DETECTION OF PROSTETHIC INFECTION AT REVISION ARTHROPLASTY
 (Basic Project)

Duration of the project:
1. 5. 2009 – 30. 4. 2012

Principal investigator / researcher:
Prof. Andrej Cör, PhD

Collaborating parties / partners:
• University of Primorska Faculty of Health Sciences
• Ortopedic Hospital Valdoltra
• Institute of Public Health Koper

COMBINATION OF ELECTROGENE IMMUNE THERAPY WITH INTERLEUKIN-12 AND IRRADITION FOR TREATMENT OF EXPERIMENTAL TUMORS
(Basic Project)

Duration of the project:
1. 5. 2009 – 30. 4. 2012

Principal investigator / researcher:
Associate Prof. Maja Cemažar, PhD

Collaborating parties / partners:
• University of Primorska Faculty of Health Sciences
• Institute of Oncology Ljubljana

Background.
Electroporation is a physical method that uses electric pulses applied to cells or tissues to reorganize structure of the cell membrane. Cell membranes thus become permeable to molecules which otherwise can not cross it. Lately, electroporation is getting consideration also in gene therapy, where it is used as gene delivery method (electrogene therapy). In treatment of cancer, electrogene therapy can be used for electrotransfer of therapeutic genes directly to tumor or into other tissues for systemic release of therapeutic proteins acting on distant tumors. One of promising therapeutic genes that were already tested in electrogene therapy is interleukin-12 (IL-12). IL-12 stimulates innate immunity, by activating natural killer cells to produce interferon-g and adaptive immunity by stimulating differentiation of T lymphocytes Th1 response). In addition, IL-12 has also antiangiogenic properties. Combination of gene therapy with IL-12 for systemic release of IL-12 in combination with radiotherapy is a new field that has not been yet evaluated for treatment of cutaneous and subcutaneous as well as deeper seeded tumors. Therefore, our hypothesis is that electrotransfection of IL-12 into muscle using optimized parameters is efficient electrogene therapy that has therapeutic effect as single therapy and has radiosensitizing effect in combination with radiotherapy.

Methods.
Measurements of in vitro cytotoxicity of recombinant IL-12 and testing of its radiosensitizing effect. In vivo transfection of tumors with electroporation and plasmid DNA encoding IL-12. Intratumoral and peritumoral application of electrogene therapy will be tested in solid subcutaneous tumors and antitumor effectiveness determined. In vivo transfection of muscles with plasmid DNA encoding IL-12 for systemic release of IL-12. Antitumor effectiveness in solid subcutaneous tumors and lung metastases will be determined. Qualitative and quantitative analysis of transfection and expression of IL-12. Determination of antitumor effectiveness of systemic and local combined electrogene immune therapy with IL-12 and radiotherapy.

Relevance and potential impact of the results.
The proposed project is designed as a preclinical project that involves experiments in cell cultures and animal and human tumor models. The results can contribute to the use of electrogene immunotherapy as an adjuvant therapy to radiotherapy. If successful, local potentiation of radiation response can lead to the use of lower irradiation doses and thus lower side effects. Combination of radiotherapy and systemic electrogene immunotherapy can in addition to enhanced local effect, result in systemic effect on micrometastases and has also prophylactic effect.

Preparation and validation of therapeutic plasmids without selection gene for antibiotic resistance for cancer gene therapy using inducible and tissue-specific promoters (basic research project)

Duration of the project:
1.7.2011 – 30.6.2014

Principal investigator / researcher:
Associate Prof. Maja Cemažar, PhD

Collaborating parties / partners:
• University of Primorska Faculty of Health Sciences
• Institute of Oncology Ljubljana
• National Institute of Biology Ljubljana
• University of Ljubljana, Biotechnical Faculty

Background.
Gene therapy is becoming increasingly more important in cancer therapy. In recent years, non-viral approaches for transfection have become very attractive. One of the goals of researchers dealing with gene therapy or DNA vaccination is to develop safe and efficient systems for gene transfer into the target cells. Namely, this topic is still one the major hurdles in the progress of different approaches in gene therapy and is crucial factor for success of the therapy. Other factors that should be considered for development of gene therapy are the choice of appropriate therapeutic gene, regulation of its expression and administration route. In the proposed project, two of these factors will be addresses for the use in cancer gene therapy: safety of plasmid DNA by preparation of plasmid DNA without marker for antibiotic resistance and regulation of therapeutic gene expression by linking the therapeutic gene to cellular promoters to achieve either stress induced (p21 and radiation) or physiological regulation (tissues specific promoters for endothelial and muscle cells and fibroblsats) of expression.  In addition, possible horizontal gene transfer between recombinant plasmid DNA or transgenic bacteria and commensal naturally occuring bacteria will be evaluated.
Therefore, our hypothesis is that prepared plasmids encoding therapeutic genes under the control of tissue specific and inducible promoters without gene for antibiotic resistance are safe vectors for gene therapy with also regulated expression.
Methods. Plasmids encoding reporter or therapeutic genes under the control of tissue specific or inducible promoters will be prepared by standard molecular biology techniques and according to the instructions of the producer of ORT® plasmid in order to prepare plasmids without gene for antibiotic resistance. Appropriate bacterial strains will be transformed with the plasmids to produce larger quantities of endotoxin free plasmid to be used in further experiments. The correctness of plasmids will be evaluated by gel electrophoresis and sequencing.
To evaluate horizontal gene transfer, PCR assays and other standard laboratory procedures used for studying horizontal gene transfer as well as standard laboratory procedures used for preparing biofilm cultures will be used. To determine the effects of prepared plasmids on cell in cultures, standard test for proliferation, antiangiogenic and antimetastatic potential will be used.
Antitumor effectiveness and histological analysis will be determined on tumor models in vivo by tumor growth delay assay, tumor control probability assay and immunohistochemistry.
Execution and management of the project. The experiments will be performed at the laboratories of 5 partners participating in the project. All necessary equipment for molecular biology, cell cultures, immunocytochemistry, and animals experiments is available. The researchers involved in the project have vast experience in the field of plasmid preparation, electroporation as a drug and gene delivery method and radiobiology.
The management and coordination of the project will be assured by regular meetings and constant communication with partners on the project.
Relevance and potential impact of the results. The proposed project is designed as a preclinical project that addresses important issues regarding safe use of cancer gene therapy in clinical setting. The results of the project, if successful, may have impact on design and execution of cancer gene therapy trials in Slovenia and worldwide.

Antiangiogenic gene therapy of cancer using electroporation and magnetic nanoparticles as targeted delivery systems (basic research project)

Duration of the project:
1.7.2011 – 30.6.2014

Principal investigator / researcher:
Prof. Gregor Serša, PhD

Collaborating parties / partners:
• Institute of Oncology Ljubljana
• R&D Center for Nanotechnologies in the Field of Magnetic Materials and Composites Ljubljana
• University of Primorska Faculty of Health Sciences
• University of Ljubljana, Faculty of Electrical Engineering

Abstract
The aim of the project is the development of new gene therapy approaches, based on anti-angiogenic mechanisms, for treatment of malignant melanoma and sarcoma. The new plasmids coding for anti-angiogenesis of tumors will be tested in experimental tumors by non-viral delivery systems; electroporation and magnetic nanoparticles. For better translation into the clinics, combined modality treatment with radiotherapy will be tested.
The project is based on the construction of new plasmids coding for miRNA against endoglin (CD105) and melanoma cell adhesion molecule (MCAM/CD146), both specific for endothelial cells in tumors and melanoma cells capable of invasion. These specific targets will be used for the treatment of two variants of melanoma tumors in experimental animals, B16F1 melanoma with low metastatic potential and B16F10 with high metastatic potential. Because of some cross-specificity with other tumor type’s sarcoma SA-1 model will also be used. In addition to that, plasmid AMEP already prepared and validated to some extent by our industrial partner will be used. It also has anti-angiogenic and direct cytotoxic effect to melanoma cells and tumors.
As plasmid delivery systems, non viral techniques will be used; electroporation and magnetic nanoparticles. Electroporation is already established gene delivery method for transfection of tumors, muscle and skin (electrogene therapy). Nanotechnology for gene delivery is in its early developmental phase for use of magnetic nanoparticles (magnetofection). This approach is being developed in our research group, and we want to test and implement it in cancer treatment.
With electrogene therapy and magnetofection the transfection efficiency of constructed plasmids as well as plasmid AMEP in cells in vitro and the effect of the plasmids on cell’s proliferation, adhesion and invasion will be performed. Anti-angiogenic potential of constructed plasmids will be tested in vitro by angiogenesis assay (tube formation assay). Based on these results we will proceed in the investigation of anti-angiogenic and antitumor effects by intravital microscopy in window chamber. These data will provide evidence on anti-angiogenic effectiveness of the constructed plasmids coding for miRNAs in vivo, as well as their antitumor effectiveness on melanoma and sarcoma tumor models.
In vivo, on experimentally induced tumors and metastases, antitumor effectiveness of the constructed plasmids coding for miRNAs against CD105 and CD146 as well as plasmid AMEP will be tested. By intratumoral electrogene therapy or magnetofection, direct effects on established tumors will be tested. Also intramuscular transfection of plasmid AMEP is foreseen in order to test the possibility of systemic shedding of the protein in circulation and its effect on local tumor growth and metastasis. Again, both transfection methods will be used; electrogene therapy and magnetofection. All these experiments will provide starting point for further experiments combined with radiotherapy.
Anti-angiogenic therapies are already being combined with radiotherapy in treatment of cancer, however anti-angiogenic gene therapy as radiosenzitizing therapy is still in early stage of development. Therefore the constructed plasmids and plasmid AMEP will be tested as adjuvant gene therapy to radiotherapy. The best treatment combinations with either single or repetitive gene therapy combined with either single or fractionated radiation regimen will be explored. The predominant tumor model used will be melanoma.
The results will provide evidence on newly constructed plasmids coding for miRNA against CD105 and CD146 and plasmid AMEP on their antitumor effectiveness on melanoma and sarcoma tumors. Their antitumor effectiveness with or without combined modality treatment with radiotherapy will provide basis for translation of the knowledge into clinical trials.

»RAZKORAK« Longitudinal study in competence potential of university graduates and a gap between graduates' competences and labour market requirements in technology, education and health (basic research project)

Duration of the project:
1.7.2011 – 30.6.2014

Principal investigator / researcher:
Associate Prof. Istenic Starcic Andreja, PhD

Collaborating parties / partners:
• Educational Research Institute Ljubljana
• Faculty of Art Ljubljana
• University of Primorska Faculty of Education Koper
• University of Primorska Faculty of Health Sciences Izola
• University of Ljubljana, Faculty of Civil and Geodetic Engineering Ljubljana
• University of Ljubljana, Faculty of Electrical Engineering Ljubljana

Abstract
The competence concept, it’s modelling, needs assessment and evaluation is challenging research in education as also transdisciplinary research. Competences in contemporary society can no longer be described as fixed set of skills; they represent a dynamic combination of abilities, skills and knowledge. Competences are context dependent and reflect person’s potential realised in different contexts. It is being developed in the processes of learning, education, training and work based activities. Witt in Lehmann identify in the internal dimension which is reflecting the individual characteristics and external dimension which is reflecting demands of working processes (2001). Boyantzis defines competences as individual abilities which reflect motives, values, self-image, social roles, skills and knowledge which individual is applying and are connected with the results in the work place (1982, 2008). Competences as actualised potentials which individual apply in a concrete situation. Klieme in Leutner define competences and as context-specific cognitive dispositions that are acquired and needed to successfuly cope with certain situations or tasks in specific domains (Klieme in Leutner 2006). In the education the demands for accountability shift the focus on the competency based curriculum (Wise, 1979; Slavin, 2002). Mclelland as alternative to measurement of general cognitive abilities identifies testing of competency with the criterion sampling with the testing of authentic tasks from real life work based environments (1973). Competencies in bologna competency based study programmes provide a framework for curriculum design. Longitudinal survey within old and bologna programs will provide longitudinal assessment data during study, after graduation in employment to identify how the competences have been developed, how students perceive professional competences, how learning environment influence students approaches to learning and competence development, how competences are actualised when graduates enter labour market and get employed. Assessment of competences are also an important focus of this study. Within the proposed research will measurement of small segment of generic competences take place. The level of competency acquired will be in the focus of measurement. In this way the topic of this research will also be a psychometric measurement of competencies. The instruments will be designed and developed also as an example how to design competency assessment instruments and as a case to be applied within different disciplines and subject specific competencies. Purpose: (1) The development of instruments for needs assessment and evaluation of competences of graduates. (2) State of the art and analysis of the competences divide among competence needs (in work environments, university and students themselves) and actualised competences (of graduates in the selected fields of technology, education, psychology and health). Objective: (1) Instruments for competence potential assessment and the analyse of divide among needs and realised competences; Instruments will support the competence management in different areas. (2) Longitudinal analyses competence potential in technology in the field of civil engineering, geodesy and electrical engineering, in education, psychology and in health. Participants will be student, graduates and employers. The analysis will cover state of the art and divide/disparity between stakeholders. In the study, different methodological approaches will be combined – a survey study (quantitative approach) will be combined with selected qualitative research methods. A combined approach is necessary to obtain a holistic view about the research object. Quantitative data will allow for generalizability, whereas qualitative data will bring a deeper and informationally richer insight, and possibly provide some findings that could not be foreseen at the beginning of the study.

Duration of the project:
1.10.2010 – 30.9.2012

Principal investigator / researcher:
Assist. Prof. Kirbiš Andrej, PhD

Collaborating parties / partners:
• National Institute of Biology Ljubljana
• University of Primorska Faculty of Health Sciences Izola
• University of Ljubljana, Biotechnical Faculty Ljubljana
• University of Ljubljana, Veterinary Faculty