spectrometric data obtained with this technique are particularly suitable to be interpreted by chemometric methods such as PCA (Principal Component Analysis) as regards data exploration and SIMCA (Soft Independent Models of Class Analogy) for the classification. As a first approach, some issues related to the field of cultural heritage were discussed with a particular attention to the differentiation of binders used in pictorial field. A marker of egg tempera the phosphoric acid esterified, a pyrolysis product of lecithin, was determined using HMDS (hexamethyldisilazane) rather than the TMAH (tetramethylammonium hydroxide) as a derivatizing reagent. The validity of analytical pyrolysis as tool to characterize and classify different types of bacteria was verified. The FAMEs chromatographic profiles represent an important tool for the bacterial identification. Because of the complexity of the chromatograms, it was possible to characterize the bacteria only according to their genus, while the differentiation at the species level has been achieved by means of chemometric analysis. To perform this study, normalized areas peaks relevant to fatty acids were taken into account. Chemometric methods were applied to experimental datasets. The obtained results demonstrate the effectiveness of analytical pyrolysis and chemometric analysis for the rapid characterization of bacterial species. Application to a samples of bacterial (Pseudomonas Mendocina), fungal (Pleorotus ostreatus) and mixed- biofilms was also performed. A comparison with the chromatographic profiles established the possibility to: • Differentiate the bacterial and fungal biofilms according to the (FAMEs) profile. • Characterize the fungal biofilm by means the typical pattern of pyrolytic fragments derived from saccharides present in the cell wall. • Individuate the markers of bacterial and fungal biofilm in the same mixed-biofilm sample.

Synthesis and aplication of linear and macrocyclic nitrogen ligand

Linear and macrocyclic nitrogen ligands have been found wide application during the years. Nitrogen has a much strong association with transition-metal ions because the electron pair is partucularly available for complexing purposes. We started our investigation with the synthesis of new chiral perazamacrocycles containing four pyrrole rings. This ligand was synthesized by the [2+2]condensation of (R,R)-diaminocyclohexane and dipirranedialdehydes and was tested, after a complexation with Cu(OAc)2, in Henry reactions. The best yields (96%) and higher ee’s (96%) were obtained when the meso-substituent on the dipyrrandialdehyde was a methyl group. The positive influence of the pyrrole-containing macrocyclic structure on the efficiency/enantioselectivity of the catalytic system was demonstrated by comparison with the Henry reactions performed using analogous ligands. Henry product was obtain in good yield but only 73% of ee, when the dialdehyde unit was replaced by a triheteroaromatic dialdehye (furan-pyrrol-furan). Another well known macrocyclic ligand is calix[4]pyrrole. We decided to investigate, in collaboration with Neier’s group, the metal-coordinating properties of calix[2]pyrrole[2]pyrrolidine compounds obtained by the reduction of calix[4]pyrrole. We focused our attention on the reduction conditions, and tested different Pd supported (charcoal, grafite) catalysts at different condition. Concerning the synthesis of linear polyamine ligands. We focused our attention to the synthesis of 2-heteroaryl- and 2,5-diheteroarylpyrrolidines. The reductive amination reaction of diarylketones and aryl-substitutedketo-aldehydes with different chiral amines was exploited to prepare a small library of diastereo-enriched substituted pyrrolidines. We have also described a new synthetic route to 1,2-disubstituted 1,2,3,4-tetrahydropyrrole[1,2-a]pyrazines, which involves the diastereoselective addition of Grignard reagents to chiral oxazolidines. The best diastereoselectivity (98:2) was dependent on the nature of both the chiral auxiliary, (S)-1-phenylglycinol, and the nature of the organometallic reagent (MeMgBr).

Development and characterization of micromachined devices for separation techniques

Nowadays microfluidic is becoming an important technology in many chemical and biological processes and analysis applications. The potential to replace large-scale conventional laboratory instrumentation with miniaturized and self-contained systems, (called lab-on-a-chip (LOC) or point-of-care-testing (POCT)), offers a variety of advantages such as low reagent consumption, faster analysis speeds, and the capability of operating in a massively parallel scale in order to achieve high-throughput. Micro-electro-mechanical-systems (MEMS) technologies enable both the fabrication of miniaturized system and the possibility of developing compact and portable systems. The work described in this dissertation is towards the development of micromachined separation devices for both high-speed gas chromatography (HSGC) and gravitational field-flow fractionation (GrFFF) using MEMS technologies. Concerning the HSGC, a complete platform of three MEMS-based GC core components (injector, separation column and detector) is designed, fabricated and characterized. The microinjector consists of a set of pneumatically driven microvalves, based on a polymeric actuating membrane. Experimental results demonstrate that the microinjector is able to guarantee low dead volumes, fast actuation time, a wide operating temperature range and high chemical inertness. The microcolumn consists of an all-silicon microcolumn having a nearly circular cross-section channel. The extensive characterization has produced separation performances very close to the theoretical ideal expectations. A thermal conductivity detector (TCD) is chosen as most proper detector to be miniaturized since the volume reduction of the detector chamber results in increased mass and reduced dead volumes. The microTDC shows a good sensitivity and a very wide dynamic range. Finally a feasibility study for miniaturizing a channel suited for GrFFF is performed. The proposed GrFFF microchannel is at early stage of development, but represents a first step for the realization of a highly portable and potentially low-cost POCT device for biomedical applications.