Department of Molecular Virology

 

 

Project PN-II-RU-TE-2014-4-2502

The role of E6/E7 HPV16 oncogenes in chromatin remodelling through components of NuRD complex (MBD2, MBD3)

 

Other Projects

1. MOLECULAR MARKERS AS PREDICTORS OF POST-THERAPEUTIC EVOLUTION AND PROGNOSIS IN DIFFERENTIAL THYROID CARCINOMA MANAGEMENT [Summary]
2. THE ROLE OF S100A4 AND MAP4K4 FACTORS IN THE PROGRESSION OF PANCREATIC DUCTAL ADENOCARCINOMA (S100MAP)[Summary]
3. CELLULAR AND MOLECULAR MECHANISMS INVOLVED IN CERVIX SEVERE DISPLASIA REGRESSION - please follow this link for details  [Summary]
4. DIAGNOSIS MARKERS AND MANAGEMENT CRITERIA FOR HUMAN PAPILOMAVIRUS CERVIX INFECTION [Summary]
5. EXPERIMENTAL STUDIES ON TRANSFECTED HUMAN MESENCHYMAL STEM CELLS WITH A VIEW TO DEVELOPING AN INNOVATIVE THERAPEUTIC STRATEGY OF GLIOBLASTOMA [Summary]
6. PROMOTION OF ROMANIAN PSYCHIATRIC GENETICS RESEARCH THROUGH INTERNATIONAL PARTNERSHIP IN GENETIC AND EPIGENETIC STUDIES OF BIPOLAR DISORDER [Summary]
7. THE ROLE OF THE EPIGENETIC INFORMATION IN ONCOGENESIS: DIAGNOSIS AND THERAPEUTIC ATTITUDE IMPLICATIONS [Summary]
8. INTEGRATION OF MOLECULAR BIOLOGY TECHNIQUES AND CYTOGENETIC ASSAYS FOR TUMOR SPLENOMEGALIA DIAGNOSIS, ETHIOPATOGENIC FACTORS, PROGNOSTIC AND THERAPEUTIC IMPLICATIONS [Summary]

 

MOLECULAR MARKERS AS PREDICTORS OF POST-THERAPEUTIC EVOLUTION AND PROGNOSIS IN DIFFERENTIAL THYROID CARCINOMA MANAGEMENT

Acronym: - GENITIR
PNCDI2 – 135-2012
CO - “C.I. PARHON” NATIONAL INSTITUTE OF ENDOCRINOLOGY
P1 - STEFAN S. NICOLAU INSTITUTE OF VIROLOGY, BUCHAREST (Anca Botezatu, PhD)
P2 - THE INSTITUTE OF CELLULAR BIOLOGY AND PATHOLOGY N. SIMIONESCU

Phase I: dec. 2012: Patients enrollment, biochemical and hormonal profiling, database set-up, creating a biological and nucleic acid samples collection
Budget: 150.000 lei
Phase II: dec. 2013
Genetic analysis - BRAF and RAS mutation, gene methylation and serum proteic markers in patients with differentiated thyroid carcinoma
Budget: 135.000 lei
Phase III: dec.12.2014
RET/PTC1 and RET/PTC3 rearrangements, specific miRNA and tissue proteic markers in patients with differentiated thyroid carcinoma and controls
Budget: 157.500 lei
Phase IV: dec.2015
RET/PTC1 and RET/PTC3 rearrangements, specific miRNA and tissue proteic markers in patients with differentiated thyroid carcinoma and controls
Budget: 96.537 lei
Phase V: dec.2016
Development of an improved diagnosis protocol for the management of differentiated thyroid carcinoma based on specific molecular markers
Budget: 160.963 lei
Summary. The concept of this project is to find genetic, epigenetic and protein molecular markers that can be predictors for formation and progression of thyroid differentiated cancer by investigating, in a multidisciplinary consortium, a representative group of Romanian population with this pathology. The study aims also to analyze the actual state of thyroid differentiated carcinoma in Romania, its prevalence regarding age, sex and regions in Romania.
The recent thyroid cancer management guidelines published by the American Thyroid Association (ATA) recommend a rather individualized management approach. ATA guidelines associate postoperative staging based on the clinical-pathological AJCC/UICC staging system with a novel, still not validated classification, comprising low, intermediate and high risk stratification, in order to assess risk for recurrence and mortality and allow early therapeutic decision making regarding radioactive iodine ablation and degree of TSH suppression. In low-risk and in intermediate/high-risk subjects, the risk stratification can be early achieved by clinical, biological and imagistic ongoing assessment within the first two years of follow-up.
The major objective of the project: Improvement of the diagnostic and follow-up protocols of differentiated thyroid cancer with new markers for a better treatment outcome, prognosis and quality of life
Specific objectives:
- Determining the prevalence of differentiated thyroid carcinoma (DTC) in Romanian population according to age, sex and geographical area;
- Determining the prevalence of BRAF, RAS mutations and RET/PTC1, RET/PTC3 rearrangements, in differentiated thyroid carcinoma patients in Romanian Population;
- Identification of methylated genes associated to formation, progression and treatment response in differentiated thyroid carcinoma;
- Establishing the miRNA specific profiling in differentiated thyroid carcinoma;
- Identification of a specific panel of protein markers for differentiated thyroid carcinoma (altered gene products, angiogenesis markers);
- Setting-up of a Thyroid cancer register based on existing platform BIOMAT-ENDO;
- Setting-up biological samples collections (tumor, nucleic acids, blood samples);
- Setting-up of a personalized microarray chip for genes involved in tumorigenesis and progression of differentiated thyroid carcinoma.

THE ROLE OF S100A4 AND MAP4K4 FACTORS IN THE PROGRESSION OF PANCREATIC DUCTAL ADENOCARCINOMA (S100MAP)

Acronym: - S100MAP
PNCDI2 90-2012
CO – INSTITUTUL CLINIC FUNDEN
P1 -SC RNTECH SRL
P2 - THE INSTITUTE OF CELLULAR BIOLOGY AND PATHOLOGY N. SIMIONESCU
P3 - STEFAN S. NICOLAU INSTITUTE OF VIROLOGY, BUCHAREST (Anca Botezatu, PhD)
P4 INSTITUTUL ONCOLOGIC “PROF.DR. AL. TRESTIOREANU”

Phase I: dec.2012
Generating a database and biological samples ductal pancreatic adenocarcinoma tissue and in silico modeling
Budget: 270.000 lei
Phase II: dec. 2013
Adjusting the MAP4K4 and S100A4 genes
Budget: 110.000 lei
Phase III: dec.12.2014
Exploring signaling pathways in PDAC cell lines and / or tissues to identify new therapeutic targets. Examination of protein expression and regulation of MAP4K4 and S100A4 in pancreatic stellate cell lines and PDAC cell lines
Budget: 78.000 lei
Phase IV: dec.2015
Exploring signaling pathways in PDAC cell lines and / or tissues to identify new therapeutic targets. Examination of protein expression and regulation of MAP4K4 and S100A4 in pancreatic stellate cell lines and PDAC cell lines
Budget: 58.000 lei
Phase V: july.2016
Validating the 3D system for a variety of medicines
Budget: 134.000 lei

Summary. Pancreatic cancer is the fourth leading cause of cancer death and has a very poor prognosis; the five year survival rate of less than 5%. The molecular mechanisms of this disease remain poorly understood. Pancreatic ductal adenocarcinoma is highly resistant to conventional treatment modalities such as systemic chemo- and radiation therapy. The current compendium of validated genetic mutations has provided a multistep model for the initiation and progression of pancreatic adenocarcinoma that is typified by the near-universal and early occurrence of activating mutations in KRAS and frequent later-stage inactivation of p16INK4A, p53, and/or SMAD4. Pancreatic cancer is not only a disease with a genetic background, but also is correlated to epigenetic alterations such as DNA methylation and histone modifications, leading to an altered gene expression. Altered gene expression due to changes in the compact chromatin structure and posttranslational histone modifications has often been considered as a significant contributing factor for tumor development. Progression of pancreatic cancer, which has a very poor prognosis, is found to be associated with a host of changes in gene expression. Chromatin status is modulated by epigenetic mechanisms, making DNA more or less accessible to the transcriptional machinery. There are five major mechanisms known that alter chromatin architecture: DNA methylation, post-translational histone modifications, use of histone variants, chromatin remodeling and incorporation of non-coding RNA into chromatin.

The major objective of the project is to analyze the MLL gene family members’ involvement in pancreatic cancer and neuroendocrine pancreatic tumors.

Specific objectives:
- Genomic and epigenomic studies of MLL members family (mutations and genes promoters methylation) associated with pancreatic cancer and metastasis (samples from primary tumor / metastasis and cell culture mutations screening).
- Characterization of chromatin remodeling complexes formed by interaction of MLL genes, H3K4 3me and RNA Pol II with specific activated genes and factor involved in transcription inactivation (H3K27 and H3K9 3me), using chromatin immunoprecipitation technique.
- Evaluation of mutant variants of MLL genes effects on transcription activation of specific controlled genes in vitro models (pancreatic cancer cell lines/ pancreatic neuroendocrine tumors lines), and they relation with invasion and metastasis.

CELLULAR AND MOLECULAR MECHANISMS INVOLVED IN CERVIX SEVERE DISPLASIA REGRESSION

Acronym: - SENCOL
PNCDI2 – 40030
CO - STEFAN S. NICOLAU INSTITUTE OF VIROLOGY, BUCHAREST (Gabriela Anton, PhD)
P1–„GRIGORE T. POPA” UNIVERSITY OF MEDICINE AND PHARMACY, IASI
P2–FUNDENI CLINICAL INSTITUTE, BUCHAREST
P3–„CAROL DAVILA” UNIVERSITY OF MEDICINE AND PHARMACY, BUCHAREST

Summary. The activity developed in the partnership of this frame project, proposes to realize and consolidate an integrated research network. The domain of interest is to define and to identify the cellular pathways contributing to the regression of severe uterine cervix lesions, and to translate the results into clinical practice. This proposal belongs to the 4.1.1 direction – the development of the biological human systems knowledge.

HPV persistent infections represent the main, but not the sufficient cause for the development of most squamous cervix carcinomas and intermediate conditions like cervical intraepithelial neoplasia (CIN). In most immunocompetent women, CIN 1 lesions regress without any medical procedure. A small fraction of this lesions progress to CIN2/3 dysplasia and lead to transformation. However many authors report spontaneous regression in this type of cases in a percent of 6 – 50%. The mechanism through this intraepithelial lesions HPV- associated regress is not yet completely understood. The regression of the severe dysplasia is a complex phenomenon, determined through apoptotic and senescence pathways. It is clearly that the accelerated proliferation is insufficient for carcinogenesis and the proliferative cells need to elude the apoptosis and senescence process in order to manifest the malignant phenotype. In exchange, the spontaneous regression is characterized by the loss of the malignant phenotype, so the tumor cells will be the target of the apoptosis process. Like apoptosis, the senescence is a stress responsive mechanism, but instead of leading to cellular death, it stops the cellular proliferation.

The aim of this research project is to decode the cellular/molecular basis of regression for some high – grade HPV- induced lesions (CIN), and to understand the mechanisms which govern the biological systems for the optimization of the cancer prognostic.
The specific objectives are:
(1) to create an experimental model in order to induce the senescence/ apoptosis process (through molecular and genetic engineering techniques) in viral transformed cell line cultures
(2) to identify, using microarray techniques, the gene clusters implicated in regression
(3) to validate the cellular pathways involved in regression related to the apoptosis/senescence process (using tissue microarray techniques from patients with severe dysplasia).

DIAGNOSIS MARKERS AND MANAGEMENT CRITERIA FOR HUMAN PAPILOMAVIRUS CERVIX INFECTION

Acronym: – VIROGYN
CEEX – M1- 119/2006
CO–STEFAN S. NICOLAU INSTITUTE OF VIROLOGY, BUCHAREST (Gabriela Anton, PhD)
P1–FILANTROPIA CLINICAL HOSPITAL, BUCHAREST
P2–„GRIGORE T. POPA” UNIVERSITY OF MEDICINE AND PHARMACY, IASI.
P3-UNIVERSITY OF MEDICINE AND PHARMACY, CRAIOVA
P4–„CAROL DAVILA” UNIVERSITY OF MEDICINE AND PHARMACY, BUCHAREST
P5–FUNDENI CLINICAL INSTITUTE, BUCHAREST
P6–UNIVERSITY OF BUCHAREST

Summary. The molecular epidemiologic data showed in an unequivocal manner that the human papillomaviruses (HPVs) are the etiologic agent of this disease. There is not a consensus regarding the inclusion of some genotypes with low prevalence in a special risk group. The genital infection with HPV must be managed as a higher risk (Hr) factor which predispose to the cervical cancer development. In this infection, the certainly diagnosis is done by PCR, so, it is not surprisingly that at the international, but also the national level, there are numerous studies focused on the value of the high specificity grade enabled by these assays, in order to eliminate cyto/colposcopic discordances. It is generally admitted that the HrHPV infection is not sufficient for the cervical cancer pathogenesis, other complementary conditions being involved The reason that certain infections are persistent may depend on:

• the inter-individual variations in the capacity to elicit an adequate immune response;
• the persistence and the clonal viral heterogeneity; - the viral oncogenes interference with the tumour suppressor proteins;
• the epigenetic events.

In this context we intend:

• to establish the prevalence and the incidence of the HPV genotypes circulating in different area of Romania
• to verify the accuracy of the cytologic diagnosis on thin layer preparations ( Thin Prep), as compared with classical techniques;
• to evaluate the consistency relation between the immunocytochemical determination of the Hr HPV L1 proteins and the PCR detection of the corresponding viral genome, in order to monitories the evolution;
• to quantify the expression of some cell and viral genes involved into the inter-individual variations of the immune response, in the viral persistence and in the progression of the genital lesions;
• to correlate the obtained results in order to improve the monitoring of the lesions and management of the viral infection

EXPERIMENTAL STUDIES ON TRANSFECTED HUMAN MESENCHYMAL STEM CELLS WITH A VIEW TO DEVELOPING AN INNOVATIVE THERAPEUTIC STRATEGY OF GLIOBLASTOMA

Acronim of the project: GLIOSTEMCEL
CEEX -M1- 176/2006
CO-„BAGDASAR- ARSENI” EMERGENCY CLINICAL HOSPITAL, BUCHAREST
P1–„VICTOR BABES” UNIVERSITY OF MEDICINE AND PHARMACY, TIMISOARA
P2–„STEFAN S. NICOLAU” INSTITUTE OF VIROLOGY, BUCHAREST (Gabriela Anton, PhD)
P3–NATIONAL INSTITUTE OF RESEARCH AND DEVELOPMENT IN BIOLOGICAL SCIENCES, BUCHAREST
P4-UNIVERSITY OF BUCHAREST

Summary. The purpose of this project is to develop an alternative glioblastoma therapy by using stem cells isolated from patients with malignant cerebral tumors. Research performed in the last years evidenced an extremely interesting property of the neural stem cells and of the mesenchimal stem cells, the one to "trace" the cerebral tumoral malignant cells along their route of metastatic insemination.

As part of this study we intend to transfect the mezenchimal stem cells with cytokine - the interferon synthesis gene (INF beta), which proved to have apoptotic action on the glioblastoma cells. First, we will establish, by in vitro studies, the manner in which the interferon beta secreting stem cells interact with the glioblastoma cells (cultures obtained from the tumoral fragments drawn off during the surgery) . In an experimental model, the tropism towards the glioblastoma cells and the apoptotic effect of interferon beta on the cancerous cells will be subsequently followed up.

PROMOTION OF ROMANIAN PSYCHIATRIC GENETICS RESEARCH THROUGH INTERNATIONAL PARTNERSHIP IN GENETIC AND EPIGENETIC STUDIES OF BIPOLAR DISORDER

Acronim: BPGEN
CEEX – M3 – 122/2006
CO–„ALEXANDRU OBREGIA” PSYCHIATRY CLINICAL HOSPITAL, BUCHAREST
P1-„STEFAN S. NICOLAU” INSTITUTE OF VIROLOGY, BUCHAREST (Carmen Diaconu, PhD, Gabriela Anton, PhD)

Summary. The first objective of the project is the extention of the Romanian involvement in international genetics projects through adding the biological molecular dimension to the clinical-biometric cooperation and the setting up of a Romanian genetics research group capable to participate in international collaborative projects.

The second objective of the project is the informational and technological transfer through the training of Romanian researchers in genetic techniques (genome scan, secventiation, identification of new genes) and epigenetics [the microarray techniques DMH (differential methylation hybridization); MSPCR (Methylation Specific PCR)] in Germany. The practical use of these methods will occur in a pilot study intended to replicate some candidate genes for bipolar (manic-depressive) disorder (particularly G72 gene) and to explore their methylation pattern in a Romanian patient sample partially recruited during the current project.

The above mentioned objectives will be achieved through the collaboration among a research unit based in the biggest psychiatric hospital of Romania (SCPDAO-GENPSI), an institute of the Romanian Academy (IVN) and three institutes of the University of Bonn (Germany) [Institute of Medical Genetics (C1), Institute of Human Genetics (C2), Institute of Neuropathology (C3)] and the Central Institute of Mental Health of Mannheim, Germany (C4). Moreover, the collection of DNA from a Romanian sample of bipolar patients and normal controls is intended; this sample will be integrated in a large European sample for genetic study of bipolar disorder and will serve to replicating genes identified in other European samples.

Likewise, the project is compatible with the thematic field 1.1.2. since it will apply non-invasive methods (DNA methylation and genome scan methods) in an exploratoy study to detect putative epigenetic and genetic causes of bipolar disorder and to develop the competence of the Romanian researchers in using these techniques

THE ROLE OF THE EPIGENETIC INFORMATION IN ONCOGENESIS: DIAGNOSIS AND THERAPEUTIC ATTITUDE IMPLICATIONS

Acronym: REN (Romanian Epigenetics Network)
CEEX – M1- 117/2006
CO-UNIVERSITY OF BUCHAREST
P1-„STEFAN S. NICOLAU” INSTITUTE OF VIROLOGY, BUCHAREST (Gabriela Anton, PhD)
P2-NATIONAL INSTITUTE OF RESEARCH AND DEVELOPMENT IN BIOLOGICAL SCIENCES, BUCHARESTI
P3–„CF2” GENERAL HOSPITAL
P4–INSTITUTE OF COMPARATIVE MEDICINE, BUCHAREST

Summary. Cancer is one of the disease models that present a great interest for the specialists in epigenetics, especially nowadays, when it became a major thread in European countries, including our country too. In spite the enormous efforts invested in its genetic causes, clinical strategies are still not efficient from both diagnosis and therapeutic points of view. The newly emerged scenario of oncogenesis recognised both genetic and epigenetic factors involved in the molecular mechanisms of cancer. The study of the epigenetic hallmarks in cancer is even more interesting as it reveals also the prognosis and the early risk assessment potential. Another innovative aspect regarding the epigenetic approach is linked with the molecular targets envisaged by new therapeutic drugs in cancer. Tackling the chromatin remodelling components and the enzymes which catalyse their modification reactions represents a new more effective strategy based on the reversibility of epigenetic changes as compared with the dramatically fixed genetic (mutations) ones. Epigenetic domain is therefore an exciting area of research linked with cancer diagnosis and treatment. It needs urgent standardization which is proposed for study by grouping experts in different fields of biology: biochemists, geneticists, cell and developmental biology, in order to gather a great amount of data explaining the link between the chromatin structure dynamics, altered gene expression and specific interactions with new anticancer drugs.

A collaborative network of academic institutes is therefore propose by this project to use specific multidisciplinary and complex methodology experience in order to develop further a Romanian epigenetic network, which will be able to integrate into the European epigenetic research by tackling the study of the genomic instability from the point of view of genetic, epigenetic, biochemical aspects when involved into the breast tumorigenesis. Therefore, DNA methylation and histone modifications (methylation, acetylation/deacetylation) by specific native enzymes will be the major focus of the research for demonstrating their roles in maintaining a healthy course of life and avoiding the pathological pathways of gene expression, as well their therapeutic potential based on chromatin conformation restauration and hence the normal gene expression in tumor cells.

INTEGRATION OF MOLECULAR BIOLOGY TECHNIQUES AND CYTOGENETIC ASSAYS FOR TUMOR SPLENOMEGALIA DIAGNOSIS, ETHIOPATOGENIC FACTORS, PROGNOSTIC AND THERAPEUTIC IMPLICATIONS

Acronym – GENCITOSPLEN
CEEX –M1- 96/2006
CO–„COLTEA” CLINICAL HOSPITAL, BUCHAREST
P1–„STEFAN S. NICOLAU” INSTITUTE OF VIROLOGY, BUCHAREST (Principal Investigator: Gabriela Anton, PhD)

Summary. The aim of the present study is represented by a new ethiopathogenic and diagnosis approach in spleen related lymphomas, with the integration of new diagnosis criteria in current medical practice, and corroboration of these criteria with the post- treatment prognostic and evolution. The purpose of the study is to introduce molecular genetics markers (with high specificity for diagnosis and prognosis) in current NHL classification criteria (p53 gene mutations, Cyclin D1 gene expression , t(11;18)(q21;q21), fusion protein API2- MALT1, t(9;14) and del 7q). Lymphomas diagnosis approaches represent a constant research interest for various work groups organized in international consortia and excellence networks.

A second objective of the project is to correlate the viral ethiopathogenic implications (HCV, HVB, γherpesviruses- EBV and Kaposi sarcoma associated herpesvirus) with the lymphomas’ genesis of and prognostic evaluation. The molecular/genetic alterations and viral factors involved in lymphomagenesis have a great impact in post-therapeutic approaches by introducing a personalized therapy. Finally, the study will evaluate costs versus advantages in Romanian healthcare system using modern diagnosis and evaluation protocols at national level.