National Science Centre

Project implemented as part of a scientific consortium. The project leader is Mirosław Mossakowski Institute of Experimental and Clinical Medicine of the Polish Academy of Sciences and the Partner - the Medical University of Warsaw, 1st Faculty of Medicine. Gilles de la Tourette Syndrome (GTS) is a neuropsychiatric disorder of children and adults of unknown cause whose main symptoms are tics and mental disorders. The severity of tics decreases after the age of 15. Only 1/5 of people have tics persistently severe in adulthood. The cause of tics recovery in some people is unknown. There is currently no diagnostic test to confirm a clinical diagnosis of GTS. Genetic factors play a major role in the pathogenesis of the disease. Autism is an early childhood developmental disorder that causes disturbances in social functioning, speech disorders, and movement stereotypes. Some patients with GTS have autistic features, and both diseases show common symptoms. The biological basis for autism is not established. The aim of the study is to determine the genetic basis of the disease in three selected groups: 1. in families with GTS with a very clear family history of the disease; 2. in families where GTS and autism occur in closely related people; and 3. in adults with GTS and severe tics (which may indicate the presence of an additional genetic factor). In the research, we plan to use the whole genome sequencing (WGS) method that has not been used so far in the study of GTS substrate, thanks to which in the obtained raw sequencing results it will be possible to search for DNA copy number variants (CNV), regions of heterozygosity loss ( loss of heterozygosity (LOH), rare genetic variants (mutations) and identification of causative mutations by using the latest bioinformatics algorithms, comparing the genome sequences of healthy and sick family members and patients with different clinical phenotypes of GTS (including severe adult GTS vs mild form in children). Three groups will be used in the research: 1. eight families, in each of which there are at least 5 people diagnosed with GTS or other tics (40 people from each family will be tested: 3 sick people + 2 healthy people, most often parents); 2. four families with coexistence of GTS and autism (20 people will be tested: 4 people with GTS, 4 people with autism and 12 healthy family members); and 3rd group of adult patients with GTS and severe tics (30 people). The total study group will include 90 people. As the population of children with GTS in Poland amounts to 60,000 people, and adults - about 3,500 people, the size of the study groups is representative and gives a chance to determine the genetic basis of the disease, especially as groups 1 and 2 relate to family forms, and group 3 - severe forms GTS. In the research, we plan to use the whole genome sequencing (WGS) method that has not been used so far in GTS research. Both the copy number variants (CNV) and point changes of the DNA sequence will be searched for in the obtained raw genome readings. The obtained list of variants will be subject to further, in-depth bioinformatics analysis using the latest algorithms and available tools. The analysis will aim to identify potential causative variants, especially those that may affect the structure of protein products. The bioinformatic analysis will each time include a comparison of the genomes of sick and healthy people within the family, sick people between families and sick people with sequences stored in public databases. The analysis will additionally use the database of the sequence of the entire exome> 100 people from the Polish population that we had prepared as part of another project. Selected genomic variants will be confirmed by the Sangerian sequencing method or MLPA (Multiplex Ligation-dependent Probe Amplification), and their occurrence will be checked as broadly as possible in proband families. The project is the first attempt in Poland to understand the genetic basis of GTS and to establish possible relationships between the genotype and the clinical phenotype of the disease. The uniqueness of the study lies in the use of a modern method of whole genome sequencing (WGS) and advanced bioinformatics methods using data obtained from ethnically homogeneous and fully clinically described groups of patients and healthy people. One study will look at both broad genome rearrangements and point changes in DNA sequences, and in particular causative mutations. We expect that the project will lead to the identification of new genes and/or loci related to GTS, determination of common genes / loci for neuropsychiatric diseases such as autism and GTS, determination of the genetic basis determining the variable course of GTS, identification of biochemical and/or signaling pathways at the level of cellularity, and will also enable the determination of genomic markers of the disease and, in the future, the development of new methods of GTS treatment.

Pituitary adenomas are tumors that arise from the anterior lobe of the pituitary gland and act as the central regulator of hormonal homeostasis in the body. Pituitary tumors account for 10-15% of all intracranial neoplasms, the vast majority of which are adenomas. They are usually benign lesions, but there may also be malignant forms with poor prognosis, in which early tumor detection and implementation of neurosurgical treatment significantly improves the prognosis. To date, there is no good biomarker for malignant pituitary adenomas [1,2,6]. MicroRNAs are small, non-coding RNA molecules that can influence the regulation of genes. The literature confirmed changes in microRNA expression in various types of neoplasms [4,5]. Recent studies suggest microRNA deregulation also in the pathogenesis of pituitary adenomas [3]. The current project aims to identify new microRNAs that could serve in the future as a new marker for invasiveness of pituitary adenomas using Next Generation Sequencing (NGS). Another goal of this project is to investigate the regulatory role of selected microRNAs having binding sites in the 3 'UTR of genes for cell cycle proteins. To date, little is known about the regulatory role of the selected microRNAs and their impact on the level of cell cycle protein expression in pituitary adenomas. It is worth noting that the increased expression of cyclins, which is the subject of the research in this project, is often correlated with the increased malignancy of neoplasms, invasiveness and metastasis formation in many neoplasms [7]. Overall, this project aims to investigate the effects of epigenetic factors on cell cycle protein expression and in vitro invasiveness potential in model pituitary adenoma cell lines. At the same time, the aim of the research is to find new microRNA genes, the change in expression of which could be confirmed in the preliminary studies. The results of the experiments included in the project will be of great cognitive value as they may help to understand the still unexplained influence of microRNA on the expression of cell cycle proteins in human pituitary adenomas. In addition, the implementation of this research may help in the discovery of completely new cellular disorders leading to the inhibition or progression of neoplastic cells, and, consequently, determine the direction of searching for new therapeutic targets in the treatment of human pituitary adenomas.

High-throughput DNA sequencing results obtained from patients with inherited retinal diseases revealed a huge number of genetic variants of unknown clinical significance. The urgent need for further molecular studies to verify the pathogenicity of these variants crosses with the lack of access to the retina, the part of the eye directly affected by the disease. In view of the new findings pointing to the expression of human retinal genes also in the skin, we believe that skin cells may be a good model for studying the processing of mutant genes responsible for the development of retinal dystrophy. In this study, we intend to focus on the ABCA4 gene, which is one of the main genes of the retina and is responsible for the pathomechanism of many of its inherited diseases. Our main goal is to determine the composition of ABCA4 transcripts and to study the expression of this gene at the mRNA and protein level in hair follicles, keratinocytes and fibroblasts. For the purpose of this task, primary cultures of keratinocytes and fibroblasts will be established from the collected skin biopsies. Our next goal will be to compare the experimental data to select and implement the most appropriate cell type to study the metabolism of the mutant ABCA4 gene in patients. In order to achieve the presented goals, a qualitative analysis of transcripts will be carried out using the technique of rapid amplification of cDNA ends (RACE) and cDNA sequencing. Quantitative expression of the ABCA4 gene at the mRNA and protein level will be measured using real-time PCR and Western Blot methods, respectively. The expected effect of this work will be the analysis of the influence of the ABCA4 mutation on its processing and verification of the pathogenicity of these changes in the intracellular environment. In addition, ABCA4 transcript sequences in various types of skin cells will be identified for the first time. We are convinced that the model proposed in these studies will be successfully implemented to analyze mutations also of other retinal genes involved in the development of a broad spectrum of posterior ocular dystrophy and may be the starting point for further research aimed at discovering the role of ABCA4 in the skin.

According to the World Health Organization (WHO), malignant tumors are the leading cause of death worldwide, causing approximately 8.2 million deaths annually. Each year, approximately 14 million new cases of malignant neoplasm are diagnosed. According to the latest reports from the International Agency for Research on Cancer (IARC), the annual number of cancer cases is expected to increase from 14 million in 2012 to 22 million over the next two decades. In Poland, malignant neoplasms are the second most common cause of death, after cardiovascular diseases. While mortality from cardiovascular disease has decreased significantly in recent years, cancer mortality has significantly increased. Indeed, mortality due to cancer in 2008 was higher in Poland than in 1980, constituting 25.8% of all deaths, while mortality due to cardiovascular diseases in 2008 was lower by about 39% compared to 1980. An important fact is that both the incidence and mortality due to cancer in Poland are at one of the highest levels among all EU countries. According to the latest report from the National Cancer Registry, in 2012 there were over 152,000 newly diagnosed people with malignant neoplasms and over 94,000 deaths related to these diseases. Classic forms of therapy, such as surgery, chemotherapy and radiotherapy, are currently only partially effective in the fight against cancer. Despite numerous advances in genetics and molecular biology, virology, chemistry, and pharmacology, cancers continue to successfully avoid treatment. However, thanks to increasing innovation, scientific discoveries and technological progress, the understanding of cancer is undergoing a profound transformation. While still in its infancy, so-called personalized medicine is emerging as a new paradigm in cancer treatment. Part of this new paradigm is the understanding of cancer as a systemic disease in which there is an intense dialogue between the cancer and the host, especially the host's immune system. It should be noted that in the light of the current state of knowledge, the ability of the human immune system to specifically recognize cancer-related molecules makes it an extremely precise tool and highly suitable for personalizing cancer treatments. This is particularly true for cancers associated with infection by viruses such as HCV, HBV or HPV, which, according to the WHO, are responsible for almost 20% of cancer deaths in low- and middle-income countries. Science Magazine called cancer immunotherapy "Breakthrough of 2013". Indeed, immuno-oncology is one of the most vigorously developing areas of cancer research today, and raises great hopes for a cure for these debilitating diseases. It is anticipated that in the next decade, immunotherapies will be the mainstay of treatment in 60% of cancer types. Before the immune system can be used to its fullest effectiveness in the fight against cancer; however, several challenges need to be dealt with. These challenges can be divided into two main areas: 1. understanding the mechanisms of cancer escape from the immune system; 2. identification of the most effective anti-cancer tools derived from the immune system. There is ample evidence that cancer employs at least several different mechanisms to avoid being controlled by the immune system. One of these mechanisms is based on the increase in oxidative stress conditions and high levels of reactive oxygen species (ROS) such as hydrogen peroxide within the tumor mass. Another important element is the conditions of reduced oxygen availability in the neoplastic environment, called hypoxia.Under such conditions, numerous anti-cancer mechanisms in the cells of the immune system are switched off. Therefore, it becomes important to understand how the cells of the immune system can adapt to such a hostile environment. This is one of the objectives of this proposal. Natural killer (NK) cells are part of the human immune system and act on the borderline between innate and acquired immunity. Numerous publications suggest that the proper functioning of NK cells plays an important role in the fight against viral infections and cancer. Therefore, intensive research is currently being carried out to successfully use these cells as a therapeutic tool in the fight against cancer. Unfortunately, the anti-tumor activity of NK cells can be suppressed by high levels of oxidative stress or the tumor's hypoxic environment, which in turn can seriously hinder their use in oncology. For this reason, in our project we try to understand in detail the mechanisms that take place in NK cells under conditions of oxidative stress and hypoxia. On this basis, we want to select mechanisms that can potentially protect NK cells against the inhibitory effects of oxidative stress and hypoxia. In the final stage of the project, we intend to create NK cells that will be able to overcome the inhibitory effect of the tumor environment and effectively kill the tumor cells. In this project, we will use a modern RNA sequencing technique to capture changes in NK cells subjected to oxidative stress and hypoxia. The conducted research will provide us with a wide range of information on changes in gene expression in NK cells under the conditions associated with neoplasms. From the range of information available, we will select those that will be of importance in the fight against cancer cells by NK cells. Based on the acquired knowledge, we will modify NK cells so that they can effectively recognize and kill cancer cells under the hostile conditions of oxidative stress and hypoxia. By deciding on such an ambitious project, we want to contribute to the identification and understanding of the molecular mechanisms regulating the activity of NK cells in the tumor environment, and then use the acquired knowledge to create NK cells, resistant to the inhibitory action of tumors. The knowledge we gain may in the future lead to the improvement of therapeutic methods that use modified NK cells as a therapy against cancer. This therapy can also be used in other human diseases.

Chronic myeloid leukemia (CML) is a model neoplastic disease; research into this disease has resulted in breakthrough discoveries and led to the development of targeted therapy in oncology; for the first time in this disease, a genetic change was associated with a neoplastic process, and for the first time in this cancer, success was achieved with the use of targeted therapy with tyrosine kinase inhibitors (TKI). Unfortunately, a significant proportion of patients develop resistance to such therapy; another problem is the lack of complete cure due to their insensitivity to the therapy of leukemic stem cells. CML begins with hematopoietic stem cells, which, after a genetic change - the formation of the Philadelphia chromosome as a result of a balanced translocation between chromosomes 9 and 22 - t (9; 22) (q34; q11) and neoplastic transformation, transform into leukemic stem cells. Leukemic stem cells represent one of the most important obstacles to curing chronic myeloid leukemia. Since they are insensitive to tyrosine kinase inhibitors (TKIs), therapy with imatinib or next-generation inhibitors, unfortunately, does not eliminate these cells. In disease evolution, leukemic stem cells show genetic instability and acquire secondary genetic changes. These may be macro-scale phenomena at the level of the cell's genome, such as additional translocations or deletions of chromosomal fragments, they may be micro-changes, such as point mutations, which may, however, have equally serious consequences - e.g. acquisition of resistance to tyrosine kinase inhibitors. Interestingly, these chromosomal changes are often accompanied by a strong shortening of telomeres. Telomeres as nucleoprotein structures located at the ends of eukaryotic chromosomes are one of the key elements responsible for maintaining genomic stability. The importance of this discovery, in particular with regard to the understanding of the fundamental mechanisms of the functioning of organisms at the cellular level, as well as the relationship of telomere disorders with cancer or the aging process, was recognized with the 2009 Nobel Prize in Physiology and Medicine for E. Blackburn, C. Greider and J. Szostak for the discovery "how chromosomes are protected by telomeres and the enzyme telomerase". The discovery of the possibility of elongation of telomere via an alternative pathway based on the mechanisms of recombination, or finding the lack of expression or activity of telomerase in neoplastic cells, indicates, much more than previously thought, the complexity of the mechanisms controlling the telomeric complex. Understanding these molecular mechanisms is therefore important not only for the very fact of understanding the causes of massive chromosomal damage, but also for the use of telomere changes for diagnostic or prognostic purposes. The existing studies as well as our own preliminary results clearly indicate a strong relationship of this process with changes taking place within telomeres. In a preliminary analysis of leukemia cells from patients in different phases of the CSF, we observed differences in telomere length with no or very low expression of TERC and TERT genes (telomerase subunits), and differences in the expression levels of some of the shell-associated proteins (telomere associated). Thus, the obtained preliminary results allowed for the formulation of a hypothesis concerning the active role of the telomeric complex in drug resistance and disease progression. A detailed understanding of the mechanisms responsible for the increasing genomic instability of chronic myeloid leukemia cells, especially leukemic stem cells, which increases with disease progression, could help in the treatment of patients with disease progression. It could also contribute to our understanding of the role of telomeres and the telomere complex in cancer.

Hypothesis: Accumulating evidence suggests that redox imbalance selectively kills cancer cells, and that some antioxidant enzymes are important therapeutic targets. Thioredoxins (TXN), and members of the same family of peroxyredoxin (PRDX) proteins are antioxidant enzymes that ensure redox balance is maintained. Our preliminary studies showed significantly elevated levels of reactive oxygen species in B-cell acute lymphoblastic leukemia (B-ALL) cells. Moreover, both in B-ALL cell lines and in patients' lymphoblasts, we observed an increased level of expression of antioxidant enzymes from the TXN family. The expression level of these enzymes increased in patients in the relapse stage. Moreover, in B-ALL lines we observed that PRDX1 promotes the proliferation of leukemic cells. We suspect that enzymes from the TXN family are one of the factors improving the survival of leukemic cells under oxidative stress conditions and may be new therapeutic targets. Research objective: In this project we will investigate the effectiveness of a new pro-oxidative strategy in the treatment of B-ALL. The most important goal of the project is to validate PRDX1 and other TXN family enzymes as potential new therapeutic targets for B-ALL. We also plan to investigate how PRDX1 supports the proliferation and survival of B-ALL cells. In addition, we would like to assess whether inhibitors of the TXN family of enzymes sensitize B-ALL cells to the chemotherapeutic agents used to treat B-ALL. Research method: To achieve the above goals, we plan to use several research models: B-ALL cell lines (NAM-6 and SEMK2), clinical material collected from B-ALL patients (bone marrow lymphoblasts, serum), and an in vivo mouse model. Three research tasks will be carried out: Task 1. Validation of PRDX1 and other antioxidant enzymes from the TXN family as therapeutic targets in B-ALL in cell line models. 1A. To study the effects of inhibition of PRDX1 expression on B-ALL cell survival and proliferation. 1B. To test whether the inhibition of leukemic cell proliferation resulting from blockade of PRDX1 expression depends on cysteines. 1C. To study the transcriptional response of leukemic cells to the inhibition of PRDX1 expression using high-throughput genetic methods. 1D. Study of the effectiveness of therapies combining inhibitors of antioxidant enzymes from the TXN family with chemotherapeutic agents used in the treatment of B-ALL. Task 2. Examination of the biomarkers of oxidative stress and the expression of antioxidant enzymes from the TXN family in the material collected from patients with B-ALL. 2 A. Collection of material from patients and clinical base of adults and children diagnosed with B-ALL 2 B. Assessment of biomarkers of oxidative stress in serum from B-ALL 2 patients C. Evaluation of the expression level of TXN family enzymes in B-ALL 2 patients D. Statistical analysis of redox parameters in relation to clinical data Task 3. Investigation of the effects of blocking the TXN family antioxidant enzymes in vivo in a mouse model of human leukemia. Effect of the results: B-ALL is the most common pediatric neoplasm. Classical chemotherapy is almost exclusively used to treat B-ALL. Most patients respond well to remission inducing therapy, but relapse, often refractory to treatment, occurs in approximately 20% of patients. Better understanding of the biology of B-ALL and the molecular mechanisms responsible for the proliferation and survival of lymphoblasts, in particular the role of antioxidant enzymes from the TXN family, will improve the effectiveness of existing treatments and may indicate new targets in B-ALL therapy. As a peroxide metabolizing enzyme, PRDX1 has important signaling functions.Additionally, under oxidative stress, PRDX1 can act as a chaperone. Not all functions of the PRDX1 are dependent on the peroxide removal activity. So far it is unclear whether the anti-apoptotic function of PRDX1 depends on cysteines and activity related to the metabolism of peroxides. In this project we will try to answer this question. A better understanding of the anti-apoptotic activity of PRDX1 will contribute to the development of new, more selective inhibitors of this enzyme.

In recent years, an inexplicable increase in the incidence of hematopoietic neoplasms has been observed. Currently, the most commonly diagnosed type of leukemia is chronic lymphocytic leukemia (CLL) - neoplastic growth originating from B lymphocytes. The course of chronic lymphocytic leukemia shows significant differences - in some patients the cancer develops slowly and does not require treatment, in others the disease progresses rapidly, drug resistance develops and the disease requires treatment with multi-drug regimens. Due to the advanced age of patients (in Poland, the median age at diagnosis is 72 years), it is extremely important to develop safe therapeutic regimens with the lowest possible side effects. In the haematological community in recent years, the postulate of creating a chemotherapy-free therapy has been repeatedly undertaken. Many physicians are inclined to use targeted therapy in combination with specific inhibitors of signal transduction pathways. The development of such therapies should be preceded by thorough research at the level of cell biology. One example of targeted therapy is the widely used anti-CD20 monoclonal antibody. By binding to the CD20 antigen present on tumor cells, these antibodies activate a number of mechanisms involving the cells of the immune system and leading to tumor elimination. The binding of the antibody to the CD20 molecule causes direct killing of the tumor cell by activating the protein cascade, the so-called complement system and programmed cell death. Therapy with anti-CD20 antibodies also involves the mechanisms of non-specific immune response, such as the cytotoxic effect of NK cells and phagocytosis, i.e. the absorption and digestion of neoplastic cells by macrophages. Anti-CD20 antibodies are well tolerated and have few side effects and are successfully used in multi-drug regimens, but when used alone, they rarely provide a complete cure. Therefore, for years, attempts have been made to increase the effectiveness of anti-CD20 antibodies by combining them with new drugs used in oncology. The results of our experiments published in international scientific journals indicate that the use of certain new compounds used or undergoing clinical trials in hemato-oncology leads to the sensitization of neoplastic cells of established cell lines to the action of anti-CD20 antibodies. Thanks to the cooperation with the Institute of Hematology and Transfusion Medicine in Warsaw, we confirmed our observations in samples obtained from patients with CLL. The next stage of our research should therefore be to verify these observations in an animal model. This is also the purpose of this project. We plan to create mouse models that will allow us to comprehensively assess the therapeutic potential of the combinations we propose. Mouse models, although far from a perfect representation of reality, will allow us to identify favorable and unfavorable interactions between drugs tested by us in the in vitro system. In addition, the project involves the creation of a specialized platform that allows the assessment of parameters important for anti-CD20 antibody therapy. The combination of information obtained from in vitro studies and studies in the mouse model will allow us to obtain a more complete picture of the effectiveness of the combinations we propose. The results obtained may therefore be a premise for undertaking clinical trials of the most promising combinations.

The early stages of tumorigenesis as well as tumor progression and metastasis are strongly associated with the inflammatory response. After tumor formation, cancer cells contribute to the development of chronic inflammation by stimulating infiltration of pro-inflammatory cells of the immune system. The tumor-infiltrating immune cells stimulate angiogenesis, increase the permeability of blood vessels, as well as enhance tumor cell proliferation and colonization of distant organs. The formation of new blood vessels is crucial for tumor development, and the process is heavily regulated by inflammation developing in the tumor microenvironment. The interaction between the CD200 ligand and its CD200R receptor regulates the immune response and the inflammatory response in the course of infection, autoimmune diseases and cancer. The exact mechanism by which the CD200R-dependent signaling pathway works is not fully understood. We recently showed that the CD200R signaling pathway regulates arteriogenesis (the process of enlarging existing arteries), restoring normal blood flow, and increasing blood vessel lumen. Moreover, in preliminary studies, we were able to show that CD200R regulates angiogenesis in a mouse tumor model. CD200-deficient mice were characterized by a more intense blood supply to matrix implants containing melanoma cells, as well as an increased size of newly formed blood vessels. Importantly, vascular parameters correlated with an increased fraction of pro-inflammatory, undifferentiated macrophages and a sub-population of pro-angiogenic T lymphocytes. We hypothesize that the CD200R signaling pathway inhibits neoplastic angiogenesis. We will investigate the effect of CD200R modulation on the development of blood vessels in matrigel implants with B78 murine melanoma cells. This model has many advantages: it allows for a very reproducible examination of angiogenesis in relatively small tumors. Moreover, it enables the modification of the tumor microenvironment (e.g. the use of inhibitors, growth factors, cell transfer). We will use mice with a disabled CD200R pathway (Cd200 - / -) and wild-type mice. We will also study the effect of increasing CD200R stimulation (agonist antibody) and blockade of CD200R (soluble CD200R-Fc receptor). The use of flow cytometry, histological analysis, as well as depletion and adoptive transfer of selected immune cells iv vivo, will enable us to identify populations responsible for the regulation of tumor angiogenesis, dependent on CD200R. In addition, we will examine the profile of secreted inflammatory factors regulated by the CD200R. Immune cells expressing CD200R and possessing pro-angiogenic properties will be isolated and tested for the secretion of pro-inflammatory factors. The expression analysis of genes regulated by CD200R will also be performed. The results of these studies will be verified in the model of capillary formation by embryonic endothelial cells (in vitro) and in matrix implants with neoplastic cells (in vivo). Currently, the knowledge about the influence of immune cells on a developing neoplasm is used in the immunotherapy of neoplastic diseases. Modulation of the activity of individual populations of immune cells leads to a marked improvement in the cure rate. Yet a large proportion of these complex interactions remain unexplained. Understanding the mechanisms by which the CD200R regulates inflammation and angiogenesis in the tumor microenvironment may be of great importance in understanding the course of cancer formation and progression. It may also contribute to increasing the effectiveness of immunotherapy in neoplastic diseases.

Chronic myeloid leukemia (CML) is a model cancer that has been studied for many years, but the genetic changes responsible for disease progression are poorly understood. The main goal of the project is to detect aberrations in the genome and epigenome of CSF responsible for the unfavorable course of the disease, i.e. resistance to targeted therapy and / or progression to the acute phase, using high-throughput techniques. The next goal is to investigate the potential biological role of the aberrations found and to attempt experimental blocking in in vitro models. In the proposed project, the search for new genetic and epigenetic aberrations in CSF will be performed using the technology of enriching DNA libraries with specific sequences (exomal and CpG islands) and then high-throughput sequencing. In the next stage, the biological role of selected aberrations in disease progression and drug resistance (including gene silencing, directional mutagenesis) and an attempt to experimentally block the detected targets with the use of computer modeling and aptamer synthesis will be examined on the in vitro models of CMF. Despite the increasing use of high-throughput sequencing, many cancers remain poorly characterized, as exemplified by chronic myeloid leukemia, especially in terms of lesions responsible for disease progression. Understanding new genetic and epigenetic changes in CML and examining their biological significance in cellular models may contribute to a better understanding of the pathogenesis of CML. and to develop new and better therapies for adversely affected patients, as well as therapies which, by hitting cancer stem cells, will give a chance for a complete cure. The implementation of the HARMONIA grant would enable not only the continuation but a significant extension of the long-term cooperation between the project manager (Dr. T. Stokłosa) and the foreign partner (Dr. T. Skórski). A team of contractors including scientists (geneticists, biotechnologists and bioinformatics) and clinicians and a modern workshop for genetic research (NGS platform) recently available at the Medical University of Warsaw, on the one hand, and on the other hand, the possibility of translational research on in vitro models, including computer modeling and synthesis of new molecules with a foreign partner and its vast experience and expertise in research on the molecular pathogenesis of leukemias give a real chance to make important discoveries. The HARMONIA grant would provide a unique opportunity to obtain synergy between the research carried out by both partners. This could contribute to a better understanding of the pathogenesis of CML and in the future, hope for better therapy for the sick. In the long term, the results obtained in the implementation of the grant could constitute an introduction to further translational research on in vivo models. An additional benefit would be short scientific internships for the implementation of research tasks by young team members at a foreign partner's, which would contribute to the exchange of experiences and research methods.

Monozygotic twins are formed from the division of one egg fertilized by one sperm. Hence, MZTs have been used to be regarded as identical both physically and genetically. Nevertheless, an increasing number of reports clearly indicate the occurrence of phenotypic differences between MZTs, including differences in sick-healthy status. Assuming that the genomes and epigenomes of identical twins are almost identical, it is much easier to pinpoint the differences and relate them to a feature that has occurred in only one of the twins than with other or no kinship. The aim of the present project is to identify genetic and/or epigenetic changes underlying the phenotypic differences observed between monozygotic twins, in a setting where one of the twins within the same pair is chronically ill and the other is healthy.