Study of oncosuppressive properties of RNASET2, the first human member of the Rh/T2/S family of glycoproteins.

During the three years of my PhD, I have been working on RNASET2, the first human member of the widespread family of T2 ribonucleases. We began to study this gene since it maps in 6q27, a region frequently deleted in many solid neoplasms and haematological malignancies, including ovarian carcinomas, which represent our model. The frequent alteration of this genomic region suggests the presence of one or more tumour suppressor genes. An in-depth study of the promoter, the 5’ and 3’ flanking regions and the coding region showed no inactivating mutations; nevertheless RNASET2 is down-regulated at the RNA level in many primary tumours and cell lines, suggesting a possible role as tumour suppressor gene. RNASET2 showed no influence on cell growth in vitro, but in vivo experimental data revealed a role in the control of tumorigenesis and metastatization for this gene: RNASET2-expressing cells formed smaller tumours in mice respect to the control cells, transfected with the vector only; besides they gave rise to a lower number of metastasis. This tumour suppressive property was demonstrated to be not correlated with the ribonuclease activity of the protein, since tumours derived from cells transfected with a version of the gene mutated at the catalytic site were similar to those obtained with wild-type RNASET2-expressing cells. In order to better characterize the enzyme and its expression, we raised a new polyclonal antibody directed against RNASET2, called anti-hRNASE-2, since the pre-existing antibodies R63 and antihRNASE were not able to recognize the native protein; during this PhD work we set the best conditions for its use in different techniques. We also improved our knowledge about subcellular localization and catalytic activity of the protein: in particular we demonstrated that the proteolytic forms of RNASET2 are located in the lysosomes and are catalytically active, proving that they represent the mature forms of the ribonuclease. Next we started a characterization of RNASET2 in human tissues, performing immunohistochemical analyses on archival specimens, formalin-fixed and paraffin-embedded. We observed a moderate to strong expression of RNASET2 in many tissues, in particular in secretory epithelia of the gastrointestinal tract; besides macrophages, when present, showed a strong positivity in all tissues examined. We also analyzed a panel of ovarian carcinomas, which represents our model, comparing RNASET2 expression with normal and benign specimens from the same histological origin; in fact, we observed a decreasing expression of the protein during grading of ovarian carcinomas, from borderline serous tumours to undifferentiated carcinomas; this trend suggested a possible role of RNASET2 in the evolution of ovarian tumour and in particular we hypothesize its involvement in cell differentiation. In order to understand the possible mechanisms by which RNASET2 exerts its tumour suppressive function, finally we performed a new in vivo experiment in the nude mouse model, inoculating clones from an ovarian highly-metastatic cell line, Hey3MET2, transfected with a control vector or with RNASET2-expressing constructs; this experiment confirmed the role of RNASET2 in control of cell growth in vivo. We progressed performing histological and histochemical analyses on the tumours isolated from athymic mice treated, to highlight possible differences: RNASET2-expressing tumours appeared more differentiated and organized and we observed remarkable differences in the composition and organization of extracellular matrix within the tumours. In conclusion, we bring here evidences about the double life of RNASET2: the ribonuclease activity, due to the proteolytic forms localized to the lysosomes, and the tumour suppressive role, probably carried out by the secreted form. We still have to understand the mechanism by which the protein influences cell growth, but further analyses on tumours derived by the in vivo experiment will help

Conical waves in nonlinear optics and applications.

The quest for light has always challenged man and constitutes one of our fundamental activity since the dawn of conscience. Being at the base of our perception of the world, the presence of light and the intelligence of our surroundings are always linked: since the prehistoric era man felt the need to produce that light which could keep him in the adequate condition to percept the ambient. Accompanied by firelights, oil lamps, wax candles and electric bulbs, man encountered a crescendo of possibility to extend his capability to stay active and clever in front of reality. The ultimate light source shining in the hands of man was invented in the fifties by Charles H. Townes: the possibility to use coherent laser light constituted a revolution in this challenge and since that moment an extremely wide horizon of phenomena began to be accessible to the perception of our eyes. Beyond new horizons of perception, laser sources extended the use of light as a tool for matter manipulation with results previously impossible to achieve, so that our everyday life is now filled with devices based and realized with laser technology. This goal has been achieved because the interaction between laser light and matter ranges in a surprisingly wider extent than the simple heating. One of the dreams in this challenge, is to obtain a light packet perfectly controllable and solid in the interaction with matter which could act as a light particle or bullet. This work is inserted in this challenge, and collects a variety of experiences and observations suggesting new perspectives for this hunt. The results I obtained during these last three years of research are intrinsically linked to the method and idea of knowledge proposed by professor Paolo Di Trapani. My work takes advantage of the experience and skill of several people coming from different corners of the world, joined by a fruitful friendship in the adventure of contemporary research. The common scientific interest of this crew is the physics of nonlinear conical waves, which are particular wave packets with more complex spectral structure than the familiar Gaussian laser pulse. The history of the conquer of this precious conceptual tool by the members of the VINO team is not trivial and gathers contributions from several events. My wish was to exploit these waves as tools for possible applications, and a consistent part of the present dissertation relies on this will. On the other hand I was also witness of other important unpredicted experiences all reported in this dissertation. The exposition of my work can’t follow the chronological flow of the events, since I have to choose a more conceptual narration to keep a higher degree of comprehension for the reader. This thesis begins with the introduction in the first chapter of some basic modeling tools of nonlinear optics phenomena which can be found on several books. At the end of this chapter I devote some space to the light soliton which is a nonlinear wave relying on the usual Gaussian approach of laser phenomena. Actually this is not the theme of my research, which is rather an escape from this approach. In the second chapter I introduce monochromatic conical waves as a first fugue from this theme. Chapter three and four exploit in detail my research on this waves. Chapter five introduces polychromatic conical waves and refers to their spontaneous formation in filamentation of ultrashort pulses. In chapter six I will enter in the details of different phenomena observed in filamentation processes; ironically my thesis ends in chapter seven reporting the first ever observed two dimensional stable soliton propagating in bulk media

Wildlife diversity and their interaction with people for sustainable use ion the semi arid areas of Northern Mount Meru ecosystem (Tanzania).

The wildlife diversity in the Mount Meru ecosystem in northern Tanzania, and the interaction of the wildlife with the local population, composed of Maasai, Waarusha and Meru ethnic communities, was studied through a field research between May 2005 and May 2007. The status of wildlife resources, their presence, distribution, seasonal trends and migration patterns were one of the main focus during the research, using two ground based survey techniques, road transects (by vehicle) and foot transects (walking transects). The second emphasis was placed on the major interactions between wildlife and people, and the attitude of local communities towards conservation of wildlife resources in their village lands. This complex was analyzed through a questionnaire survey. The results showed a high wildlife diversity in the northern dry lands of Mount Meru ecosystem. The abundance of wildlife, their trends and migration patterns were highly influenced by the seasons of the year. The findings suggested that the two methods used were most suitable for surveying wildlife within such environments. More species were recorded along the foot transects than in the road transects. The study also revealed that the only remaining wildlife migration corridor that links Arusha National Park with the northern savannah plains is threatened getting blocked. This corridor on the northern slopes of Mount Meru runs through the lands of Kisimiri villages. In order to avoid Arusha National Park on Mount Meru becoming an isolated protected area, the corridor needs urgent conservation measures by involving local communities. The three resident ethnic groups were involved in the questionnaire survey, i.e. Maasai, Waarusha and Meru. Wildlife damage was considered as the major interaction between people and wildlife. The level and kind of damages differed among the three ethnic groups, depending on their particular economic activities, being pastoralism, agropastoralism and peasant farming respectively. Also the people’s attitude on willingness to manage and own the wildlife resources within the village lands was highly biased by the economic activities performed by the ethnic group. The study suggests for the effective conservation of the wildlife within this area in general, and the safeguarding of the migration corridor in particular, a specific community based wildlife conservation program should be developed. The direct involvement of local communities in the decision making process regarding the use of the wildlife resources in the area could help to gain more confidence on wildlife management among the local population, and thus will enhance the conservation objectives

Photosensitizers against tumour cells and bacteria.

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Water quality control in coastal aquaculture system: a Tuscany fish farm as a case study.

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Molecular and functional characterization of the newly identified interaction between the Rett syndrome-associated factor MeCP2 and the pro-apoptotic factor HIPK2.

RTT Syndrome is an X-linked neuronal progressive disorder causing mental retardation mostly in females. MeCP2, a methyl-DNA-binding protein that represses transcription by modulating chromatin structure, is mutated in about 80% of classical RTT-patients. The rather large number of patients with a normal MeCP2 gene and the lack of an apparent genotype-phenotype correlation, suggests that other genes may be involved in the onset of this neurological disorder. Based on these considerations, we reasoned that the identification of novel MeCP2 interacting factors might help to refine the comprehension of the molecular mechanism(s) involved in RTT syndrome. Thus, we performed a yeast two hybrid screening using MeCP2 as a bait. We identified the homeodomaininteracting kinase 2 (HIPK2) as a new MeCP2 interactor. HIPK2 belongs to a family of nuclear serine/threonine kinases originally cloned due to their ability to interact with homeodomain transcription factors. HIPK2, the most studied of this family, binds also to a series of proteins involved in the regulation of cell survival and proliferation, including the oncosoppressor p53. In the present study, we have confirmed the physical interaction between MeCP2 and HIPK2 and, by in vitro phosphorylation assays, we have demonstrated that HIPK2 is able to phosphorylate MeCP2. Furthermore, we have mapped the specific MeCP2 site targeted by HIPK2. At the functional level, we have demonstrated that the ectopic expression of MeCP2 leads to apoptosis in different cell lines and that this effect is increased by the presence of HIPK2. Importantly, the serine residue on MeCP2 which is phosphorylated by HIPK2 in vitro, is required for the cooperation between the two proteins in inducing apoptosis, strongly suggesting that this residue is also an in vivo target of HIPK2. In conclusion our findings indicate that MeCP2 belongs to the HIPK2 apoptotic pathway and clearly suggest a novel role for MeCP2 in programmed cell death that might provide novel valuable insights into Rett syndrome

Est project in teleosts: an approach for molecular biomarker research.

The scarcity of the genomic resources for some fish species, in spite of their commercial interest, could retard the positive effects that modern biotechnology can offer to aquaculture industry. Then an effort should be made to reduce, as far as it concerns genomic resources, the gap that separates farming species from “model organisms". In this thesis I have reported the construction and the analysis of eight cDNA libraries obtained from different tissues of three teleosts: D. labrax, P. fluviatilis and T. thynnus. A total of 13618 clones were generated from Expressed Sequence Tags and these were searched for homology in the GeneBank with BlastN and BlastX programs. In order to understand the molecular composition of the tissues analyzed, the expression profiles of the identified clones in these cDNA libraries were analyzed. Therefore, in this study have been identified 293 genes expressed in the liver of D. labrax, 258 genes expressed in three libraries (liver, liver vs brain and brain vs liver) of P. fluviatilis and 2094 genes expressed in four libraries (ovary, testis, liver and normalized ovary) of T. thynnus. EST cataloguing and profiling of these teleosts will set the basis for functional genomic research in these species, but will also serve for comparative and environmental genomics, for the identification of polymorphic markers useful, for example, to survey the disease resistance of fish, for the discovery of new molecular markers of exposure