Assessment of SNPs for linkage mapping in Eucalyptus: construction of a consensus SNP/microsatellite map from two unrelated pedigrees

Financial support. Brazilian Ministry of Science and Technology (CNPq Grant 577047-2008-6), FAP-DF NEXTREE Grant 193.000.570/2009 and EMBRAPA Macroprogram 2 project grant 02.07.01.004.

Synthetic biology: a novel approach for the construction of industrial microorganisms

The recent achievement of synthesising a functioning bacterial chromosome marks a coming of age for engineering living organisms. In the future this should allow the construction of novel organisms to help solve the problems facing the human race, including health care, food, energy and environmental protection. In this minireview, the current state of the field is described and the role of synthetic biology in biotechnology in the short and medium term is discussed. It is particularly aimed at the needs of food technologists, nutritionists and other biotechnologists, who might not be aware of the potential significance of synthetic biology to the research and development in their fields. The potential of synthetic biology to produce interesting new polyketide compounds is discussed in detail.

Towards the construction of a validated numerical system to study the mesoscale dynamics of the North East Atlantic (2003-2006)

Máster en Oceanografía

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.

Construction of polynomial spline spaces over quadtree and octree T-meshes

[EN]We present a new strategy for constructing tensor product spline spaces over quadtree and octree T-meshes. The proposed technique includes some simple rules for inferring local knot vectors to define spline blending functions. These rules allow to obtain for a given T-mesh a set of cubic spline functions that span a space with nice properties: it can reproduce cubic polynomials, the functions are C2-continuous, linearly independent, and spaces spanned by nested T-meshes are also nested. In order to span spaces with these properties applying the proposed rules, the T-mesh should fulfill the only requirement of being a 0-balanced quadtree or octree. ..

Construction of polynomial spline spaces over quadtree and octree T-meshes

[EN]We present a new strategy for constructing tensor product spline spaces over quadtree and octree T-meshes. The proposed technique includes some simple rules for inferring local knot vectors to define spline blending functions. These rules allow to obtain for a given T-mesh a set of cubic spline functions that span a space with nice properties: it can reproduce cubic polynomials, the functions are C2-continuous, linearly independent, and spaces spanned by nested T-meshes are also nested. In order to span spaces with these properties applying the proposed rules, the T-mesh should fulfill the only requirement of being a 0-balanced quadtree or octree. ..

Nearrings and a construction of triply factorized groups

In this thesis a connection between triply factorised groups and nearrings is investigated. A group G is called triply factorised by its subgroups A, B, and M, if G = AM = BM = AB, where M is normal in G and the intersection of A and B with M is trivial. There is a well-known connection between triply factorised groups and radical rings. If the adjoint group of a radical ring operates on its additive group, the semidirect product of those two groups is triply factorised. On the other hand, if G = AM = BM = AB is a triply factorised group with abelian subgroups A, B, and M, G can be constructed from a suitable radical ring, if the intersection of A and B is trivial. In these triply factorised groups the normal subgroup M is always abelian. In this thesis the construction of triply factorised groups is generalised using nearrings instead of radical rings. Nearrings are a generalisation of rings in the sense that their additive groups need not be abelian and only one distributive law holds. Furthermore, it is shown that every triply factorised group G = AM = BM = AB can be constructed from a nearring if A and B intersect trivially. Moreover, the structure of nearrings is investigated in detail. Especially local nearrings are investigated, since they are important for the construction of triply factorised groups. Given an arbitrary p-group N, a method to construct a local nearring is presented, such that the triply factorised group constructed from this nearring contains N as a subgroup of the normal subgroup M. Finally all local nearrings with dihedral groups of units are classified. It turns out that these nearrings are always finite and their order does not exceed 16.

The construction of TRIGA-TRAP and direct high-precision Penning trap mass measurements on rare-earth elements and americium

Nuclear masses are an important quantity to study nuclear structure since they reflect the sum of all nucleonic interactions. Many experimental possibilities exist to precisely measure masses, out of which the Penning trap is the tool to reach the highest precision. Moreover, absolute mass measurements can be performed using carbon, the atomic-mass standard, as a reference. The new double-Penning trap mass spectrometer TRIGA-TRAP has been installed and commissioned within this thesis work, which is the very first experimental setup of this kind located at a nuclear reactor. New technical developments have been carried out such as a reliable non-resonant laser ablation ion source for the production of carbon cluster ions and are still continued, like a non-destructive ion detection technique for single-ion measurements. Neutron-rich fission products will be available by the reactor that are important for nuclear astrophysics, especially the r-process. Prior to the on-line coupling to the reactor, TRIGA-TRAP already performed off-line mass measurements on stable and long-lived isotopes and will continue this program. The main focus within this thesis was on certain rare-earth nuclides in the well-established region of deformation around N~90. Another field of interest are mass measurements on actinoids to test mass models and to provide direct links to the mass standard. Within this thesis, the mass of 241-Am could be measured directly for the first time.

Negotiating conservation: the construction of meaningful spaces in a world heritage debate

According to one’s personal biography, social background and the resultant degree of affectedness, a person has certain ideas about the meaning of, in our example, a World Heritage Site (WHS), what he or she can expect from it and what his or her relation to it can and should be. The handling of potentially different meaningful spaces is decisive, when it comes to the negotiation of pathways towards the sustainable development of a WHS region. Due to the fact that – in a pluralistic world – multiple realities exist, they have to be taken seriously and adequately addressed. In this article we identified the ways the Jungfrau-Aletsch- WHS was constructed by exploring the visual and verbal representations of the WHS during the decision-making process (1998-2001). The results demonstrate that in the visual representations (images), the WHS was to a large extent presented as an unspoiled natural environment similar to a touristy promotion brochure. Such a ‘picture-book’-like portrait has no direct link to the population’s daily needs, their questions and anxieties about the consequences of a WHS label. By contrast, the verbal representations (articles, letters-to-the-editor, comments) were dominated by issues concerning the economic development of the region, fears of disappropriation, and different views on nature. Whereas visual and verbal representations to a large extent differ significantly, their combination might have contributed to the final decision of the majority of people concerned to support the application for inscription of the Jungfrau-Aletsch-Bietschhorn region into the World Heritage list. The prominence of economic arguments and narratives about intergenerational responsibility in the verbal representations and their combination with the aesthetic appeal of the natural environment in the visual representations might have built a common meaningful space for one part of the population.

Bowel function remains subjectively unchanged after ileal resection for construction of continent ileal reservoirs

Construction of a continent catheterizable urinary reservoir or an orthotopic bladder substitute requires substantial bowel resection, which can cause changes in bowel transit time. The reported incidence of chronic diarrhea after ileocecal resection is about 20%. Studies assessing bowel function after resection of 55-60 cm of ileum without compromising the ileocecal valve are scarce, and long-term results have not been reported.