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.

Topological analysis of singularity loci for serial and parallel manipulators

Singularities of robot manipulators have been intensely studied in the last decades by researchers of many fields. Serial singularities produce some local loss of dexterity of the manipulator, therefore it might be desirable to search for singularityfree trajectories in the jointspace. On the other hand, parallel singularities are very dangerous for parallel manipulators, for they may provoke the local loss of platform control, and jeopardize the structural integrity of links or actuators. It is therefore utterly important to avoid parallel singularities, while operating a parallel machine. Furthermore, there might be some configurations of a parallel manipulators that are allowed by the constraints, but nevertheless are unreachable by any feasible path. The present work proposes a numerical procedure based upon Morse theory, an important branch of differential topology. Such procedure counts and identify the singularity-free regions that are cut by the singularity locus out of the configuration space, and the disjoint regions composing the configuration space of a parallel manipulator. Moreover, given any two configurations of a manipulator, a feasible or a singularity-free path connecting them can always be found, or it can be proved that none exists. Examples of applications to 3R and 6R serial manipulators, to 3UPS and 3UPU parallel wrists, to 3UPU parallel translational manipulators, and to 3RRR planar manipulators are reported in the work.

Sustained-release formulations for compounds underlying intestinal drug efflux

ZusammenfassungDie Sekretion von Arzneistoffen aus Darmzellen zurück ins Darmlumen, die durch intestinale Transporter wie P-Glykoprotein (P-GP) vermittelt wird, stellt eine bekannte Quelle für unvollständige und variable Bioverfügbarkeiten und für Interaktionen mit anderen Arzneimitteln und Nahrungsbestandteilen dar. Dennoch liegen bisher keine Veröffentlichungen vor, die sich mit daraus resultierenden Konsequenzen für die Entwicklung neuer peroraler Darreichungsformen befassen. Ziel der vorliegenden Arbeit war es, deutlich zu machen, dass dem Auftreten von intestinalen Sekretionsphänomenen bei der Entwicklung von Retardarzneimitteln Rechnung getragen werden muss.Dazu wurden effektive Permeabilitäten für den Modellarzneistoff Talinolol in unterschiedlichen Darmabschnitten anhand eines Rattendarmperfusionsmodells bestimmt.Des weiteren wurde eine Retardformulierung für den Modellarzneistoff Talinolol entwickelt. Dabei wurde gezeigt, dass die Verwendung unterschiedlicher Puffer als Wirkstofffreisetzungmedien zur Ausbildung unterschiedlicher Talinolol-Kristallstrukturen führt.Die neu entwickelten Retardmatrixtabletten wurden mit Hilfe des Pharmakokinetik-Computersoftwareprogrammes Gastro Plus® evaluiert. Das Zusammenspiel von verlangsamter Wirkstofffreigabe aus der Arzneiform und intestinaler Sekretion führte zu einer deutlich verringerten Bioverfügbarkeit der Modellsubstanz Talinolol aus der Retardformulierung im Vergleich zu schnellfreisetzenden Arzneiformen.Daher sollte der Einfluß intestinaler sekretorischer Transporter wie P-GP bei der Entwicklung von Retardarzneiformen unbedingt berücksichtigt werden.

Epidemiological and functional characterization of Streptococcus pneumoniae pili

Streptococcus pneumoniae is an important life threatening human pathogen causing agent of invasive diseases such as otitis media, pneumonia, sepsis and meningitis, but is also a common inhabitant of the respiratory tract of children and healthy adults. Likewise most streptococci, S. pneumoniae decorates its surface with adhesive pili, composed of covalently linked subunits and involved in the attachment to epithelial cells and virulence. The pneumococcal pili are encoded by two genomic regions, pilus islet 1 (PI-1), and pilus islet-2 (PI-2), which are present in about 30% and 16% of the pneumococcal strains, respectively. PI-1 exists in three clonally related variants, whereas PI-2 is highly conserved. The presence of the islets does not correlate with the serotype of the strains, but with the genotype (as determined by Multi Locus Sequence Typing). The prevalence of PI-1 and PI-2 positive strains is similar in isolates from invasive disease and carriage. To better dissect a possible association between PIs presence and disease we evaluated the distribution of the two PIs in a panel of 113 acute otitis media (AOM) clinical isolates from Israel. PI-1 was present in 30.1% (N=34) of the isolates tested, and PI-2 in 7% (N=8). We found that 50% of the PI-1 positive isolates belonged to the international clones Spain9V-3 (ST156) and Taiwan19F-14 (ST236), and that PI-2 was not present in the absence of Pl-1. In conclusion, there was no correlation between PIs presence and AOM, and, in general, the observed differences in PIs prevalence are strictly dependent upon regional differences in the distribution of the clones. Finally, in the AOM collection the prevalence of PI-1 was higher among antibiotic resistant isolates, confirming previous indications obtained by the in silico analysis of the MLST database collection. Since the pilus-1 subunits were shown to confer protection in mouse models of infection both in active and passive immunization studies, and were regarded as potential candidates for a new generation of protein-based vaccines, the functional characterization was mainly focused on S. pneumoniae pilus -1 components. The pneumococcal pilus-1 is composed of three subunits, RrgA, RrgB and RrgC, each stabilized by intra-molecular isopeptide bonds and covalently polymerized by means of inter-molecular isopeptide bonds to form an extended fibre. The pilus shaft is a multimeric structure mainly composed by the RrgB backbone subunit. The minor ancillary proteins are located at the tip and at the base of the pilus, where they have been proposed to act as the major adhesin (RrgA) and as the pilus anchor (RrgC), respectively. RrgA is protective in in vivo mouse models, and exists in two variants (clades I and II). Mapping of the sequence variability onto the RrgA structure predicted from X-ray data showed that the diversity was restricted to the “head” of the protein, which contains the putative binding domains, whereas the elongated “stalk” was mostly conserved. To investigate whether this variability could influence the adhesive capacity of RrgA and to map the regions important for binding, two full-length protein variants and three recombinant RrgA portions were tested for adhesion to lung epithelial cells and to purified extracellular matrix (ECM) components. The two RrgA variants displayed similar binding abilities, whereas none of the recombinant fragments adhered at levels comparable to those of the full-length protein, suggesting that proper folding and structural arrangement are crucial to retain protein functionality. Furthermore, the two RrgA variants were shown to be cross-reactive in vitro and cross-protective in vivo in a murine model of passive immunization. Taken together, these data indicate that the region implicated in adhesion and the functional epitopes responsible for the protective ability of RrgA may be conserved and that the considerable level of variation found within the “head” domain of RrgA may have been generated by immunologic pressure without impairing the functional integrity of the pilus.

Bifidobacterium - Human Host Interaction: Role of Human Plasminogen

Bifidobacterium is an important genus of the human gastrointestinal microbiota, affecting several host physiological features. Despite the numerous Bifidobacterium related health-promoting activities, there is still a dearth of information about the molecular mechanisms at the basis of the interaction between this microorganism and the host. Bacterial surface associated proteins may play an important role in this interaction because of their ability to intervene with host molecules, as recently reported for the host protein plasminogen. Plasminogen is the zymogen of the trypsin-like serine protease plasmin, an enzyme with a broad substrate specificity. Aim of this thesis is to deepen the knowledge about the interaction between Bifidobacterium and the human plasminogen system and its role in the Bifidobacterium-host interaction process. As a bifidobacterial model, B. animalis subsp. lactis BI07 has been used because of its large usage in dairy and pharmaceutical preparations. We started from the molecular characterization of the interaction between plasminogen and one bifidobacterial plasminogen receptor, DnaK, a cell wall protein showing high affinity for plasminogen, and went on with the study of the impact of intestinal environmental factors, such as bile salts and inflammation, on the plasminogen-mediated Bifidobacterium-host interaction. According to our in vitro findings, by enhancing the activation of the bifidobacterial bound plasminogen to plasmin, the host inflammatory response results in the decrease of the bifidobacterial adhesion to the host enterocytes, favouring bacterial migration to the luminal compartment. Conversely, in the absence of inflammation, plasminogen acts as a molecular bridge between host enterocytes and bifidobacteria, enhancing Bifidobacterium adhesion. Furthermore, adaptation to physiological concentrations of bile salts enhances the capability of this microorganism to interact with the host plasminogen system. The host plasminogen system thus represents an important and flexible tool used by bifidobacteria in the cross-talk with the host.

Impact of folate absorption and transport for nutrition and drug targeting

In this thesis, interactions of folic acid with tea and tea components at the level of intestinal absorption have been investigated. Firstly, the interaction between folic acid and tea as well as tea catechins was studied in vitro, using Caco-2 cell monolayers and secondly, a clinical trial was designed and carried out. In addition, targeting of folic acid conjugated nanoparticles to FR expressing Caco-2 cells was studied in order to evaluate the principle of nutrient-receptor-coupled transport for drug targeting. In the first part of this work, it was shown that EGCG and ECG (gallated catechins) inhibit folic acid uptake (IC50 of 34.8 and 30.8 µmol/L) comparable to MTX (methotrexate) under these experimental conditions. Moreover, commercial green and black tea extracts inhibited folic acid uptake with IC50 values of approximately 7.5 and 3.6 mg/mL, respectively. These results clearly indicate an interaction between folic acid and green tea catechins at the level of intestinal uptake. The mechanism responsible for the inhibition process might be the inhibition of the influx transport routes for folates such as via RFC and/or PCFT. For understanding the in vivo relevance of this in vitro interaction, a phase one, open-labeled, randomized, cross-over clinical study in seven healthy volunteers was designed. For the 0.4 mg folic acid dose, the mean Cmax decreased by 39.2% and 38.6% and the mean AUC0 decreased by 26.6% and 17.9% by green tea and black tea, respectively. For the 5 mg folic acid dose, the mean Cmax decreased by 27.4% and mean AUC0 decreased by 39.9% when taken with green tea. The results of the clinical study confirm the interaction between tea and folic acid in vivo leading to lower bioavailabilities of folic acid. In the second part of the thesis, targeting studies using folic acid conjugated nanoparticles were conducted. Folic acid conjugated nanoparticles were shown to be internalized by the cell via FR (folate receptor) mediated endocytosis. DNA block copolymer micelles equipped with 2, 11 and 28 folic acid units respectively were applied on FR expressing Caco-2 cells. There was a direct proportion in the amount of internalized nanoparticle and the number of folic acid units on the periphery of the nanoparticle. To sum up, throughout this thesis, the importance of folic acid for nutrition and nutrient and drug related interactions of folic acid at intestinal level was shown. Furthermore, significance of FRs in targeting for cancer chemotherapy was demonstrated in in vitro cell culture experiments. Folic acid conjugated DNA block copolymer micelles were suggested as efficient nanoparticles for targeted drug delivery.

Identifizierung und Charakterisierung von Intermediärfilament-Organisatoren in Caenorhabditis elegans

Intermediärfilamente (IFs) sind neben Mikrotubuli und Aktinfilamenten die dritte filamentäre Komponente des Zytoskeletts. Sie wirken als mechanische Stabilisatoren, sind außerdem an Zelldifferenzierung, Proliferation und Apoptose beteiligt und tragen zu Zellpolarität bei. IFs sind dynamische Strukturen, die zelltypspezifisch in unterschiedlichen Anordnungen und Abundanzen vorkommen und von Signalkaskaden beeinflusst werden. Die zugrundeliegenden molekularen Mechanismen dieser fein abgestimmten Prozesse sind weitgehend unbekannt. In dieser Arbeit sollte deswegen ein Tiermodell entwickelt werden, um Regulatoren der IF-(Netzwerk)-Organisation in vivo zu untersuchen und zu identifizieren. Dazu wurde C. elegans ausgewählt, da es sich hierbei um einen genetisch gut charakterisierten und leicht manipulierbaren Organismus handelt, in dessen Genom elf Gene für zytoplasmatische IFs kodieren. Zunächst wurden stabil transgene C. elegans-Linien generiert, die fluoreszierende IFs exprimieren. Es konnte gezeigt werden, dass das darmspezifische IFB-2::CFP im Bereich des apikalen Junktionskomplex verankert ist und nahezu vollständig im subapikalen Terminalgeflecht der Enterozyten lokalisiert, das als Teil der endotube besonders stabil und widerstandsfähig ist. Wenn diese Tiere mit dsRNA gegen das ebenfalls im Terminalgeflecht exprimierte IF ifc-2 behandelt wurden, entwickelten sich blasenförmige Ausstülpungen des Darmlumens, die auf eine Schwächung der rigiden und formgebenden endotube hinwiesen und damit einen direkten in vivo-Beweis für die stressprotektive Funktion des intestinalen IF-Netzwerks lieferten. Die leichte Detektierbarkeit des IFB-2::CFP-Musters wurde in einem optischen Screen ausgenutzt, bei dem nach chemischer Mutagenese nach Veränderungen im IF-Muster gefahndet wurde. Hierbei wurden drei Mutanten isoliert. In Komplementationsanalysen stellte sich heraus, dass es sich in zwei Fällen um Allele desselben Gens handelt. Die Identifizierung der betroffenen Gene gelang durch eine PCR-basierte Kartierung von single nucleotide polymorphisms nach Verpaarung mit dem Hawaii-Stamm (snp-mapping) und anschließender RNAi-Analyse der Einzelgene in den identifizierten Chromosomenabschnitten. Im einen Fall handelte es sich um das sma-5-Gen, einer Serin/Threonin-Kinase mit Homologie zu den MAP-Kinasen MAPK7/ERK5 der Säuger. Hier wurden, ebenso wie beim ifc-2 (RNAi)-Phänotyp, progressive blasenförmige Ausstülpungen des Darmlumens beobachtet. Die beiden anderen Allele tragen Mutationen in einem bisher nicht näher charakterisierten Gen. In diesen Würmern kommt es zu einem vollständigen Auflösung des IFB-2::CFP-Netzwerks mit prominenten Akkumulationen um die apikalen Junktionen. Das Darmlumen ist stellenweise geweitet und das elektronendichte Terminalgeflecht fehlt fast vollständig, die Integrität des Darmepithels ist jedoch nicht kompromittiert. Die anderen IFs des Terminalgeflechts sind ebenfalls fehlverteilt, und die intestinale Expression von Aktin ist stark reduziert. Expressionskonstrukte des Gens zeigten weiterhin, dass es darmspezifisch synthetisiert wird und mit den IFs im Terminalgeflecht kolokalisiert. Das Protein ist, ähnlich wie das IF-assoziierte Filaggrin der Säuger ausgesprochen histidinreich. Es enthält außerdem eine Prolin-reiche Domäne, die Teil einer potentiellen Aktin-Bindedomäne ist. Auf Grund all dieser Eigenschaften wird die Bezeichnung IFO-1 (intermediate filament organizer) für das neue Protein vorgeschlagen, das möglicherweise als struktureller Zytoskelett-Linker wirkt. Die vorgestellten Ergebnisse untermauern die Bedeutung von C. elegans für die Identifizierung von Faktoren, die IF-Netzwerke regulieren, und die Möglichkeit, Defekte im lebenden Gesamtorganismus zu bestimmen.

On the development of novel cocaine-analogues for in vivo imaging of the dopamine transporter status

The present thesis is concerned with the development of novel cocaine-derived dopamine transporter ligands for the non-invasive exploration of the striatal and extra-striatal dopamine transporter (DAT) in living systems. The presynaptic dopamine transporter acquires an important function within the mediation of dopaminergic signal transduction. Its availability can serve as a measure for the overall integrity of the dopaminergic system. The DAT is upregulated in early Parkinson’s disease (PD), resulting in an increased availability of DAT-binding sites in the striatal DAT domains. Thereby, DAT imaging has become an important routine diagnostic tool for the early diagnosis of PD in patients, as well as for the differentiation of PD from symptomatically similar medical conditions. Furthermore, the dopaminergic system is involved in a variety of psychiatric diseases. In this regard, DAT-selective imaging agents may provide detailed insights into the scientific understanding of the biochemical background of both, the progress as well as the origins of the symptoms. DAT-imaging may also contribute to the determination of the dopaminergic therapeutic response for a given medication and thereby contribute to more convenient conditions for the patient. From an imaging point of view, the former demands a high availability of the radioactive probe to facilitate broad application of the modality, whereas the latter profits from short-lived probes, suitable for multi-injection studies. Therefore, labelling with longer-lived 18F-fluoride and in particular the generator nuclide 68Ga is worthwhile for clinical routine imaging. In contrast, the introduction of a 11C-label is a prerequisite for detailed scientific studies of neuronal interactions. The development of suitable DAT-ligands for medical imaging has often been complicated by the mixed binding profile of many compounds that that interact with the DAT. Other drawbacks have included high non-specific binding, extensive metabolism and slow accumulation in the DAT-rich brain areas. However, some recent examples have partially overcome the mentioned complications. Based on the structural speciality of these leads, novel ligand structures were designed and successfully synthesised in the present work. A structure activity relationship (SAR) study was conducted wherein the new structural modifications were examined for their influence on DAT-affinity and selectivity. Two of the compounds showed improvements in in vitro affinity for the DAT as well as selectivity versus the serotonin transporter (SERT) and norepinephrine transporter (NET). The main effort was focussed on the high-affinity candidate PR04.MZ, which was subsequently labelled with 18F and 11C in high yield. An initial pharmacological characterisation of PR04.MZ in rodents revealed highly specific binding to the target brain structures. As a result of low non-specific binding, the DAT-rich striatal area was clearly visualised by autoradiography and µPET. Furthermore, the radioactivity uptake into the DAT-rich brain regions was rapid and indicated fast binding equilibrium. No radioactive metabolite was found in the rat brain. [18F]PR04.MZ and [11C]PR04.MZ were compared in the primate brain and the plasma metabolism was studied. It was found that the ligands specifically visualise the DAT in high and low density in the primate brain. The activity uptake was rapid and quantitative evaluation by Logan graphical analysis and simplified reference tissue model was possible after a scanning time of 30 min. These results further reflect the good characteristics of PR04.MZ as a selective ligand of the neuronal DAT. To pursue 68Ga-labelling of the DAT, initial synthetic studies were performed as part of the present thesis. Thereby, a concept for the convenient preparation of novel bifunctional chelators (BFCs) was developed. Furthermore, the suitability of novel 1,4,7-triazacyclononane based N3S3-type BFCs for biomolecule-chelator conjugates of sufficient lipophilicity for the penetration of the blood-brain-barrier was elucidated.

The silent survival of Staphylococcus aureus phagocyted by HL-60 derived neutrophils

Staphylococcus aureus is a Gram positive pathogen that causes various human infections and represents one of the most common causes of bacteremia. S. aureus is able to invade a variety of non-professional phagocytes and that can survive engulfment by neutrophils, producing both secreted and surface components that compromise innate immune responses. In the contest of our study we evaluated the functional activity of vaccine specific antibodies by opsonophagocytosis killing assay (OPKA). Interestingly a low level of killing of the staphylococcal cells has been observed. In the meanwhile intracellular survival studies showed that S. aureus persisted inside phagocytes for several hours until a burst of growth after 5 hours in the supernatant. These data suggest that the strong ability of S. aureus to survive in the phagocytes could be the cause of the low killing measured by OPKA. Moreover parallel studies on HL-60 cells infected with S. aureus done by using transmission electron microscopy (TEM) interestingly showed that staphylococcal cells have an intracellular localization (endosomal vacuoles) and that they are able not only to maintain the integrity of their membrane but also to replicate inside vacuolar compartments. Finally in order to generate 3D volume of whole bacteria when present inside neutrophilic vacuoles, we collected a series of tomographic two-dimensional (2D) images by using a transmission electron microscope, generating 5 different tomograms. The three-dimensional reconstruction reveals the presence of intact bacteria within neutrophil vacuoles. The S. aureus membrane appears completely undamaged and integral in contrast with the physiological process of phagosytosis through vacuoles progression. S. aureus bacteria show a homogenous distribution of the density in all the three dimensions (X, Y, Z). All these evidences definitely explain the ability of the pathogen to survive inside the endosomal vacuoles and should be the cause of the low killing level.

Sparse Signal Representation of Ultrasonic Signals for Structural Health Monitoring Applications

Assessment of the integrity of structural components is of great importance for aerospace systems, land and marine transportation, civil infrastructures and other biological and mechanical applications. Guided waves (GWs) based inspections are an attractive mean for structural health monitoring. In this thesis, the study and development of techniques for GW ultrasound signal analysis and compression in the context of non-destructive testing of structures will be presented. In guided wave inspections, it is necessary to address the problem of the dispersion compensation. A signal processing approach based on frequency warping was adopted. Such operator maps the frequencies axis through a function derived by the group velocity of the test material and it is used to remove the dependence on the travelled distance from the acquired signals. Such processing strategy was fruitfully applied for impact location and damage localization tasks in composite and aluminum panels. It has been shown that, basing on this processing tool, low power embedded system for GW structural monitoring can be implemented. Finally, a new procedure based on Compressive Sensing has been developed and applied for data reduction. Such procedure has also a beneficial effect in enhancing the accuracy of structural defects localization. This algorithm uses the convolutive model of the propagation of ultrasonic guided waves which takes advantage of a sparse signal representation in the warped frequency domain. The recovery from the compressed samples is based on an alternating minimization procedure which achieves both an accurate reconstruction of the ultrasonic signal and a precise estimation of waves time of flight. Such information is used to feed hyperbolic or elliptic localization procedures, for accurate impact or damage localization.

Flagellin:allergen fusion proteins as novel vaccines for the treatment of severe type I allergies

Over the last decades the prevalence of food allergies has continually increased on a world wide scale. While there are effective treatments available for bee and wasp venom allergic patients, there is currently no established therapy for the treatment of severe food allergies. Aim of the project was to genetically fuse different food allergens with the immune modulating Toll-like receptor 5 (TLR5)-ligand flagellin and to test these constructs for their immune modulatory capacities both in vitro and in vivo. Chicken ovalbumin (Ova) as model antigen, Pru p 3, and Ara h 2 the respective major allergens from peach and peanut were used as allergens. The potential vaccine candidates were characterized by protein biochemical methods (purity, folding, endotoxin contaminations). Moreover, their immune modulating effects on cell culture lines (TLR5-receptor activation) and primary mouse immune cells (myeloid and plasmacytoid dendritic cells) were investigated. Additionally, the prophylactic and therapeutic use of the flagellin Ova fusion protein (rflaA:Ova) were investigated in a mouse model of intestinal allergy. In myeloid dendritic cells (mDC) stimulation with the fusion proteins led to a strong cell activation and cytokine secretion. Here, the fusion proteins proved to be a much stronger stimulus than the equimolar amount of both proteins provided alone or as a mixture. Noteworthy, stimulation with rflaA:Ova induced the secretion of the anti-inflammatory cytokine IL-10 from mDC. In co-culture experiments this IL-10 secretion suppressed the Ova-induced secretion of Th1 and Th2 cytokines from Ova-specific CD4 T cells. Using MyD88-deficient mDC this repression of cytokine secretion was shown to be TLR-dependent. Finally, the potency of the rflaA:Ova construct was investigated in a mouse model of Ova-induced intestinal allergy. In a prophylactic vaccination approach rflaA:Ova was shown to prevent the establishment of the intestinal allergy and all associated symptoms (weight loss, temperature drop, soft faeces). This fusion protein-mediated protection was accompanied by a reduced T cell activation, and reduced Th2 cytokines in intestinal homogenates. These effects were paralleled by a strong induction of Ova-specific IgG2a antibodies in rflaA:Ova-vaccinated sera, while Ova-specific IgE antibody production was significantly reduced. Therapeutic vaccination with rflaA:Ova reduced allergic symptoms and T cell activation but did not influence weight loss and antibody production. In all in vivo experiments vaccination with both proteins either provided alone or as a mixture did not have comparable effects. Future experiments aim at elucidating the mechanism and further optimization of the therapeutic vaccination approach. The results presented in this thesis demonstrate, that fusion proteins containing flagellin have strong immune modulatory capacities both in vitro and in vivo. Therefore, such constructs are promising vaccine candidates for the therapy of type I allergies.

The behaviour of osteocyte dendritic processes in bone under cyclic load (il comportamento dei processi dendritici degli osteociti nel tessuto osseo sottoposti a carico ciclico)

Bone is continually being removed and replaced through the actions of basic multicellular units (BMU). This constant upkeep is necessary to remove microdamage formed naturally due to fatigue and thus maintain the integrity of the bone. The repair process in bone is targeted, meaning that a BMU travels directly to the site of damage and repairs it. It is still unclear how targeted remodelling is stimulated and directed but it is highly likely that osteocytes play a role. A number of theories have been advanced to explain the microcrack osteocyte interaction but no complete mechanism has been demonstrated. Osteocytes are connected to each other by dendritic processes. The “scissors model" proposed that the rupture of these processes where they cross microcracks signals the degree of damage and the urgency of the necessary repair. In its original form it was proposed that under applied compressive loading, microcrack faces will be pressed together and undergo relative shear movement. If this movement is greater than the width of an osteocyte process, then the process will be cut in a “scissors like" motion, releasing RANKL, a cytokine known to be essential in the formation of osteoclasts from pre-osteoclasts. The main aim of this thesis was to investigate this theoretical model with a specific focus on microscopy and finite element modelling. Previous studies had proved that cyclic stress was necessary for osteocyte process rupture to occur. This was a divergence from the original “scissors model" which had proposed that the cutting of cell material occurred in one single action. The present thesis is the first study to show fatigue failure in cellular processes spanning naturally occurring cracks and it's the first study to estimate the cyclic strain range and relate it to the number of cycles to failure, for any type of cell. Rupture due to shear movement was ruled out as microcrack closing never occurred, as a result of plastic deformation of the bone. Fatigue failure was found to occur due to cyclic tensile stress in the locality of the damage. The strain range necessary for osteocyte process rupture was quantified. It was found that the lower the process strain range the greater the number of cycles to cell process failure. FEM modelling allowed to predict stress in the vicinity of an osteocyte process and to analyse its interaction with the bone surrounding it: simulations revealed evident creep effects in bone during cyclic loading. This thesis confirms and dismisses aspects of the “scissors model". The observations support the model as a viable mechanism of microcrack detection by the osteocyte network, albeit in a slightly modified form where cyclic loading is necessary and the method of rupture is fatigue failure due to cyclic tensile motion. An in depth study was performed focusing on microscopy analysis of naturally occurring cracks in bone and FEM simulation analysis of an osteocyte process spanning a microcrack in bone under cyclic load.

Citizen science as a tool for the environmental quality assessment of the Mediterranean coastal habitats

The growing need to assess the environmental status of the Mediterranean coastal marine habitats and the large availability of data collected by Reef Check Italia onlus (RCI) volunteers suggest the possibility to develop innovative and reliable indices that may support decision makers in applying conservation strategies. The aims of this study were to check the reliability of data collected by RCI volunteers, analyse the spatial and temporal distribution of RCI available data, resume the knowledge on the biology and ecology of the monitored species, and develop innovative indices to asses the ecological quality of Mediterranean subtidal rocky shores and coralligenous habitats. Subtidal rocky shores and coralligenous were chosen because these are the habitats more attractive for divers; therefore mlst data are referring to them, moreover subtidal rocky bottom are strongly affected by coastal urbanisation, land use, fishing and tourist activities, that increase pollution, turbidity and sedimentation. Non-indigenous species (NIS) have been recognized as a major threat to the integrity of Mediterranean native communities because of their proliferation, spread and impact on resident communities. Monitoring of NIS’ spreading dynamics at the basin spatial scale is difficult but urgent. According to a field test, the training provided by RCI appears adequate to obtain reliable data by volunteers. Based on data collected by RCI volunteers, three main categories of indices were developed: indices based on species diversity, indices on the occurrence non-indigenous species, and indices on species sensitive toward physical, chemical and biological disturbances. As case studies, indices were applied to stretches of coastline defined according to management criteria (province territories and marine protected areas). The assessments of ecological quality in the Tavolara Marine Protected Area using the species sensitivities index were consisten with those previously obtained with traditional methods.

The effect of high-energy extracorporeal shock waves on hyaline cartilage of adult rats in vivo

The aim of this study was to determine if extracorporeal shock wave therapy (ESWT) in vivo affects the structural integrity of articular cartilage. A single bout of ESWT (1500 shock waves of 0.5 mJ/mm(2)) was applied to femoral heads of 18 adult Sprague-Dawley rats. Two sham-treated animals served as controls. Cartilage of each femoral head was harvested at 1, 4, or 10 weeks after ESWT (n = 6 per treatment group) and scored on safranin-O-stained sections. Expression of tenascin-C and chitinase 3-like protein 1 (Chi3L1) was analyzed by immunohistochemistry. Quantitative real-time polymerase chain reaction (PCR) was used to examine collagen (II)alpha(1) (COL2A1) expression and chondrocyte morphology was investigated by transmission electron microscopy no changes in Mankin scores were observed after ESWT. Positive immunostaining for tenascin-C and Chi3L1 was found up to 10 weeks after ESWT in experimental but not in control cartilage. COL2A1 mRNA was increased in samples 1 and 4 weeks after ESWT. Alterations found on the ultrastructural level showed expansion of the rough-surfaced endoplasmatic reticulum, detachment of the cell membrane and necrotic chondrocytes. Extracorporeal shock waves caused alterations of hyaline cartilage on a molecular and ultrastructural level that were distinctly different from control. Similar changes were described before in the very early phase of osteoarthritis (OA). High-energy ESWT might therefore cause degenerative changes in hyaline cartilage as they are found in initial OA.

TNF suppresses acute intestinal inflammation by inducing local glucocorticoid synthesis

Although tumor necrosis factor (alpha) (TNF) exerts proinflammatory activities in a variety of diseases, including inflammatory bowel disease, there is increasing evidence for antiinflammatory actions of TNF. In contrast, glucocorticoids (GCs) are steroid hormones that suppress inflammation, at least in part by regulating the expression and action of TNF. We report that TNF induces extraadrenal production of immunoregulatory GCs in the intestinal mucosa during acute intestinal inflammation. The absence of TNF results in a lack of colonic GC synthesis and exacerbation of dextran sodium sulfate-induced colitis. TNF seems to promote local steroidogenesis by directly inducing steroidogenic enzymes in intestinal epithelial cells. Therapeutic administration of TNF induces GC synthesis in oxazolone-induced colitis and ameliorates intestinal inflammation, whereas inhibition of intestinal GC synthesis abrogates the therapeutic effect of TNF. These data show that TNF suppresses the pathogenesis of acute intestinal inflammation by promoting local steroidogenesis.