NATIONAL CENTRE FOR INFORMATION AND DOCUMENTATION

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ADVANCES IN BULGARIAN SCIENCE

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Advances in Bulgarian Science

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Published by National Centre for Information and Documentation 52 A G. M. Dimitrov Blvd 1125 Sofia, Bulgaria, Phone: +359 2 817 38 62 http: www.nacid-bg.net e-mail: [email protected]

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ISSN: 1312-6164

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NATIONAL SCIENTIFIC PROGRAMS – AN INSTRUMENT FOR STIMULATION OF RESEARCH AND INNOVATION

National scientific programs in Bulgaria play the part of specialized priorities in the field of science, aiming at increasing the competitiveness of the national economy. They are approved as relevant to the European Framework Programs for development of science and technology and are in conformity with the requirements of the European Commission on reciprocity with regard to participation of other countries. The main characteristics of the national programs are: creation of conditions for sustainable economic development; improvement of the environment for research and innovation; fostering the conditions for regional science and innovation development; improvement of the effective support of the research work; binding to the priorities of the European Union. National scientific programs are expected to bind horizontally different national and regional scientific programs and initiatives and to create conditions for coherent environment between different national, regional, and European policies. An open model for coordination of scientific research and innovation as well as other activities connected with the development of the human resource is achieved through effective development of national scientific programs. Areas of competency are being defined, synergetic effect is being achieved, and technology clusters are being formed. The applicable instruments are from the themati- cally orientated programs in the priority fields for the country, via reciprocity of the national programs at regional and European levels up to building up joint program initiatives on the principle of mutually agreed priorities. The topic of the present issue of „Advances in Bulgarian Science“ is the national scientific program „Genomics“. Genomics is a new science concerning with the analysis of genetic information coded in the genomes of a living being, including those of a human being. It won recognition during the last decade in connection with the development of the international Human Genome Mega Project, the first stage of which was successfully completed in 2000. Genomics has disclosed unexpected prospects for deciphering of genetic information and using it in favour of a man. During the recent years a Functional Genomics subprogram was started within the framework pro- grams for scientific research of the European Union. Its aim is to finance strategic integral projects in the field of genomics, as well as to unite efforts and improve qualifications of scientists from European countries working in this field. In view of the strategic place of genomics for the development of medical, biological, agrarian sciences and pharmaceutics during the next decade, national programs on genomics have already been started in most of the European countries. Giving an account of the fact that Bulgarian science is a part of the European science, the national Genomics Program should be considered as a projection of the European program reflecting specific for Bulgaria problems and interests. The aim of the program is to accelerate the development of genomics, using the existing and getting new knowledge on genomes of important for the country living organisms (bacteria, plants, animals and man). The efforts should be directed to creation of conditions for increasing the quality of the public health and life of the Bulgarians and preservation of biodiversity in the country. The program realizes its aims through integrated projects in genome analysis, bioinformatics and new genome markers.

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MAIN OBJECTIVES

NACID is a governmental institution affiliated to the Ministry of Education and Science. NACID collects, processes, maintains and disseminates reference and analytical information to support the national policy in the field of education, science and innovation as well as to support Bulgarian research bodies, individual researchers and SMEs.

PRIMARY FIELDS OF ACTIVITIES:

Processing and disseminating bibliographic, reference data and analytical information. Maintaining specialized databases of scientific production and research resources in Bulgaria. Providing information about national, European and trans–European research programs. Providing information to support the process of harmonization of the Bulgarian education and research legislation with European Union ones. Performing the role of institutional contact point of the Sixth Framework Program in Bulgaria.

INFORMATION PRODUCTS AND SERVICES

NACID offers a large range of information products, including: • Subject profiles; • Reviews; • Bibliographic references; NACID offers a great variety of information services through its own databases as well as information brokerage to external databases to the following locally maintained own databases: „Bulgarian Science“ Databases

„SIRENA“, R&D reports and dissertations – more than 75 000 records „Scientific and Technical Publications in Bulgaria“ – more than 2 000 records „Who is Who in Bulgarian Science“– more than 5 500 records „Papers“ – more than 120 records „ Science and Industry“ Databases „Partnership for Innovation and Development“ – information about the national Research units. „Knowledge for Innovations and Development“ – information about Bulgarian R&D activities.

The information brokerage services are available upon request, providing access to over 1200 databases from major international commercial host centers, thus providing the research community with a wealth of S&T information. Central Research and Technical Library services: – Library collections – more than 4 million items/reference – Lending of library materials; books, monographs, serials, dissertations, CD ROM, DVD etc., – Electronic Document Delivery; – Online access to the library catalogues since 1980; – Interlibrary loan; – Searching in electronic catalogues and databases; – More than 9000 users per year.

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CONTENTS ○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○ NATIONAL SCIENTIFIC PROGRAMMES WITH EUROPEAN DIMENSIONS 7

PLANT GENOMICS RESEARCH IN BULGARIA – PRESENT STATUS, PROBLEMS AND DIRECTIONS OF DEVELOPMENT 7

FAMILIAL COLORECTAL AND ENDOMETRIAL CANCER 15

BIOCHEMICAL DIAGNOSIS OF LYSOSMAL STORAGE DISEASES IN BULGARIA 19

PRENATAL DIAGNOSIS OF THE MOST COMMON HUMAN

ANEUPLOIDIES WITH DNA ANALYSIS 22 ○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○ BULGARIAN ADDED VALUE TO ERA 24

THE STEPHAN ANGELOFF INSTITUTE OF MICROBIOLOGY – CENTER OF BULGARIAN MICROBIOLOGICAL SCIENCE 24

MARITSA VEGETABLE CROPS RESEARCH INSTITUTE: 75 YEARS OF SERVICE 38

MOLECULAR MEDICINE CENTER – NOVEL CENTER FOR BIOMEDICAL RESEARCH IN BULGARIA 47

GENETIC SERVICE FOR CYSTIC FIBROSIS IN BULGARIA 51

NEUROPSYCHIATRIC GENETICS RESEARCH GROUP 52 ○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○ MADE IN BULGARIA WITH EUROPEAN SUPPORT 56

SELECTIVE SCREENING FOR DIAGNOSTICS OF INHERITED METABOLIC DISEASES IN BULGARIA 56

DNA ANALYSIS FOR SOME OF THE NEUROMUSCULAR DISORDERS IN BULGARIA 58

METHODS AND TECHNOLOGIES FOR MANUFACTURE OF ENZYMES – ALPHA-AMYLASE, XYLANASE, CELLULASE 62

GRANULATED APPLE AND CITRUS PECTINS 62

PHYTEX – NEW SOLUTIONS FOR EXTRACTION OF BOTANICALS 63

TRANSPLANTATION OF ANIMAL EMBRYOS IN BULGARIA 64 ○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○ EQUAL IN EUROPEAN RESEARCH AREA 66

BULGARIAN VIPs 66

AWARDS 73

ARTICLES 78

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National Scientific Programmes with Europian Dimension

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NATIONAL SCIENTIFIC PROGRAMMES WITH EUROPEAN DIMENSIONS

PLANT GENOMICS RESEARCH IN BULGARIA – PRESENT STATUS, PROBLEMS AND DIRECTIONS OF DEVELOPMENT

Atanas Atanassov, Elena Marinova, Elena Todorovska, Ivan Atanassov, Rositza Bachvarova, Tzvetanka Hvarleva – AgroBioInstitute, Sofia, Bulgaria Aglika Edreva, Kostadin Gecheff – „Doncho Kostov“ Institute of Genetics, Bulgarian Academy of Sciences, Sofia, Bulgaria Dimitrina Kostova, IZK „Maritsa“, Plovdiv, Bulgaria Ilia Denev, Tsanko Gechev – Plovdiv University, Plovdiv, Bulgaria Ivan Todorov – Institute of Agriculture and Seed Science „Obraztsov Chiflik“, Rousse, Bulgaria Natasha Kovacheva – Institute of Oil Rose, Kazanlak, Bulgaria Nikolai Tsenov – Dobroudja Agricultural Institute, General Toshevo, Bulgaria

The beginning of the 21st century marks mine and implementation of adequate and suc- the era of functional genomics. The genomics cessful mid– to long–term national directions research is driving the development and rapid and priorities of genomics research will be crucial progress of a number of „omics“ technologies for further development of Bulgarian agricul- directed to the analysis and deduction of gene tural and plant sciences as well as providing of (one or many) function in a short time, at the the necessary support for implementation of ad- transcript (transcriptomics: parallel analysis of vance practices for product quality control and gene expression), protein (proteomics: analysis of „modern production“ technologies. polypeptide complement), and metabolite (metabolomics: parallel analysis of a range of I. CURRENT STATUS AND PROBLEMS OF OR- primary and secondary metabolites) levels. As a GANIZATION, FUNDING AND PERFORMING OF result plant genomics leads not only to rapid PLANT GENOMICS RESEARCH IN BULGARIA expansion of our understanding of how genes In 2001 the Ministry of Education and Sci- determine plant function, but also promote con- ence applied and the Council of Ministers selected solidation of diverse research areas, bringing to- six priority National Scientific Programs. Among them gether biological, agricultural and ecological stud- was „GENOMICS“, relevant to EC FP6 priorities for ies. The genomics studies is relating with substan- scientific and technological development and con- tial and elevating rate of funding, infrastructure sidering traditionally good practices and research development and advance training correlated with capacities of the country. The Decision was that increasing international cooperation and concen- funds would be annually covered by the Budget in tration of research capacity. At the same time the frame of group „Research“ equally by the the level of performed genomics research largely Ministry of Education and Science (National Science determines the rate and capacity of nation–wide Fund)(NSF) and Ministry of Agriculture and For- technology development, transfer and applica- estry (MAF). tions in large areas of agricultural and food in- The National program was launched in 2003. dustries. This is particularly important for coun- The main advantage of this program is to join the tries like Bulgaria, possessing very limited resources best for the moment researchers from different for development of plant genomics. Thus, deter- Bulgarian institutions in the field of plant genomics.

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Main achievements: economically important plant species and have been Large scale genotyping of a number of extensively used in the breeding programs. The SNP crops and respective EU cooperation; (Single Nucleotide Polymorphisms) markers are the Advanced and extended transgenic and next wave highly perspective di–allelic DNA mark- GMO management researches; ers that already have found multiple applications Internet accessible Bulgarian plant genomics in plant genotyping, gene diversity studies, mutation database; detection analyses, QTL and MAS. Started advance extension services for GMO The main directions of studies proposed by and genetic authenticity analysis; the ABI research group in this multidisciplinary Started first in the country genome wide project are as follows: mapping projects. Genotyping of economically important lines Main problems: and cultivars by SSR markers; Insufficient rate of government–backed Development of an experimental proto- funding for genomics research; col for detection of SNP polymorphisms and its No clear vision and implementation of the application in genotyping analyses of economically long–term government–backed program for plant important lines and cultivars; genomics; Selection of core SSR and SNP marker Still underdeveloped national industry and sets sufficient for determination of genetic identity small demands for modern biotech products and of economically important for Bulgaria crops; technologies; Characterization of the segregating map- No infrastructure for „omics“ technolo- ping populations by SSR markers and mapping of gies. loci for the important agronomic traits, Mapping of genes, specifically expressed in petals, and those re- The most urgent needs and measures: sponsible for important agronomic traits by SSR Establishment of „minimal“ infrastructure markers; for the application of „omics“ technologies, provid- Selection of appropriate SSR and SNP ing countrywide services at competitive prices. markers for MAS.

II. CURRENT PROJECTS SUPPORTED BY THE NSF, II.1.2. Partner: Dobroudja Agricultural Institute PROGRAM „GENOMICS“ – G. Toshevo

II.1. MOLECULAR MARKERS FOR GENETIC AU- Program Module Leader: Associate Prof. PhD THENTICITY, MARKER ASSISTED SELECTION Nikolai Tsenov AND PRODUCTION OF CERTIFIED PLANTING Key words: wheat, sunflower, hybridization, abiotic MATERIAL FROM ECONOMICALLY IMPORTANT stress, DNA, SSR–markers CROPS (wheat, grape, sunflower, rose) Varieties and breeding lines of wheat and sun- flower are included as the object of study. The aim II.1.1. Partner: AgroBioInstitute – Sofia of the project is an optimal organization of experi- mental research work on the use of molecular mark- Program Module Leader: Acad. DSc. Atanas ers. The Laboratory of Molecular Biology is sug- Atanassov gested to be equipped with the necessary Key words: molecular markers, wheat, grape, sun- consumables for DNA isolation. Analyses of this DNA flower, rose, genotyping, QTL, marker assisted selec- are performed in ABI. The results obtained are pro- tion, mapping cessed simultaneously in both institutes via Internet. The SSR – (Simple Sequence Repeast) mark- The experimental research work on sunflower ers are one of the most developed DNA markers includes: and have been extensively exploited in genotyping Genotyping by 10 microsatellite mark- and gene diversity studies. Recently, reference ers of parental lines included in own and foreign microsatellite maps have been developed from most hybrids;

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National Scientific Programmes with Europian Dimension

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Development of a segregating population Key words: V. vinifera, inbreeding, hybridization, by crossing lines, which carry genes for resistance seedlessness, resistance, molecular markers to downey mildew and broomrape; Analysis of the population by microsatellite Having in mind fast development of elabora- markers; tion and application of high effective molecular Genetic analyses of materials with regard markers in plant genomics it is considered to form to the microsattelite markers used. consortium connected with research and practical The experimental research work on wheat application of molecular markers to Vitis vinifera. includes: Genotyping by 30 microsatellite markers The main objectives of the consortium are as of promising breeding lines and economically im- follows: portant varieties; • Fast development and application of high Developing of a segregating population by technological methods for control and evaluation crossing lines resistant and susceptible to frost; of quality and origin of planting material in agricul- Analysis of segregating population by ture; microsatellite markers corresponding to loci com- • Routine application of molecular markers in paratively evenly distributed in the wheat genome. selection of vine, starting with registration of lines and cultivars and intellectual property protection; Within the project’s framework it is expected • Marker assisted selection of polygenian to introduce efficient use of markers in breeding symptoms conditioned by great number of loci/ work as a result of establishment of the necessary genes. conditions and organization for their application. The following practical results are expected: The following activities take place in IASS Determining markers, which characterize „Obraztsov Chiflik“ Rousse: economically important lines and varieties; • Maintenance and phenotype characteriza- Determining the genetic identity of the tion of collections of lines and vine cultivars; planting material; • Passportization of cultivars and vine forms Developing of method for quantitative on the basis of molecular markers; analysis of the homogeneity of the wheat. • Joint research with ABI on: (i) genotyping of collections of lines and cultivars on the basis of II.1.3. Partner: Institute of Oil Rose – Kazanlak SSR and SNP markers; (ii) analyzing of segregation Program Module Leader: Natasha Kovacheva populations by SSR and SNP markers; (iii) process- Key words: oil rose, molecular markers, genotyping, ing of experimental results. cross–breeding, segregating populations, gene map- ping. II.2. IDENTIFICATION OF KEY GENES CONTRIB- The aim of this study is to genotype sorts UTING TO THE INCREASED RESISTANCE OF IM- from oil – bearing rose and to determinespecific PORTANT CROPS AGAINST BROOMRAPE genes. WEEDS (OROBANCHE SPP.) Cross–breeding materials including different genotypes are used. Methods of Sequence Re- Project leader: Ilia Denev, „Paisii Hilendarski“ Plovdiv peats and Single Nuclear Polymorphism for evalua- University, Plovdiv tion and characterization of determined genes, dis- tillations, gas–chromatography and organonoleptic II.2.1. Partner: AgroBioInstitute – Sofia analysis of oils are used. Program Module Leader: Dr. Rositza Bachvarova II.1.4. Partner: Institute of Agriculture and Seed Key words: Genomic bases of plant pathology, new Science „Obraztsov Chiflik“, Rousse genomic markers, Arabidopsis mutants, differential Program Module Leader: Prof. DSc. Ivan Todorov display, Orobanche spp., resistance

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It is well known that broomrapes sistance to fungal diseases and high quality of grape (Orobanchaceae) are obligatory root parasites of and wine. The established segregation population, plants. About 70% of agricultural lands in South- obtained after self–pollination of plants of culti- ern Europe and Mediterranean are infested by var Storgozia, gives unique possibility for direct iden- several broomrape species, which parasitize impor- tification of QTL locies conferring resistance to fungi tant crops, thus causing tremendous agricultural and economically important traits. losses. Currently there are no effective, inexpensive The proposed integrated project involves a control measures for broomrape that can be ap- complex (phenotypic, molecular and metabolic) char- plied in case of large scale outbreak. Genetic engi- acterization of the above–mentioned segregation neering of resistance mechanisms is the most prom- population. Core mapping of the populations with ising solution, but it requires knowledge of funda- microsatellite markers, combined with phenotypic mental aspects of host–parasite interaction that is and metabolic analysis of the plants, will provide currently lacking. The project aims to focus investi- identification and characterization of QTLs related gations on haustorium penetration, because it pre- to the fungal resistance and important agronomic cedes parasitism and accompanying crop damages. traits, as well as selection of the range of molecu- Arabidopsis has been chosen as a host for Orobanche, lar markers linked to the QTLs. The obtained re- because of its sequenced genome and readily avail- sults will be further incorporated into the respec- able mutant populations. Several lines with enhanced tive breeding programs. The project development resistance have been selected. Differential display will provide an excellent opportunity for initiation to identity genes involved in host–parasitic interac- of complex genomics research in the county, fo- tions both in resistant mutants and wild–type hosts cused on the pressing problems of Bulgarian agri- have been used. Results are expected for starting culture and industry. the characterization of resistance mechanisms. All genes identified as contributing to the host–para- II.4. GENETIC VARIABILITY IN MUTANT BAR- site interaction will be characterized as to copy LEY LINES INDUCED BY IONIZING RADIATION number and expression pattern. The presence of Project leader: Prof. DSc. Kostadin Gecheff, Insti- related genes in economically important Orobanche tute of Genetics, Bulgarian Academy of Sciences, host plants will be determined. Sofia, Bulgaria

II.3. MOLECULAR MARKERS FOR QTL ANALY- II.4.1. Partner: AgroBioInstitute, Sofia SIS OF FUNGAL DISEASE RESISTANCE FACTORS Program Module Leader: Dr. Todorovska, Dr. Elena AND IMPORTANT AGRONOMIC TRAITS IN Georgieva GRAPEVINE Genetic diversity achieved by interspecies crosses or by induced mutagenesis underlies im- Project leader: Acad. DSc Atanas Atanassov provement of the plant species. Effective use of Program Module Leader: Acad. DSc Atanas experimentally induced mutants in breeding pro- Atanassov grams depends a lot on the nature of the in- Key words: grapewine, fungal diseases’ resistance, duced mutations. The main goal of the project is segregating population, molecular markers, genome to reveal the cytogenetic and molecular nature mapping, QTL–analyses, metabolite analysis of ionizing radiation–induced mutational alterations Grapevine is an economically important crop in a stock of originally produced mutant barley in Bulgaria, but also with growing requirements for lines. Three main assumptions for the genetic the quality of production. The development of grape- nature of the radiation–induced mutational vine cultivars resistant to fungal pathogens (pow- changes of breeding value are put forward in this dery mildew, downy mildew and grey mould) is of project: these changes are due to point (gene) great importance for both improving of viticulture mutations; they are linked with the induction of and wine quality and production as well as reduc- structural chromosomal alterations; such type of ing of fungicide4 spray volumes. The cultivar changes is provoked by chromosome breaks in Storgozia created in the last century combines re- the vicinity of the genes, responsible for the trait

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○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○ of interest. The expected results are going to profiling of PCD coupled with reverse genetics, in contribute towards elucidation of one important which by extensive microarray analysis we will iden- but underestimated problem, concerning the char- tify genes that are transcriptionally regulated dur- acter of the initial mutation events induced by ing PCD and those genes will be functionally stud- ionizing radiation in plant genome and for better ied by the knock out technology or by generating and directed utilization of genetic variability in the plants with altered expression of the genes. contemporary breeding practice. Three PhD students will be assigned to carry out the experiments in order to develop highly II.5. MOLECULAR BIOLOGY AND GENETICS OF qualified young Bulgarian specialists in the field of PROGRAMMED CELL DEATH IN ARABIDOPSIS Genomics. THALIANA, FNSF–2018, 2005 – 2006, Project leader: Dr. Tsanko Gechev II.5.1. Partner: Plovdiv University ‘Paisii Keywords: PCD hydrogen peroxide, AAL–toxin, Hilendarski“, Plovdiv transcriptome analysis Program Module Leader: Dr. Tsanko Gechev Programmed cell death (PCD) is a geneti- Programmed cell death (PCD) is a geneti- cally controlled suicidal process in which cells self– cally controlled suicidal process in which cells self– destruct upon certain cell death stimuli, selectively destruct upon certain cell death stimuli, selectively removing in this way unwanted cells. This process is removing in this way unwanted cells. This process is an essential part of plant development as well as an essential part of plant development as well as of many plant responses to abiotic stress and patho- of many plant responses to abiotic stress and patho- gens. Examples of developmentally controlled PCD gens. Examples of developmentally controlled PCD can be found during development of the embryo, can be found during development of the embryo, nucellar degeneration, maturation of tracheal ele- nuclear degeneration, maturation of tracheal ele- ments, and epidermal trichomes, formation of lace ments, and epidermal trichomes, formation of lace leaf shape, and leaf senescence. A type of PCD is leaf shape, and leaf senescence. A type of PCD is also the hypersensitive response (HR) – a defense also the hypersensitive response (HR) – a defense reaction in which plant cells in and around the site reaction in which plant cells in and around the site of pathogen contact sacrifice themselves in order of pathogen contact sacrifice themselves in order to physically restrict the spread of the pathogen. to physically restrict the spread of the pathogen. On the other hand, some necrotrofic pathogens On the other hand, some necrotrofic pathogens can secrete toxins that trigger PCD in healthy tis- can secrete toxins that trigger PCD in healthy tis- sues so that the pathogens can feed on the death sues so that the pathogens can feed on the death tissues. Unwanted PCD can be caused also by some tissues. Unwanted PCD can be caused also by some environmental factors like cold, salt, and heat stress. environmental factors like cold, salt, and heat stress. Thus, PCD is not only an essential part of plant Thus, PCD is not only an essential part of plant development and beneficial for defense against development and beneficial for defense against pathogens, but can be an unwanted and even dan- pathogens, but can be an unwanted and even dan- gerous plant process. While the process is well studied gerous plant process. While the process is well studied in animals, little is known about the mechanisms in animals, little is known about the mechanisms and genes involved in plant PCD. and genes involved in plant PCD. The main aim of the proposed project is to The main aim of the proposed project is to unravel the mechanisms behind the plant PCD by unravel the mechanisms behind the plant PCD by discovering genes involved in the regulation and discovering genes involved in the regulation and execution of PCD in the model species Arabidopsis execution of PCD in the model species Arabidopsis thaliana. thaliana. To realize that goal two experimental ap- To realize that goal two experimental ap- proaches will be used: (1) forward genetics, in which proaches will be used: (1) forward genetics, in which we will screen for mutations in genes that render we will screen for mutations in genes that render plants altered in PCD responses, and (2) expression plants altered in PCD responses, and (2) expression

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profiling of PCD coupled with reverse genetics, in be determined, and the activity, molecular het- which by extensive microarray analysis we will iden- erogeneity, and content of main components of

tify genes that are transcriptionally regulated dur- enzymatic and non–enzymatic H202–scavenging ing PCD and those genes will be functionally stud- network will be estimated: catalase, guaiacol per- ied by the knock out technology or by generating oxidase, ascorbate peroxidase, glutathione per- plants with altered expression of the genes. oxidase, ascorbic acid, glutathione, etc. The impor- Three PhD students will be assigned to carry tance of chloroplasts as a site of generation

out the experiments in order to develop highly and scavenging of H2O2, and the extremely scarce qualified young Bulgarian specialists in the field of data on their role in PCD, the photosynthetic Genomics. performance and the photochemical efficiency The ultimate goal would be integration of all of photosystem II will be studied. Our hypoth- data into a model explaining the com – plexity of esis is that failure of PCD in A.thaliana mutants plant PCD. can be related to maintaining of a lower pool of ROS in chloroplasts through enhancement of II.5.2. Partner: Institute of Genetics, Bulgarian alternative routes consuming excess electrons, Academy of Sciences, Sofia such as photorespiration and Mehler reaction. MODULE: BIOCHEMICAL AND PHYSIOLOGICAL Results of both fundamental and applied im- CHARACTERISTICS OF MUTANT ARABIDOPSIS portance, contributing to a deeper knowledge

THALIANA PLANTS ALTERED IN THE H202–IN- of PCD in plants, can be expected. DUCED PROGRAMMED CELL DEATH Program Module Leader: Prof. DSc. Aglika Edreva II.5.3. Partner: Institute of Vegetable Crops Key words: Programmed cell death (PCD); „Maritsa“, Plovdiv Arabidopsis thaliana (wild type and mutants); reac- MODULE: PHENOTYPE ANALYSIS OF PRO-

tive oxygen species (ROS); hydrogen peroxide (H202); GRAMMED CELL DEATH IN ARABIDOPSIS

enzymatic and non–enzymatic H202–scavengers; THALIANA chlorophyll fluorescence Program Module Leader: Dr. Dimitrina Kostova Programmed cell death (PCD), an integral Key words: PCD, pathogens, plant viruses, hyper- part of life cycle, is relatively well understood in sensitive response. animal organisms, while in plants genetical and Programmed cell death (PCD) plays an biochemical mechanisms underlying PCD remain important role during plant development and still unclear. Data point to the key importance stress responses. The hypersensitive response of reactive oxygen species (ROS) in induction (HR) is one of the most common forms of PCD. and execution of PCD, a special role being at- The purpose of HR is to physically restrict the

tributed to H202, a ROS of both signal and toxic spread of the pathogen and to generate signals properties. To gain a better insight into the bio- that switch on the so–called systemic acquired chemical events related to the regulation of resistance, a defense reaction in the distant cells PCD in plants (focusing on the role of ROS and and tissues.

particularly H2O2), a reliable model system (Gechev While a significant advance was made in terms et al. 2005) will be used. It is based on PCD of receptor–pathogen interactions, still little is known

induction by high endogenous levels of H2O2 in about the genes and signaling cascades that lead A.thaliana plants resulting from specific inhibi- to PCD. tion of catalase activity by aminotriazole (AT), The aim of the proposed project is to iden- whereas in A.thaliana mutants, although AT– tify genes that are key regulators of plant PCD supplied, PCD is compromised. Our hypothesis is and plant response to pathogens. First, a large– that the suppression of PCD in A.thaliana mu- scale screening for mutants that are altered in

tants may be related to a default in H2O2 func- PCD responses will be performed. Subsequently, those

tion or to depletion of H2O2 level due to inter- mutants as well as knock–out lines of genes impli-

ference by H202–scavenging factors. To validate cated in PCD will be tested for resistance/sensitiv-

the latter assumption, the content of H2O2 will ity to plant pathogens.

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III. INTERNATIONAL EVALUATION AND REC- which Genomics is one of the five priorities. The OMMENDATIONS establishment of the Genome Centre is consid- VIIIth International Consultative Council (ICC) ered as an appropriate step in this direction. It of AgroBioInstitute (ABI) pointed out that during aims to contribute to the efforts of Academia the last three years markers were developed, based and the private sector. The establishment of such on microsatellites as well as AFLP, for the following a key centre could lead to the generation of crops: wheat, maize, barley, grape, small fruits, oil– products and services with a significant economi- rose, sunflower and citrus. cal, social and ecological impact. As such, the Ge- Some of the research (e.g. that on grapes) nome Centre could: was done with companies, predominantly for iden- • Create close synergy between research tification and breeding purposes. Research is done policy, innovation policy and educational policy in on a contract basis, which is important for the ABI order to contribute to the whole Lisbon strategy because it provides financial support and permits for knowledge–based economy and society; publication. The latter is part of the contract but • Avoid duplication in genomics at the na- the company is allowed to patent (mainly Bulgar- tional level, bringing together life science from ad- ian patents). Improvement of the application of vanced laboratories, institutes, universities, and pri- molecular markers was recommended to map vate sector laboratories; QTL’s for the purpose of marker–assisted selec- • Accumulate a „critical mass“ of state–of– tion (MAS). the–art specialized equipment; Concerning the Genome Centre initiative of • Develop and effectively implement high– ABI it was stressed that In December 2000, plant tech methods for diagnostics, control and assess- genomics was recognized as a key priority at the ment of quality and origin of food, feed and other milestone EC–supported Versailles Conference on products. This in turn should provide the means for Agricultural research in the European Research Area. preservation and protection of traditional Bulgar- The EC expressed serious concern about lagging ian species/breeds; behind colleagues and competitors not only from • Improve the synergy with the leading in- the USA, but also from China, Japan and even Brazil. ternational research centres, companies and pro- The ICC recognizes that the Genome Centre grams (e.g. with the ERA NET program and EMBO); (GenomeC) initiative presented by the ABI properly • Assure effective technological transfer and highlights the importance of the knowledge and development of collaborative high–tech products. strategies for Genome research and technology ap- plication in the ERA–NET and the rest of the devel- On the basis of the above, the ICC makes oped world, for example the USA and Japan. the following suggestions: The ICC found that its original recommenda- At the policy level: tion for establishment of the Genome Centre dur- • The ICC supports the efforts of the ABI, ing its last meeting (two years ago) had not moved NCAS, ABT and BF of Sofia University to establish beyond its primary conception. It was recognized GenomeC in Bulgaria. For further successful de- that the current restrictive GMO law seriously ham- velopment of plant genome research it is essen- pers the establishment of the Genome Centre. tial to have strong governmental support not only ICC also endorses the view that the priority at the policy level, but also financially. The ICC of the Genome Centre should be to deal with re- particularly invites the Ministries of Science and gional species and breeds and that this should be Education, Agriculture and Forestry and Environ- done in the context of international collaboration. ment and Waters to reinforce their support to ICC understands that the Genomics Centre the GenomeC initiative. as it is presented is expected to underpin and pro- • The ICC recommends that the GenomeC mote Bulgarian strategies and ambitions in the so- be used as an instrument for Bulgarian participa- cial and economic domain. tion in international initiatives and that it strives to The development of a national program for establish synergy between different scientific fields, Genomics entirely fits the government policy in education, technology and community.

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• All operations of the GenomeC should be • Production and distribution of control and carried out with the highest possible level of qual- reference samples for local cultivars and breeds to ity. The ICC therefore invites national and interna- be used. Accreditation scheme for GMO detection tional bodies to financially support accreditation of and traceability, authenticity and phytosanitary sta- the Genome Laboratory in the ABI. The ICC notes tus of valuable plants and breeds; that this would align the GenomeC with similar • The Genome characterization of the eco- laboratories in the EU. nomically important local varieties and breeds could lead to understanding what is unique about their The ICC notes that, at the R&D level, the genomic background in order to provide informa- following examples of research projects have par- tion for biosafety purposes; ticular importance for Bulgaria: • Exploitation of the unique flora and fauna • Assisting breeding programs with molecu- for production and use of products important for lar techniques for improvement of local cultivars nutrition, pharmaceuticals, bioremediation etc. and breeds;

DNA- Markers

AFLP

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FAMILIAL COLORECTAL AND ENDOMETRIAL CANCER

Kadiyska T., PhD Laboratory of Molecular Pathology, University Hospital of Obstetrics and Gynaecology, Sofia

COLORECTAL CANCER and tissue specimens from 294 incident CRC cases Colorectal cancer (CRC) is the most com- diagnosed in the period 1998 – 2005 have been mon gastro–intestinal neoplasia. The cumulative life- collected. Family information, including the main time risk of CRC and mortality from CRC is ap- causes of morbidity and mortality of first and sec- proximately 5–6 % and 2,5 % respectively. The ond–degree relatives, was recorded and genealogi- majority of colorectal cancers are sporadic (75– cal tree prepared. A tissue and DNA CRC banks 80 %). Approximately 20–25% are determined as have been established in the Laboratory of Mo- familial CRC. Hereditary nonpolyposis colorectal lecular Pathology. cancer (HNPCC), or the Lynch syndrome is the most All patients received genetic counseling and common type of familial CRC and accounts for informed consent was obtained for data and sample approximately 1 to 5% of all colorectal cancers. collection. Patients were sub–grouped into four HNPCC is an autosomal dominant disease with a main categories: 1. HNPCC families, according to high risk for colorectal and HNPCC– related can- Amsterdam criteria; 2. Families according to cers, caused by germline mutations in DNA mis- Bethesda criteria; 3. „Family“ cases group – repre- match–repair genes (MMR). These mutations lead sented by families with one or more first–degree to genomic instability, manifested by microsatellite relatives of the proband affected by tumors at instability (MSI). The screening for MSI might have any site; 4. „Sporadic“ cases whose families did not prognostic implications. Patients with CRC whose show any other cancer cases among relatives or tumors have high frequency MSI (MSI–H) have a without sufficient family information. more favorable prognosis, independently of the stage The clinical and histological information re- at diagnosis, than patients without MSI (MSS). In corded includes patient’s age at diagnosis, tumor the treatment of CRC patients there are indica- localization, tumor stage and differentiation, pres- tions that MSI–H tumors may be resistant to the ence of synchronous or metachronous tumors. actions of certain commonly used chemotherapeu- The following four years the research project tic agents such as fluorouracil, raising the important supported by the National Science Fund aimed to possibility that chemotherapy may have different initiate the National Registry and to involve several outcomes in this group of patients. It has been genetic methods in the molecular diagnostics of found that the phenomenon of DNA instability is CRC: „Investigation of possibilities for molecular di- not confined to the setting of hereditary disease agnostics and prophylactics of hereditary colorectal and may be seen in up to 15% of CRC in those cancer in Bulgaria“. The work was further extended with no family history of disease. In the case of by a new two – year project „ Investigation of the these so–called sporadic tumors, mutation of MMR molecular genetics and clinical profiles of patients genes is relatively infrequent whereas biallelic with hereditary colorectal cancer in the Bulgarian hypermethylation of the promoter of MLH1 ap- population“. The aim of the project was to im- pears to be the most important mechanism for prove and to expand the National Registry and the inactivation of MMR genes. possibilities of early diagnostics and prophylactics.

ESTABLISHMENT OF A NATIONAL REGISTRY Microsatellite instability analysis FOR COLORECTAL CANCER In 2001 one–year research project „Micro- In 1998 a National Registry for Colorectal satellite instability analysis in Bulgarian patients with cancer was initiated in Queen Giovanna Hospital, Colorectal Cancer“ funded by the Science Fund of Sofia, Bulgaria. The medical records, blood samples Medical University–Sofia aimed to analyze the

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prevalence and the role of genetic instability test- moter methylation and the LOH sporadic cases ing in the molecular diagnostic strategy. The next was also significant (p<0.02). two years two other projects supported by the Our results supported the role of MLH1 pro- National Science Fund continued the study of MSI moter methylation as a major mechanism of MLH1 and its correlation with clinical and pathomorpho- gene inactivation in the MSI sporadic cases. We logical characteristics. Recently one–year research propose the inclusion of this method in the diag- project continued this work with the collection of nostics of CRC, as a step following the analysis for new CRC cases. MSI and prior to the screening for mutations in the A total of 150 patients with colorectal can- MMR genes. cer have been included in the study. DNA from Immunohistochemistry. MMR mutation screen- fresh blood and/or frozen normal and tumor tis- ing. Genotype – phenotype correlations sue was isolated using standard protocols. A set of The IHC showed lack of MLH1 expression in five polymorphic markers– BAT26, D2S123, D5S346, all detected tumors with methylation. Patients whose D18S35 and FGA has been selected for analysis of tumors showed hMLH1 promoter hypermethylation MSI. The repeat markers were amplified from both were not included in the screening for MMR mu- normal and tumor DNA samples and electrophore- tations. Our search for genetic alterations in hMLH1 sis was performed on an automated fluorescence and hMSH2 genes found the following variations: sequencer (ALF Express, Pharmacia). four known and two novel mutations, one unpub- We detected 25 tumours with MSI and 14 lished and four known intronic polymorphisms in with loss of heterozygosity (LOH) in at least two both hMLH1 and hMSH2 genes. of the loci analyzed. The MSI phenotype was asso- PCR amplification of all exons including flank- ciated with distinct clinicopathological features, no- ing intronic regions was performed and than ana- tably proximal tumor site, high grade and mucinous lyzed using single–strand conformation polymor- production. The LOH tumors were more frequently phism (SSCP) analysis. Samples showing an altered distally localized and no correlation was found ac- mobility pattern were then subjected to direct se- cording to the grade and mucin production. No quencing. difference was seen in sex, age of onset and stag- Our search for genetic alterations in hMLH1 ing, presence of synchronous or metachronous and hMSH2 genes found the following varia- tumours. tions: four known and two novel mutations, one unpublished and four known intronic polymor- Analysis for MLH1 promoter hypermethylation phisms in both hMLH1 and hMSH2 genes. Lack In 2001 the National Science Fund supported of MLH1 protein expression was shown in all the two–year research project for young scientists tumors with previously described pathogenic „Investigation of promoter methylation of MLH1 mutations as well as in the tumor from the and MSH2 genes on the microsatellite instability in novel frameshift mutation. colorectal cancer“. We aimed to analyze the segregation of In 25 MSI positive tumors (10 sporadic cases the novel mutation within the pedigree and cor- and 15 with family history), 13 with loss of het- related the observed clinical features to this mo- erozygosity (eight sporadic cases and five with family lecular defect on both genomic and protein lev- history) and 15 random selected sporadic MSI– els. Four tumors were analyzed for microsatellite negative cases, the methylation status of the MLH1 instability (MSI) and for MLH1 protein expression promoter has been analyzed using polymerase chain using the immunohistochemical (IHC) assay. In reaction (PCR)–based HpaII restriction assay. Sig- the three adenocarcinomas MSI and lack of the nificant association was found between this trend MLH1 protein expression were detected. The only and MSI/LOH (p<0.04). The correlation between one tubulovilous adenoma analyzed was the MLH1 promoter methylation and the MSI spo- microsatellite stable (MSS) and the MLH1 protein radic cases was highly significant (p<0.01). In this showed an intact staining. group there were no patients younger than 50 The clinical evaluation of the cases revealed years (p<0.02). The correlation between MLH1 pro- typical features of HNPCC such as early age of

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This project aims to study the molecular mecha- leading medical universities, hospitals and a NGO for nisms of developing familial EC in Bulgaria. the fulfillment of the project aims. Clinical and biological material from 150 pa- Team members: tients with EC at the age under 50 years will be Endometrial Cancer collected. These patients will be genetically char- acterized by analyses for MSI, promoter Kadiyska T., PhD; Kaneva R., PhD; Kre- hypermethylation of the MLH1 gene, immunohis- mensky I., MD, PhD – Laboratory of Molecular tochemistry and screening for mutations in the Pathology, University Hospital of Obstetrics and main MMR genes. The optimal strategy for early Gynaecology, Sofia, diagnosis and molecular diagnostics of EC will be Ivanov St., MD, PhD – Clinic of Gynaecology, proposed. The project will initiate a familial EC National Oncological Institute, Sofia, Tiufekchieva register. Information brochures about possibilities E., MD, PhD; Dimitrov R., MD, PhD; Mainkhard for early detection and prophylactics of the EC K., MD; Doganov N., MD, – University Hospital of will be prepared for patients and specialists. Obstetrics and Gynaecology, Sofia, The project brings together the research po- Vlaykova T., PhD; Galabova M., PhD – tential of a multidisciplinary team of gynaecological Department of Chemistry and Biochemistry, Trakyan surgeons, pathologists and molecular biologists from University, St. Zagora.

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BIOCHEMICAL DIAGNOSIS OF LYSOSMAL STORAGE DISEASES IN BULGARIA

Ivanka Sinigerska, MSc; Irfet Hassanova; Katerina Vladimirova, Assoc. Prof. Ivo Kremensky, MD, PhD Laboratory of Molecular Pathology, Sofia

Lysosomal storage disorders (LSD) are a of choice only in cases with previously strongly con- group of more than 40 different genetic dis- firmed enzymological or molecular diagnosis of the eases. They occur in total in about 1 in 7000– genetic defect. 8000 live births and display considerable clinical The initial approach to the diagnosis of LSD is and biochemical heterogeneity. The common fea- based on clinical features. The well–known symp- ture of these diseases is the defective activity of toms might be highly suggestive, but the correct a lysosomal protein (result of a single gene muta- diagnosis of the genetic defect is often difficult tion) that is part of a catabolic pathway. This because of the overlapping phenotypes and quite prevents the further breakdown of complex com- often is complicated by the fact that most of the pounds and leads to an intralysosomal accumula- diseases are quite rare and moreover clinically het- tion of partially degraded material. Most often erogeneous. Clinicopathological findings and identi- the deficient protein is a specific enzyme, respon- fication of abnormally stored substances may rein- sible for one step in the sequential degradation force the clinical suspicion but they are not diag- of sphingolipids, glycoproteins, mucopolysaccharides nostic. There is frequent overlap of storage mate- and glycogen. Other causes of LSD are the defi- rial between different diseases. Analysis of urinary ciency of the so–called activator protein assisting metabolites facilitates differential diagnosis of LSD, a lysosomal enzyme in its catabolic function(in pointing to the probable enzyme defect. Enzymatic GM2–AB variant), or the deficiency of a compo- diagnosis offers the chance of a definitive result nent of larger lysosomal enzyme complex (SAP and also makes possible prenatal diagnosis in fu- deficiencies). Inappropriate lysosomal enzyme tar- ture pregnancies in the family. geting (in mucolipidoses) and defective export of The enzymatic diagnosis of LSD in Bulgaria degradation products from lysosomes (in sialic acid was introduced 30 years ago by Prof. I. Kremensky, storage disease) also can lead to LSD . The result- Prof. L. Kalaydjieva and Dr. R. Varon. Till now, with ing clinical symptoms develop progressively, rang- the great help and support of Enzyme laboratory, ing from coarse facial features, bone changes and Department of Clinical Genetics – Rotterdam, the organomegaly, to neurological problems and psy- Netherlands, the number of assays has been ex- chomotor retardation. LSD can be classified de- panded and the applied procedures improved. At pending on the pathway affected and the nature present 24 enzyme tests, listed in Table 1, are per- of accumulated substrate – mucopolisaccharidoses, formed in the Laboratory of Molecular Pathology. glycoproteinoses, lipidoses. In Bulgaria the postnatal and prenatal diagnosis of A precise diagnosis is needed as early as 26 LSD, resulting from enzyme deficiency (from possible to allow adequate treatment, therapy and altogether 30 known), is possible. genetic counseling. At present there is no effective It is not practical to examine all known en- therapy for the most severe LSD, leading to pre- zyme deficits in every particular case. Appropriate mature death. Enzyme replacement therapy is lim- scheme of assays should be performed, depending ited to three diseases (Gaucher, Fabry, and MPS I) on the observed key features. A screening flow- and is quite expensive. The majority of LSD is inher- chart for biochemical diagnosis of LSD was devel- ited in an autosomal recessive manner (except X– oped in our laboratory . The goal of our work was linked Hunter, Fabry and Danone diseases) and the to detect rapidly and reliably most of the known high 25 % recurrent risk in subsequent pregnancies LSD and to offer prenatal diagnosis for them. refrain many families from having further children. More than 3000 patients, aged from sev- The prenatal diagnosis (providing selective termina- eral days up to 52 years, suspected of having LSD, tion of affected fetus is acceptable) is an action from the whole country, as well as patients from

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Advances in Bulgarian Science

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Macedonia and Serbia, have been referred to the This non–lysosomal enzyme, originating from ac- laboratory for biochemical diagnosis. tivated macrophages, has been markedly elevated Laboratory diagnosis of mucopolysaccharidoses in patients with Gaucher disease and in some is generally achieved by three sequential steps: quan- other LSD (Niemann–Pick, Krabbe, Pompe, Wolman) titative detection of excessive excretion of in a less extent. Using the flowchart and the glycosaminoglicans (GAGs) in urine followed by quali- methods already mentioned for more than 3000 tative analysis to determine the probable MPS type patients, suspected of having LSD, the correct di- and finally demonstration of the specific enzyme agnosis has been obtained on a total of 189 defect in plasma, leukocytes or cultured skin fibro- patients. In 56 cases diagnosis has been set on blasts. The GAGs were measured using own modi- metabolite level only, due to the lack of material fication of 1, 9–dimethylmethylene blue (DMB) for enzyme assay. The diagnostic rate about 8.2 method. Age–dependant reference limits were % is rather close to the value given by Prof. determined using the program REFVAL, recom- David Wenger, Jefferson Medical College, Phila- mended by the International Federation of Clinical delphia (about 8.0 %) and has to be regarded Chemistry. From 1996 to 2001 an external quality as confirmation of the usefulness of our diag- assessment scheme for accuracy control, provided nostic algorithm. Mucopo-lysaccharidoses (90 by the European Research Network for evaluation cases) are the most common disorders of diag- and improvement of screening, diagnosis and treat- nosed LSD in our laboratory – 38.5%. Significant ment of Inherited Disorders of Metabolism number of patients with Niemann–Pick (29 pa- (ERNDIM) was carried out. The results obtained tients), all from gypsy origin, as well as 27 cases were within one standard deviation, calculated from of GM1, 26 cases of MLD, 26 cases of Krabe have values reported by all 72 European laboratories par- been detected. We do not pretend that the con- ticipating in the scheme. firmed LSD diagnoses correspond to the real preva- Fractionation of GAGs has been performed lence of these disorders in Bulgarian population.For by thin layer chromatography (TLC) or one–di- 25–year period prenatal diagnosis has been at- mensional electrophoresis. Enzyme assays have been tempted in 41 cases (13 of them with the coop- carried out using syntetic fluorogenic substrates eration of Dr.W. Kleijer, Department of Clinical (4–methylumbelliferone derivatives). The patients’ Genetics, Rotterdam). The enzyme activities have range of enzyme activity has been less than 10 % been measured in cultured amniotic fluid cells from the control group range. (40 cases), cell–free amniotic fluid (2 cases) and Thin layer chromatography of oligosaccha- cord blood leukocytes (1 case). The results from rides has been applied as a first step in biochemical enzyme assay in cell–free amniotic fluid have diagnosis of glycoproteinoses, followed by confir- been confirmed by enzyme assays in cultured matory enzyme assays in the cases with abnormal amniocytes. In 11 cases of prenatal diagnosis pattern. (26.8% from all) the results have indicated af- For diagnosis of lipidoses enzyme tests rel- fected fetuses and pregnancies had to be termi- evant to clinical symptoms have been performed. nated. The diagnoses have been confirmed when Determination of plasma levels of fetal tissues were available. chitotriosidase has been used as a screening test.

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National Scientific Programmes with Europian Dimension

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Table 1. List of LSD, corresponding enzyme assayed, number of diagnosed patients and prenatal diagnoses in the Laboratory of molecular pathology.

Prenatal DISEASE ENZYME Patients Diagnoses 1 MPS I Hurler/Scheie α-Iduronidase 3 2 MPS II Hunter Iduronate 2-sulfatase 14 6

3 MPS III A Sanfilippo A Heparan Sulphamidase 7

4 MPSIII B Sanfilippo B N-acetyl α - glucosaminidase 7 2 Acetyl-CoA: α-glucosaminide 5 MPSIII C Sanfilippo C N-acetyltranspherase 6 MPS IV A Morquio AN-acetylgalactosamine 3 1 6-sulphatase 7 MPSIV B Morquio B β-Galactosidase 1

8 MPSVI Maroteaux-Lamy Arylsulphatase B81

9 MPS VII Sly β-Glucuronidase

10 Mucosulphatidosis ArylsulphataseA , ArylsulphataseB 2

11 Pompe disease α-Glucosidase 5 1

12 Aspartylglucosaminuria Aspartylglucosaminidase

13 Fucosidosis α-Fucosidase 1 3 14 a-Mannosidosis α-Mannosidase 1

15 b-Mannosidosis β -Mannosidase

16 Schindler disease α -N-acetylgalactosaminidase

17 Fabry disease α-Galactosidase 3 18 Gaucher disease β-Glucosidase 26 3

19 GM1-gangliosidosis β-Galactosidase 27 12

20 GM2-gangliosidosis (Sandhoff) Hexosaminidase A+B 4 1

21 GM2-gangliosidosis(Tay-Sachs) HexosaminidaseA 2 3

22 Krabbe disease Galactocerebrosidase 123

23 Metachromatic leukodistrophy Arylsulphatase A263 24 Nieman –Pick disease A &B Sphyngomielinase 29 1

25 Wolman disease Acid lipase 1

26 Mucolipidosis I (Sialidosis) Neuraminidase 1

27 Mucolipidosis II (I cell disease) Multiple lysosomal hydrolases 6 1

Total 189 41

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Advances in Bulgarian Science

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PRENATAL DIAGNOSIS OF THE MOST COMMON HUMAN ANEUPLOIDIES WITH DNA ANALYSIS

Silvia Andonova; Radostina Raynova,MSc; Assoc. Prof. Ivo Kremensky, MD, PhD – Laboratory of Molecular Pathology, University Hospital of Obstetrics and Gynaecology, Sofia Medical University and genetic councellers from the Department of Medical Genetics and Department of Paediatrics, Sofia Medical University, Tel: + 359-2-9172473, Fax: +359-2-9172469; E-mail: [email protected] Violeta Dimitrova; Valentina Mazneykova – University Hospital of Obstetrics and Gynaecology, Sofia

Prenatal cytogenetic analysis in high risk preg- referred for prenatal diagnosis because of one or nancies for chromosome abnormality has been more indications: increased maternal age (most fre- performed routinely since the early 1970s. Despite quent indication), previous child or a pregnancy with of the improved cell culturing and chromosome a chromosomal abnormality or multiple malforma- banding, it takes around 4 weeks from the collec- tions, elevated risk from Down syndrome second tion of the sample till the reporting of the results trimester serum screening, abnormal ultrasound scan, from conventional cytogenetic analysis. The long or parental anxiety. Most of the samples were lasting procedure have been recognized as main obtained after invasive prenatal procedure – usu- disadvantage of this analysis and was one of the ally amniocentesis or chorionic villus sampling. Ge- main reasons for the introduction of new molecu- nomic DNA was extracted from different fetal lar methods for prenatal diagnosis of common tissues – amniotic fluid samples, chorionic villi, culti- autosomal aneupoidies. vated amniotic cells, fetal tissue samples after abor- With the development of DNA technologies tions, or fetal blood cells collected after a new diagnostic method has been proposed since cordocentesis. Most of the QF–PCR results were the 1990s – indirect quantitative fluorescent PCR verified by a conventional cytogenetic analysis in a analysis (QF–PCR). It is based on the investigation parallel study. DNA samples with structural chro- of highly polymorphic DNA markers (short tandem mosomal abnormalities on other chromosomes, not repeats – STRs), located on the target chromo- included in our analysis will not be detected with somes. The use of STR markers with a high het- QF–PCR analysis.The method allows detection of erozygosity will decrease the probability of unin- maternal contamination by the presence of addi- formative results. The addition of any supplemen- tional alleles or by comparison with the genotype tal polymorphic marker per chromosome would of the mother, when possible. increase the diagnostic accuracy in most if not all A total of 4 STR markers for chromosome investigated samples. The high throughput, low cost 21 (D21S11, D21S1411, D21S1440, D21S1435), 3 – for and accuracy of this technique made it a reliable chromosome 18 (D18S535, D18S51, D18S858) and 3 method of choice for detection of the most com- – for chromosome 13 (D13S631, D13S258, D13S256) mon human aneuploidies – trisomies 21, 18 and 13 in four multiplex PCR assays were used routinely (causing Down, Edwards and Patau syndrome, re- for QF–PCR analysis in Bulgaria. These markers spectively), as well as triploidy. QF–PCR method were selected because of their high heterozygosity has been successfully applied for rapid prenatal reported in literature. Detection of the sex chro- diagnosis of aneuploidy by a number of UK and mosomes numerical disorders and particularly Turner European labs. syndrome is in progress. Since the introduction of QF–PCR analysis in Thirty of the fetus samples were diagnosed Bulgaria (2000) more than 1300 fetuses were as trisomic for chromosome 21, 8 – for chromo- diagnosed. Investigated pregnancies ranged from some 18, 3 – for chromosome 13 and 1 sample 10 to 34 weeks of gestation (most of them be- was diagnosed with triploidy with at least 2 STR tween 16 and 20 week). Pregnant women were markers (see figure 1).

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Conclusions mative for our population and are suitable for the QF–PCR technique is a reliable and rapid purposes of prenatal diagnosis in Bulgaria. alternative for prenatal diagnosis of trisomy 21 Our research was supported by grants from (Down syndrome), trisomy 18 (Edwards syndrome) Research Fund, Sofia Medical University and Na- and trisomy 13 (Patau syndrome), especially when tional Science Fund, Ministry of Education and Sci- some problems with cell cultivation arise and/or ence (MUL 1201/2002). cytogenetic analysis is impossible. QF–PCR analysis could be an option in prenatal and postnatal diag- References: nosis in cases with strong suspicion for particular Andonova S., Vazharova R., Dimitrova V., autosomal aneuploidies (including chromosomes 21, Mazneikova V., Toncheva D., Kremensky I. 18, 13) especially in small countries with restricted Introduction of the QF–PCR analysis for the resources like Bulgaria. However, because a limited purposes of prenatal diagnosis in Bulgaria—estima- number of chromosomes are involved in this study, tion of applicability of 6 STR markers on chromo- the classical cytogenetic analysis cannot be fully somes 21 and 18. replaced at this stage of our work. Our results Prenat Diagn. 2004 Mar;24(3):202–8. indicate that DNA markers we use are highly infor- PMID: 15057954 [PubMed – indexed for MEDLINE]

Figure 1: Electrophoretogram showing the QF-PCR detection of a 47,XY,+21 fetus. M – Mother, F- Father

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Advances in Bulgarian Science

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BULGARIAN ADDED VALUE TO ERA

THE STEPHAN ANGELOFF INSTITUTE OF MICROBIOLOGY – CENTER OF BULGARIAN MICROBIOLOGICAL SCIENCE

Prof. Maria Angelova, DSc; Assoc. Prof. Lyubka Doumanova, PhD; Prof. Angel Galabov,DSc, Corr. Mem. The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, 26, Acad. G. Bonchev Str., 1113 Sofia, Tel. +359 2 8701081, Fax +359 2 8700109, E-mail: [email protected]

The Institute of Microbiology was founded mans under the direct leadership of Prof. S. on March 7, 1947 by the world–famous Bulgarian Angeloff and his first collaborator Prof. Ilia scientist Prof. Stephan Angeloff, Member of the Kujumdjiev. The achievements in the study of the Bulgarian Academy of Sciences (BAS), who became etiology and the development of diagnostic meth- its first director for 15 years. Stephan Angeloff ods and immunoprophylactic preparations for these graduated from the Berlin University in 1901 as a infections were significant. Bacteria, mycoplasma and veterinary doctor. He specialized in microbiology in L forms, antibiosis and antibacterial medicinals, as the laboratory of Robert Koch in Berlin and was a well as agents causing protozoic invasions were student of his most close collaborators: Paul Ehrlich, also studied. The beginning of the investigations in . . Friedrich Loffler, and August von Wasserman. the field of infectious immunology was set by Prof. Stephan Angeloff was the scientist, who on the eve Vladimir Markov, Member of BAS. Industrial micro- of the First World War, using own original vaccine, biology started with investigations of Prof. Ignat built up a barrier on the way of the rinderpest into Emanuilov, Member of BAS, on the microbiology of the European continent. He was the founder of milk and dairy products, biochemistry and physiology the Bulgarian microbiology, the creator of a solid of lactic acid bacteria and application of bacterial scientific school in all directions of microbiology. enzymes (proteinases) in cheese making. Alongside The establishment of the Institute of Micro- with bacteria as producers of biologically active biology realized the necessity of formation of a substances, filamentous fungi were studied. About research center with national importance for car- 1964 the investigators were grouped into two prin- rying out contemporary investigations in rapidly de- cipal fields: I. Metabolism and biosynthesis of mi- veloping and prosperous branches of microbiologi- croorganisms and their regulation; II. Nature and cal science. Five units were founded at first in the regularities of the interactions between microor- Institute: bacteriological (medical, agricultural, and ganisms and the macroorganism. industrial bacteriology), protozoological, virological (in- In the field of microbial morphology electron vestigation of viruses and viral diseases in man, ani- microscopic studies of bacteria, filamentous fungi, mals, and plants), immunological, and biochemical. yeasts and host immune system cells were per- During the initial period of its development formed since 1964. The investigators in the field of (1947–1962) the Institute was connected with stud- microbial biochemistry after 1973 aimed at eluci- ies of the current after the Second World War dation at a contemporary biochemical level of the infectious pathology of domestic animals and hu- main peculiarities in the physiology and biosynthetic

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Bulgarian Added Value to ERA

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• Genetically constructed bacterial vectors approach for treatment of systemic lupus erythe- for elaborating a new generation of vaccines – matosus. classical swine plaque, etc. • Testing of different natural and synthetic Applied microbiology products for antibacterial activity. • Genetic analysis of microorganisms, used in • Bacterial resistance to antimicrobial agents. the food and pharmaceutical industries – lactoba- • Vaginal lactobacilli – molecular – genetic cilli, streptomycetes, etc. analysis and their role in the protection against • Characterization of the microbial cell re- sexually transferred infections. sponse to physiological stress. • Bacteriocins. • Selecting and characterizing of microor- • Elaborating methods for microbiological ganisms, used as starter cultures in dairy production. control of food products • Production, characterization and application range of biologically active microbial products: Virology – enzymes incl. thermo stable – superoxide • Experimental chemotherapy of viral infections. dismutase, pullulanase, innulinase, proteases, pectinases, • Development of effective anti–adenoviral amylases, TAQ– polymerase, neuraminidase, etc. chemotherapy. – organic acids, vitamins and growth factors. • Antiviral substances vs. toga– and – amino acids. flaviviruses. – biosurfactants. • New virus inhibitors from synthetic and – antibiotics . natural origin (plant, microbial, etc.). • Microbiological methods for steroid trans- • Biological response modifiers (BRM) – formation. immunomodulators, interferon inducers, antioxidants, • Cell cultures of endemic plants – a source etc. of biologically active substances for the food and • Combined effects of antiviral substances pharmaceutical industries. and BRM. • Mathematical modeling of biotechnologi- • Antiviral microbicides – screening system, de- cal processes. velopment of new generation of viral disinfectants. • Microbial ecology: • Mechanisms of action of viral inhibitors. – microbiological monitoring of soils and water. • Role of some viruses in human pathology – the role of microorganisms and surfac- (Balkan endemic nephropathy, diabetes, cervical tants in bioremediation and biodegradation. cancer). – technology for biogas production.

Infectious immunology Public health • Immunopharmacological investigation on • Virulence factors in bacteria–causative substances with natural origin (microbial, plant) and agents of socially important zoonosis (optimizing of synthetic products. diagnostics, therapy, and immunoprophylaxis). • Studying on their immunomodulating ef- • Introduction of molecular methods for fect in experimental animal models of infections early diagnostics of tuberculosis in children and and of tumors. adults. • Autoimmune disorders resulting from in- • New methods for detection of intestinal fection. infections and toxic infections in foods of animal • Formulation of the conception for physi- origin. ological autoimmunity. • Epidemiological investigation on the role of • Mechanisms of control of autoimmunity. human papilloma viruses in cervical cancer. • Investigation of the role of natural au- • Balkan endemic nephropathy – etiopatho- toantibodies in the autoimmune diseases. genesis, epidemiology, immune status. • Reestablishing the tolerance to DNA with • Immunoglobulin preparations for treatment gene–engineering chimeric molecules – a new of autoimmune diseases.

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Bulgarian Added Value to ERA

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• Chemotherapy of epidemic adenoviral kera- radioactive hybridization were included in the frame toconjunctivitis. of scientific program. Eminent scientists from Pas- • Monitoring of disinfectants as main com- teur Institutes – Paris (France), Sankt–Peterburg ponents in hospital hygiene for control of nosoco- (Russia), Cantacuzino (Romania) and The Stephan mial infections. Angeloff Institute of Microbiology Sofia (Bulgaria) • Participation in the national strategy in the were invited to deliver lectures and practical exer- control of infectious diseases – diagnostics, epide- cises. Young scientists from Bulgaria, Greece, R. miology, vaccines. Macedonia, and Romania attended the courses. They • Participation in the Commission for Eradi- were highly estimated both by the participants and cation of Poliomyelitis at the Ministry of Public Health the invited lecturers. The courses provided oppor- as part of the WHO program. tunity for future scientific collaboration, exchange • Participation in Biological Safety Expert of information and friendship. A collaboration project Council at the Permanent Commission for Preven- „Macrophage dependent immunoprotective action tion of the Population from Natural Disastres and of Candida albicans DNA“ (the project coordinator Catastrophes. Assoc. Prof. N. Ivanovska) was granted by ACIP • Participation in Research and Development program in September 2005. Institute Pasteur Paris, Expert Group on Countering the Effects of Bio- France supported five fellowships for Bulgarian logical and Chemical Terrorism, European Union. microbiologists. • Informing by mass media on the current The education activity includes also participa- for the country problems of infectious diseases. tion in Leonardo da Vinchi Programme of EC. Our scientists created innovative programmes for voca- Education activity tional training in healthcare microbiology and im- The Stephan Angeloff Institute of Microbiol- munology as well as in production of healthy sound ogy is authorised and certified by the Highest Tes- bioproducts for improvement of skills and compe- timonial Committee and the Ministry of Education tence of the staff. and Science as a national institution for training The Professors, Assoc. Professors and some and preparing of graduated students in obtaining a Junior Scientific Workers from the Institute deliver doctor degree in three fields: microbiology, virology lectures and provide experimental exercises to the and immunology. master degree students in many universities all over Three International Training Courses for young the country (as well as abroad) in more than 20 scientists were organized at the Atelie of the Institut subjects – Sofia University St. Kliment Ohridski, Medi- Pasteur at the Stephan Angeloff Institute of Mi- cal University – Sofia, University of Forestry – Sofia, crobiology: Prof. Dr. A. Zlatarov University – Bourgas, Technical .1 Molecular methods for diagnostic and taxo- University of Sofia, Technical University – Gabrovo, nomic identification of bacteria and fungi (23 Oc- Technical University – Dresden, South–West Univer- tober – 8 November 2002). sity – Blagoevgrad, University – Bitola, etc. Many of 2. Molecular methods for diagnostic and taxo- the students received their master degree after nomic identification of viruses and fungi (6 – 14 making the thesis work in different laboratories of November 2003). the Institute. For the previous 2004–year the lec- 3. Methods for DNA finger–printing and tures and exercises were 1233. genotyping of drug–resistant Mycobacterium tu- Great number of scientific workers from dif- berculosis strains“ (25–30 October, 2004). ferent departments consults many institutions (gov- The courses aimed to provide training in mo- ernmental, university, private, etc.) on problems of lecular biology methods for identification and typ- ecology and environment, public health, agriculture ing of pathogens. High level standing lectures on and food industry. Some members of the Institute molecular biology of medically important microor- are involved in the work of many expert groups, ganisms as well as seminars dedicated to the re- for example , Research and Development Expert cent breakthroughs in the field and practical train- Group on Countering the Effects of Biological and ing on DNA extraction, PCR, RT–PCR, and non– Chemical Terrorism at EC, Biological Safety Expert

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Advances in Bulgarian Science

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Council at the Permanent Commission for Preven- ment of MDR–TB and will fundamentally benefit tion of the Population from Natural Disasters and patients by more effective chemotherapy. The re- Catastrophes, Expert Council for Epidemiological searchers have the unique opportunities to get ac- Control of Infectious Diseases, Immunoprophylaxis quainted with advanced technologies and interna- and Commission for Eradication of Poliomyelitis at tional cooperation, thus contributing to improvement the Ministry of Public Health, National Agencies of of MDR–TB case detection rate in Bulgaria. Standardization and Authorization, Scientific Coun- Yersinia enterocolitica is an important caus- cils of different institutions, etc. The Institute of ative agent of intestinal lesions including acute Microbiology is the Head Office of the Balkan enteritis, enterocolitis and mesenterical lym- Society of Microbiology, Bulgarian Society of Micro- phadenitis. biology, as well as a Center for organizing national In rare cases, particularly in immuno-com- and international congresses, conferences and work- promised hosts, Y. enterocolitica can induce sys- shops in the field of microbiology. temic infections with abscesses in spleen, liver and The Director of the Institute is a member of other organs. Our investigations focus on the roles the Expert Advisory Board, the Council of Interna- of different virulence factors of Y. enterocolitica tional Union of Microbiological Societies, Council of required for pig colonization and persistence. We Federation of European Microbiological Societies, and studied the pathogenicity and immunogenicity of Council of Directors of Institute Pasteur and asso- wild–type and different lipopolysaccharide mu- ciated Institutes. He participates in the work of tant strains of Y. enterocolitica serotype O:8 and FEMS, IUMS, WHO, International Society for Antiviral Y. pseudotuberculosis, serotype O:3, using a pig oral Research, etc. model of infection. We followed in vivo the colo- During the recent years the aim of our re- nization of different organs and the histopatho- search activity was to maintain a high level of scien- logical and immunomorphological changes associ- tific programs with the modern directions in bio- ated with the infection. Also the ability of the sciences that are in close relation with the national mutant strains to resist different host defence priorities. The projects developed in the Institute of mechanisms of pigs was studied. We were able to Microbiology are financially supported by the Na- select the mutant strains, which are highly promis- tional Fund for Scientific Investigations (Ministry of ing and applicable for further construction of live Education and Science), other ministries, foundations, carrier vaccine. and Bulgarian companies (48) as well as by the Discovery and characterization of new antivi- European Union, NATO, UNESCO, and other interna- ral substances – inhibitors of viral replication – tional organizations and companies (40). products of chemical synthesis or of natural origin. Among our most important achievements of 1–(4)–Morpholinomethyl–1(1Í)– research activity we count the following: tetrahydropyrimidinone (mopyridone) is character- Intervention strategies against the develop- ized as a potent inhibitor of replication of influenza ment of drug–resistant tuberculosis. viruses A (H2N2, H3N2) and B, and of togaviruses There is an alarming increase of tuberculosis (species of genera Alphavirus and Rubivirus – ru- incidence in Bulgaria: from 25.1 per 100000 popu- bella virus) as well. A high efficacy of mopyridone lation in 1990 to 50.1 in 1998 and 48 in 2001, as was proved in experimental influenza A (H3N2) well as of MDR–TB incidence (to 8.6% of the and  infections in albino mice. The compound mani- total registered TB case load). International col- fests high activity in experimental alphavirus infec- laboration of the Department of Pathogenic Bac- tions in mice. On the base of its protective effect teria with German scientific institutions focuses on in infections with massive virus inocula and high a recent development in tuberculosis research, which values of selectivity index mopyridone could be con- is the increasing recognition of the importance of sidered as the most active anti–alphavirus sub- drug–resistant populations of Mycobacterium tu- stance described in literature. berculosis in patients. Present investigations contrib- Aporphinoid alkaloid oxoglaucin (isolated ute to understanding of the nature of and synthesized in the Center of Phytochemistry, „heteroresistance“ as a prerequisite for develop- Institute of Organic Chemistry, BAS) possesses strong

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Bulgarian Added Value to ERA

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In order to restrict the process of de- Oxidative stress in the pathogenesis of flu and velopment of drug resistance, an original scheme antioxidants. for combined administration of enteroviral in- Characterization of the role of oxidative hibitors in vivo was developed. damage („oxidative stress“) in the flu pathogen- Establishment of a markedly synergistic esis (in albino mice infected with influenza virus combination effect of the most effective anti–flu A/H3N2). Tracing out the markers of oxidative chemotherapeutic agents rimantadine–hydrochlo- stress – lipid peroxidation products, anti–oxidant ride and oseltamivir in experimental infection with enzyme activities, liver monooxigenase enzyme, en- influenza virus A/H3N2 in albino mice. dogenous antioxidant levels – as vessels–damag- Proven several effective synergistic com- ing factors. binations vs. herpes simplex viruses 1 and 2: ribavirin Proving the protective action of antioxi- + acyclovir, methotrexate + BVDU, mizoribine + dants of natural (vitamins E and C) and synthetic acyclovir, etc. origin [methyl–2,6–ditretbutylphenol (Yonol)] on Establishment of a synergistic combina- the course of influenza virus infection (with influ- tion effect of idouridine and sidofovir on vaccinia enza virus A/H3N2 in albino mice) by tracing the virus replication in vitro. changes in oxidative stress markers. Establishment of the fact that ant i– Establishment of antiviral effect of cycluridine flu chemotherapeutic agent rimantadine–hydro- vs. Flaviviruses and its efficacy in the treat- chloride significantly decreases the oxidative stress ment of the flavivirus infections in cattle (mu- markers’ values during the course of influenza cosal diseases). virus infection. The compound does not possess Establishment of anti–flaviviral activity of antioxidant properties (does not influence induc- cycluridine (a cyclis urea synthesized in Bulgarian tion of both lipid peroxidation and antioxidant laboratory): inhibitory effect on flavivirus replica- enzymes in various model systems, does not inter- tion on the model of the bovine pestivirus (BVDV), act with superoxide). Its antioxidant–like action is etiological agent of the mucosal diseases in calves, based on its inhibitory effect on influenza virus as well as a marked protective effect in experi- replication. mental BVDV infection in calves. Cold and/or immobilization stress, preced- Establishment of protective effect of ing the influenza virus (A/H3N2) infection, causes a cycluridine in lethal experimental infection with the drastic increase of oxidative damages in the body (in flavivirus of tick–born encephalitis in albino mice. This liver, lungs, blood plasma, stomach) and as a conse- study was carried out in collaboration with Prof. I. F. quence a severe course of the flu pathogenesis. Barinsky et al. (D. I. Ivanovskii Institute of Virology, Rus- sian Academy of Medical Sciences, Moscow). Results Zinc–containing complexes in influenza virus obtained are unique with the proved efficacy of this infection. chemotherapeutic agent in this especially dangerous Establishment of protective effect of zinc– infection with a high lethality in humans. containing complexes (zinc diglycinate and other Establishment of a marked chemothera- compounds) in experimental infection with influ- peutic effect of cycluridine in natural infection enza virus A/H3N2 in albino mice. of mucosal diseases – viral diarrhea in calves. Characterization of a new–type virucidal agent Development of optimal treatment course of for hand disinfection. oral administration (doses, duration, etc.). Determination of the antiviral spectrum Cycluridine is the first efficacious chemo- of the highly effective and broad–spectrum viru- therapeutic agent described in literature vs. this cidal composition, a new–type hand disinfectant, infection damaging cattle–breeding in this country Manorapid® Synergy, introduced in industrial pro- and on a global scale), and one of the first active duction and in the clinical practice (USA, Canada, agents vs. flavivirus infections at all occupying an Germany, Bulgaria). important place in the infectious pathology in hu- Large–spectrum system for in vitro screen- mans (hepatitis C included). ing of virucidal agents: Development and introduc-

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Bulgarian Added Value to ERA

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Studies on the Balkan endemic nephropathy Mechanism of action of plant substances in (BEN). experimental tumors. Role of viruses in etiology and pathogen- Significant evidence was obtained on the ef- esis of BEN: Data are obtained about the role of fect of substances of plant origin in experimental hantavirus infection in BEN as a result of tumors in mice. The mechanisms of their action on seroepidemiological investigation of BEN patients and the antitumor effector cells and on the release of by electron microscopy of surgery materials. reactive oxygen and nitrogen species and cytokines, Role of the immune system in BEN etio- participating in the antitumor defense are revealed. pathogenesis: Characterization of the immune sta- The obtained results are important for the com- tus in BEN patients by phenotyping of lymphocytes plete characterization of these plant metabolites and determination of phagocytic activity of mac- as substances stimulating the immune response in rophages by flowcytometry. malignant diseases. Determination of neopterin level (in urine) in BEN patients and its correlation with other basic Selective suppression by a chimeric antibody chlinico–laboratory markers. of DNA–specific B cells in SLE. A hybrid antibody molecule was constructed Studies on the problem viruses – diabetes. that is expected to down–regulate specifically the Diabetogenic action of herpesviruses: Rep- proliferation and differentiation of dsDNA–binding lication and damaging action of a representative B cells from lupus patients. This engineered mol- of herpesviruses selectively in Langerhans islets’ B ecule containing a monoclonal antibody against the cells was established for the first time (Aujesky human complement receptor 1 (CR1, CD35) coupled disease virus, pseudorabies virus on the model of to a peptide that mimics antigenically DNA is ex- organ culture of Langerhans islets from pigs). pected to target selectively B–cells recognizing na- Concept for diabetogenic action of neuro- tive DNA and to deliver to them a strong suppres- tropic viruses: Concept for diabetogenic action of sive signal via their inhibitory surface CR1 receptors. neurotropic viruses is formulated, based on the theory We have produced chimeric antibodies by coupling a of the paraneuronal nature of pancreatic endo- monoclonal anti–human CR1 antibody to the pep- crine cells. tide DWEYSVWLSN that mimics the antigenic epitopes Papillomaviruses in Bulgarian population. of dsDNA. The number of IgG anti–dsDNA antibod- Molecular epidemiological study by PCR on ies producing cells was reduced after treated PBMCs the role of human papillomaviruses as cancerogens with chimeric antibodies contain monoclonal anti- (precancerous states and cervical cancer) on Bul- body against the human complement receptor 1 garian population was realized for the first time. (CR1, CD35) coupled to a peptide that mimics anti- genically DNA. We show in this study that it is pos- Autoreactivity of therapeutic immunoglobulin sible to suppress selectively autoreactive B lympho- preparations. cytes by using an antibody chimera contain mono- The binding to human liver antigens of a panel clonal antibody against the human complement re- of seven different commercial IVIg preparations was ceptor 1 (CR1, CD35) coupled to a peptide that compared. The results indicated that the binding of mimics antigenically DNA. IgG molecules from IVIg to self–antigens correlated to the treatment at low pH during the production. Reactivity of human natural IgG autoantibod- The treatment of IVIg at low pH resulted in increas- ies to serum proteins is directed mainly to cir- ing of their inhibitory effect on proliferation of hu- culating immunoglobulins. man peripheral blood cells. The effects of different Natural polyreactive IgG autoantibodies are IVIg preparations on an experimental sepsis in mice known to be present in the plasma of disease– were also studied. IVIgs with high and low self– free individuals and as a result – in pooled human

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Advances in Bulgarian Science

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therapeutic intravenous immunoglobulin G (IVIg) bined approach which included amplified ribosomal preparations. Early studies have shown that that DNA restriction analysis (ARDRA), ribotyping and serum proteins are one of the groups of self– polymerase chain reaction (PCR) with species–spe- antigens to which natural autoantibodies bind. We cific oligonucleotide primers. All vaginal isolates and analyzed the spectrum of serum proteins, bound reference stains were grouped in 3 clusters in com-

by F(ab)’2 fragments of IVIg. The human serum parison to a set of 21 reference strains based on was passed through a column with immobilized the initial ARDRA results, which was confirmed by

F(ab)’2 fragments of IVIg or a column with F(ab)’2 ribotyping. Finally, the strains were subjected to PCR fragments of a human immune monoclonal IgG analysis with 7 different species–specific primers, antibody (as a negative control). The spectra of which allowed most of them to be classified as proteins in the eluates from the both affinity belonging to one of the following species: Lactoba- columns were analyzed by 2D electrophoresis and cillus fermentum, Lactobacillus helveticus, Lactoba- by blotting. The results indicate that a therapeutic cillus crispatus, Lactobacillus plantarum, and Lacto- IgG, obtained from a large group of healthy plasma bacillus gasseri. This is the first molecular–genetic donors binds to circulating immunoglobulin mol- study of vaginal lactobacilli isolated from Bulgarian ecules and to an additional group of serum pro- women, as the predominant species was L. teins. The blotting experiments confirmed that IgA, fermentum.

IgG and IgM are bound by the immobilized F(ab)’2 The cryptic plasmid pt38 (2911 bp) of fragments of IVIg. This result comes as no sur- Streptococcus thermophilus 2783, a strain isolated prise as it has been shown before that idiotype/ from Bulgarian yogurt, was subcloned and se- antiidiotype interactions take place between im- quenced. Five ORFs were identified. ORF2 would munoglobulin molecules in immunoglobulin prepa- specify a 142–amino acid protein sharing a high rations. The affinity of the binding of natural au- degree of homology with plasmid–born low–mo- toantibodies to self–antigens is known to be low, lecular–weight heat stress proteins described in a

with KD values in the micromolar range. Whether variety of S. thermophilus strains. The plasmid these interactions take place in vivo and whether pt38replicates via a rolling circle mechanism A they have biological significance obviously depends DNA region that can be transcribed into a small on the concentration of both ligands. As both the RNA (ctRNA) complementary to the leader seg- concentrations of serum IgG, IgA and IgM, and the ment of the rep (ORF1) mRNA is proposed to be kD values of idiotype/antiidiotype interactions be- involved in the control of plasmid replication. In tween them are in the micromolar range, this bind- vitro synthesis of this ctRNA was observed, and ing should take place also under physiological con- this constitutes the first report on the existence ditions in vivo. Very large doses (30 and more of such antisense RNAs, likely acting as regulatory grams) of pooled IgG are administered by the elements, in S. thermophilus plasmids. intravenous route to autoimmune patients and Physiology and resources of lactic acid bacte- thus these interactions may also be relevant to ria and health benefit solutions. To exploit the the immunomodulatory activity of the IVIg treat- biological potential of lactic acid bacteria for mak- ment. ing original Bulgarian starter cultures for whole- Identification of lactic acid bacteria from dif- some dairy products of high nutritive value we ferent ecological niches and characterization studied the unique natural biosystems that defined of antimicrobial peptides (bacteriocins) pro- the species diversity of lactic acid microflora, and duced. identified localities of biotypes of lactic acid bacte- In order to assess the important role of ria. The investigations positioned strains of vaginal lactobacilli in maintaining the vaginal health lactobacteria according to their ability to synthesize and in the prevention or therapy of bacterial biologically active substances – providers of health vaginosis a molecular–genetic study of Bulgarian benefits, as well as according to the levels of pro- vaginal lactobacilli was carried out. Twenty–two teolytic enzymes. The main achievement related to active strains were identified and grouped accord- creating multi–component symbiotic starters by in- ing to their genetic relatedness, based on a com- corporating highly productive strains of lactobacteria

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Bulgarian Added Value to ERA

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sp. NRRL B–3805 and Rhodococcus sp., respectively, phenol degrading agents which was confirmed by in a single phase organic solvent based medium. In the biochemical analyses of the first three en- both cases cells retained their viability and revealed zymes included in phenol degradation pathway. The high steroid transforming activity. The investigations sequence analysis of the specific phenol hydroxilase, were performed in collaboration with Centro de catechol dioxygenase and cis,cis–muconate cyclase Engenharia Biológica e Quimica, Instituto Superior genes leads to new possibilities for a specific re- Técnico, Lisboa, Portugal. sponse to the growing environmental pollution with Surface–active compounds. toxic aromatic compounds. It was found that nonionic detergents in- Anaerobic digestion of organic wastes is a bio- creased the permeability of yeast cells for low technology which mineralizes different in origin or- molecular substrates. On this base a simple, rapid, ganic wastes and as a result compost and biogas and mild procedure allowing determining periplasmic (energy source) are produced. The increased inter- enzyme activities in whole microbial cells was pro- est of many European countries in this biotechnol- posed. Recently the interest in microbial surfac- ogy is due to the solving of energy and environ- tants increased due to their diversity and environ- mental problems. On the basis of the research in mentally friendly nature. The effect of rhamnolipid laboratory „pseudohomogeneous“ bioreactors with biosurfactants, produced by Pseudomonas strains organic wastes from farming, food industry and on microbial growth, cell–surface properties (hy- activated sludge and different mixtures of these drophobicity and permeability), composition and fine wastes in different proportions we formulated the structures of cell membrane and wall of bacterial following main conclusions: and yeast strains and bacterial monocultures iso- For specific mixtures of organic waste biogas lated from polluted sites were studied. The bio- production could increase significantly and at the chemical and ultrastructural studies showed that same time the stability of the process is main- the biosurfactants affected the surface structures tained; of different microbes in different degree, leading The use of appropriate stimulating sub- to different changes in cell abilities. These results stances and surface–active compounds could also will elucidate the role of rhamnolipid–biosurfactants increase biogas production, but it is necessary to do on the biodegradation of low soluble compounds techno–economic calculations in order to take ef- and will extend the theoretical foundations for the ficient decisions; development of efficient technologies for Anaerobic digestion of organic wastes in bioremediation of polluted sites. a single „pseudohomogeneous“ biogas reactor does The microbial utilization of aromatic com- not significantly decrease the COD of the efflu- pounds represents a potential solution to en- ent compared to the influent, i.e. the degree of vironmental protection problems. depollution is not enough. In a „cascade“ of two The available scientific literature data con- bioreactors with different working volumes it is cerning catabolic genes in yeast species, in particu- possible to increase significantly the depollution lar genes coding for enzymes of the phenol de- effect; grading pathway is quite insufficient. The methods The community of different groups of mi- for discovering new catabolic genes, as well as genes croorganisms in the biogas reactor is sometimes involved in the bioaugmentation process of the en- influenced by some hard–to–measure environmen- vironment demonstrate the advantages of unique tal factors (such as geomagnetic activity and at- and easily identified molecular markers for the mosphere pressure) even at stable physicochemical investigation of natural microbial populations. The variables (ex. t 0C and pH in the biogas reactor), aim of our work is to contribute to more exact which is one of the reasons leading to unexpected and complete information about the diversity and „failures“ of the process. specificity of genes codyng the enzymes degrading A modified chemostat technique called titrostat aromatics. The investigated Trichosporon cutaneum was developed for selective isolation and continu- R57 yeast strain possesses an exclusive degradation ous flow cultivation of rapidly growing bacteria effectivity compared to other reported microbial utilizing toluene vapours as a single source of car-

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Bulgarian Added Value to ERA

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Annotation: We offer analysis to determine un- cryoconservation of farm–animal sperm. This en- desired microflora in foods, using up to date meth- zyme may be used in: ods, as well as to prove microorganisms to be use- Oncology (protection against tumour ap- ful and to prove their vitality. pearance; retardation of tumour progression; re- Readiness stage: Laboratory expertise. duction of cytotoxic and cardiotoxic effects of anti– tumour drugs); STUDY OF SUBSTANCES FOR ANTIMICROBIAL Cryoconservation of cells and organs; ACTIVITY AND FOR THEIR BIO–PROTECTIVE Transplantation of organs. EFFECT Readiness stage: Laboratory technology, know– Area of application: Pharmaceutical industry. how, and consultations. Annotation: We offer screening investigations of substances possessing antimicrobial activity to prove METHODS FOR THE DETECTION OF DEGRA-DA- their potential and application as bio–protectors. TIVE FUNGI Readiness stage: Laboratory expertise, consultations. Area of application: museums, libraries, leather– enterprises, shops, stores. ELECTRON–MICROSCOPIC EXPERTISE (SCAN- Annotation: Methods for detection and identifi- NING AND TRANSMISSION ELECTRON MICROS- cation of filamentous fungi included in biodegrada- COPY) FOR MORPHOLOGICAL CHARACTERIZA- tion of wide range of polymers (wood, leather, tex- TION OF WHOLE CELLS AND ULTRATHIN SEC- tile, historical records, etc.) have been developed. TIONS OF VARIOUS MICROORGANISMS Readiness stage: Laboratory expertise, consulta- Area of application: biological monitoring, medi- tions, instructions for preservation and elimination cine, biotechnology, microbial diagnostics and tax- of fungi. onomy. Annotation: A variety of electron microscopy pro- METHODS FOR THE DETECTION, ISOLATION AND tocols have been approbated and modified for spe- CHARACTERIZATION OF FOOD–BORN FUNGI cific application to different microbial taxa. The Area of application: food industry enterprises appropriate choice of protocols allows both ultra- (bakery, confectionery, sausage etc.) structural characterization of microorganism spe- Annotation: Methods for detection and identifi- cies and strains and also their changes under the cation of food–born fungi have been developed. impact of environmental factors. This can be ap- Readiness stage: Laboratory expertise, consulta- plied for expert evaluation of cellular microbe– tions, instructions for preservation and elimination host interactions or of microorganism changes in of fungi. the course of biotechnological processes. ONE–STEP HYDROLYSIS OF STARCH TO GLUCOSE Readiness stage: Laboratory expertise and scien- Area of glucose syrup application: tific consultations. food industry: confectionary, production of MICROBIAL TECHNOLOGY FOR PRODUCTION ice creams, soft drinks, baby foods, dressings; OF NOVEL ANTIOXIDANT ENZYME, Cu/Zn– ethanol production; CONTEINING SUPEROXIDE DISMUTASE (HLSOD) yeast protein for stock–breeding; Area of application: medicine, pharmaceutical and raw material for production of glucose– cosmetic industry, enzyme production industry. fructose syrups. They represent much more healthy Annotation: A highly efficient microbial technol- sweetener than sucrose and intensively replace it ogy for production of purified enzyme prepara- in confectionary. tion Cu/Zn–SOD has been elaborated. The novel Annotation: Laboratory technology includes simulta- α antioxidant enzyme is naturally glycosylated and neous action of four thermostable enzymes ( –amy- can be isolated in very few cases only. Such SOD lase, β–amylase, pullulanase and glucoamylase) with exerts a far higher pharmacological activity than similar pH and temperature optima instead of tradi- non–glycosylated enzymes. HLSOD showed a pro- tionally used 3–stage process. The hydrolysis is per- tective effect against myeloid Graffi tumour, in formed at 60°C, temperature at which starch is soluble. experimental influenza virus infection, and Readiness of technology: laboratory technology.

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Bulgarian Added Value to ERA

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DETERMINATION OF PHARMACOLOGICAL AC- BIOTECHNOLOGICAL PROCESSES FOR REMOVAL TIVITY OF BIO–SUBSTANCES OF HEAVY METALS FROM WASTE WATER AND Area of application: Pharmaceutical industry, food INDUSTRIAL SOLUTIONS industry, agriculture, etc. Area of application: The object of the biotech- Annotation: Via group of lab methods we test nological processes is connected with removal and different substances possessing bioactivity as newly concentration of copper, cobalt, iron and other heavy obtained drugs, as food additives and fodder addi- metals from waste, industrial or mining water and tives for animals; newly synthesized chemical com- following regeneration of the biosorbent resulting pounds, etc. We study the effect of these sub- to concentration and extraction of the metals in stances on different model experimental animals, solutions. on their immune systems, as well as on test micro- Annotation: The biotechnological process is char- organisms, as follows: pyrogen activity (in vitro and acterized by repeated use of sorbent (fungal bio- in vivo); toxicity (in vivo); influence on complement mass), possibility for extraction of desirable metal activity; functional activity of phagocyte cells (ph- by electrolize and high economic efficiency in com- agocyte test, killing test, oxygen and nitrite radicals parison to classical chemical methods. production); cytokine induction (interleukins, tumor Readiness stage: Laboratory technology with pos- necrotizing factor, interferon, etc.); antibacterial ac- sibility for getting patents. tivity; antifungal activity. ROSMARINIC ACID PRODUCTION BY LAVAN- Readiness stage: Established methods for lab ex- DULA VERA MM PLANT CELL SUSPENSION pertise of the above–mentioned items. Area of application: Food, cosmetic and pharma- DETERMINATION OF COLI–TITER, TOTAL NUM- ceutical industries. BER UNITS (CFU) Annotation: Rosmarinic acid possesses various bio- Area of application: Sanitary control of plant logical activities (antimicrobial, anti–inflammatory, departments, conveyors, raw materials, etc., used in antimutagenic) and also shows strong radical scav- food industry for drinks and foods production, etc, enging activities. These features reveal possibilities as well as the products themselves. for its application in food, pharmaceutical and cos- Annotation: Assessment and control of total mi- metic industries. crobial contamination is of great importance for Readiness stage: Laboratory technology and scale– quality and stability (durability) of the entire food, up to 3L bioreactor. pharmaceutical, cosmetic, etc. products. Effective in- BETALAINS BIOSYNTHESIS BY BETA VULGARIS ternal department control could be reached, using L. HAIRY ROOT CULTURE proposed methods. Area of application: Food, cosmetic and pharma- Readiness stage: Established methods for lab as- ceutical industries. sessment of hygiene and quality of the corresponding Annotation: The betalaines are water–soluble (relevant) products. natural colorants, including the red–violet betacyanins and the yellow–orange betaxanthines. METHODS FOR INCREASING OF ANTIBIOTIC AC- They possess strong radical scavenging activity and TIVITY OF INDUSTRIAL STRAINS–PRODUCERS are stable in pH and temperature area of the OF ANTIBIOTICS most food technologies, which defines them as Area of application: Pharmacy industry – for appropriate colorants in foods, cosmetics and phar- production of antibiotics for use in human and vet- maceuticals. erinary medicine. Readiness stage: Laboratory technology. Annotation: Wide range of methods (from in- duced mutagenesis to modern molecular–geneti- BIOSYNTHESIS, PROPERTIES AND BIOLOGICAL cal techniques) are applied for increasing the activ- ACTION OF EXOMANAN FROM YEASTS ity of industrial strains–producers of important an- Area of application: Stock breeding, plant protec- tibiotics used in human and veterinary medicine. tion, medicine. Readiness stage: Laboratory technology. Annotation: The proposed technology is for ob-

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taining polysaccharide manan, which, when added to chemical productions, such as: metilstyren, acetone, animal feed, inhibits intestinal colonization by patho- benzoic acid, methanol. For the first time it was gens. In vivo trials with animals show positive results established that, unlike other biodegrading agents, – decreased mortality rates, increase of productiv- this strain is capable of degrading highly toxic com- ity and improved health status, which defines the pounds isopropylbenzene and dimethylcarbinole. The polymer as a new biologically active substance for cells of the strain maintain their high activity after application in stockbreeding. The biological action covalent immobilization on polyacrilamide carriers. of manan was proved through its inhibitory effect Readiness stage: Laboratory technology, consulta- on phytopathogens, establishing its role as ecologi- tions. cally friendly means for plant protection. For its antimicrobial effect towards clinical staphylococcal CREATION AND INTRODUCTION OF THE ANTI- strains, manan can also be considered as an anti– VIRAL CHEMOTHERAPEUTIC ÀDENOSTATIN infection means for human application. COLLYRIUM® Readiness stage: Laboratory technology, tested at Area of application: medicine, antiviral chemo- semi–industrial scale. therapy. Annotation: The antiviral chemotherapeutic BIODEGRADATION OF TOXIC AROMATIC COM- Àdenostatin Collyrium® for treatment of ocular POUNDS IN WASTE WATERS viral infections with adenoviral etiology (epidemic Area of application: elaboration or optimization keratoconjunctivitis, etc.) was elaborated. It is the of technologies for detoxification of wastewaters most efficacious anti–adenoviral chemotherapeu- and soils, contaminated in the result of oil process- tic agent and the first Bulgarian antiviral chemo- ing and organic synthesis productions. therapeutic. The preparative dossier is prepared in Annotation: A yeast strain, with high effectiveness view of the interest of leading pharmaceutical com- in phenol and its toxic derivatives degradation was panies from USA and Japan. selected and characterized. It was proved, that the Readiness stage: Introduced for industrial pro- strain is capable of degrading other toxic com- duction and in clinical ophthalmologic practice. pounds, components of the wastewaters of some

MARITSA VEGETABLE CROPS RESEARCH INSTITUTE – 75 YEARS OF SERVICE

Dr Stoyka Masheva; Dr Miroslav Michov; Mr Galina Pevicharova; Dr. Dimitrina Kostova Maritsa Vegetable Crops Research Institute 32, Brezovsko shosse, 4003 Plovdiv, Bulgaria Phone number: +359 32 952296, Fax number: +359 32 960177 E mail: [email protected]

Maritsa Institute, founded in 1930, has devel- Prominent researchers from Maritsa Institute oped over the years as a leading center for veg- have created close links to agricultural universities etable research in Bulgaria. Combining scientific stud- and educational institutions to transfer modern hor- ies with practical application, the Institute has yielded ticultural methods and knowledge. Looking forward remarkable successes in different fields including to Bulgaria membership in the European Union, the development of vegetable hybrids, improvement Maritsa Institute has developed fruitful collabora- and stabilizing of local forms and accessions to tions with related institutions in member countries. high-yielded and high-quality cultivars, and amelio- The Institute will join the European Research ration of vegetable growing technologies. Area focusing on transferring European knowledge

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Institute as a leader and promoter of Vegetable 300 cultivars and F1 hybrids were created and widely Crops Improvement for Bulgaria and the Balkans. spread throughout the country. Some of them, be- cause of their valuable characteristics, were ex- INTRODUCTION ploited in foreign breeding programs. Maritsa Insti- Historical Background tute also became famous as a pioneer of new Bulgaria is a country with a long tradition in technologies in vegetable production. The unique vegetable growing, due to favorable climatic condi- and traditional Bulgarian method for vegetable tions, as well as great diversity of vegetable forms growing on “phytarion” (small areas enclosed with and accessions with unique and desirable proper- berms), was mechanized and adapted for bean, pea ties. For centuries Bulgarian gardeners were well and later for processing tomato, red pepper, and known in Europe, especially in the Danube region of potato. The Institute was also a pioneer in green- the Austrian Empire. Even today, fine restaurants in house vegetable production and constructed the Vienna buy their fresh vegetables from the Bul- first greenhouses for tomato and cucumber. Fur- garian vegetable market. Together with other ag- thermore, the Institute developed the first plant ricultural branches, vegetable growing made con- tissue culture laboratory for potato and flower siderable progress in Bulgaria at the end of the crops, established an international station for po- 19th century, enriching local traditions with modern tato breeding and cultivar maintenance with Ger- methods. However it was realized that advances in man participation, elaborated technology for bio- modern agriculture could not be achieved without logical pest control, and utilized hydroponics sys- science. As a result an Experimental Agricultural tems for vegetable growing. Station, mainly devoted to vegetables, was estab- For years the Institute has been a coordina- lished in Plovdiv on April 1, 1930. The nucleus of the tor of scientific research from several local experi- Station was built on the ground of a large private mental stations established on different climatic farm with excellent fertile soil. Later the Institute zones and soil types: in Negovan (on flowers and was named the Maritsa Vegetable Crops Research leaf vegetables), in Samokov and in Smolyan (on Institute, after the famous Maritsa river, the an- potato), in Gorna Oryahovitsa (on onion and cu- cient Hebros, which rises from the Rila Mountains, cumber). The Agricultural University of Plovdiv in- transverses the town of Plovdiv, and irrigates the vites many leading scientists from Maritsa Institute province of Tracia, until it reaches the Aegean Sea. as prominent lecturers and teachers of new gen- The first successes came soon. In 1932 Professor erations of horticulturists and agronomists in the Hristo Daskalov and his research team carried out fields of genetics, breeding, and agrotechnology. profound theoretical investigations on heterosis and Research groups of the Institute are involved in released the first F1 tomato hybrids the same year. international collaboration with corresponding insti- Due to its excellent quality, this hybrid remained in tutions in the Netherlands, Italy, France, England, Spain, production for more than 30 years. Several years Germany, Hungary, Poland, Czech Republic, countries later heterosis breeding was applied successfully to of the former Yugoslavia, Russia, and Moldova. eggplant, pepper, and cucumber. The Institute also played a significant role in elaborating the theo- Structure of the Institute retical basis for hybrid seed production and its ap- Maritsa Vegetable Crop Research Institute is plication into practice. At that time Bulgaria be- a legal entity in Bulgaria, and a Member of the came a world leader in yield and total production National Center for Agrarian Science (NCAS) un- of hybrid seeds. In 1950, total production was 10,000 der the Ministry of Agriculture and Forestry. It - 12,000 kg of tomato hybrid seeds, with yields of comprises two main Departments: (1) Breeding, 200 - 250 kg/ha. During this early stage, laborato- Variety Maintenance and Introduction and (2) Tech- ries for quality control and technological analysis nology-for-Growing. The people playing major roles were established, striving to evaluate unique local in the Breeding Department are the breeders of germplasm collected from all over the country the most important Bulgarian vegetables including and preserved in the Institute. tomato, pepper, cabbage, cucumber, potato, onion and

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garlic, bean and pea. Their research is supported by protection strategies. The team of Plant Nutrition investigations carried out in seven laboratories that directs its efforts towards investigations on bio- are included as separate units in the departments products as alternative solutions to improve nutri- having long experience in different aspects of veg- ent regime and plant growth. The team of etable technology: Vegetable Fruit Quality, Virus Agrotechnology deals with the influence of the Immunity, Plant Tissue Culture, Cytogenetics, Mo- main environment factors on yield. Current research lecular Investigation, Physiology, and Vegetable Pro- on mechanized technologies concerns analysis on cessing). input-output energy for growing vegetables in open For 75 years the Breeding Department has and protected fields, in order to decrease the en- been a coordinator and executor in the creation ergy ratio. There is an on-going project on im- and implementation of a large number of cultivars provement of greenhouse crops production tech-

and F1 hybrids of the principal vegetable crops of nology and efficiency, funded by FAO [TCP/BUL/ Bulgaria. A great diversity of local and introduced 3002(A)]. The Institute is also involved in organiza- forms and cultivars, breeding lines and populations tion of training and extension activities in the Veg- of wild species has been developed. Most of the etable Sector, part of the joint project, entitled local cultivars (Bulgaria is a secondary centre of „Strengthening of Fresh Fruit and Vegetable Mar- origin for pepper, head cabbage, bean, and onion) keting Quality Standards Control System” (PPA/ have been stabilized and improved through the 04/BG/9/1), funded by the Netherlands Ministry breeding programme of the Institute, distinguished of Economy. A significant part of Maritsa research by high nutritional value and organoleptic charac- programs integrates the efforts of scientists from teristics that make them competitive on the mar- different structural units of the Institute. ket. The Department works with a rich gene pool from these crops, variable in their morphological RESEARCH TOPICS and biological characters. There are 14 PhD scien- The priority investigations of the Institute com- tific officers, including 6 senior members. In addition, prise a number of topics as follows: there are 6 professors as associate members. The Enhancement of vegetable quality by im- scientific staff of the Department has a long-stand- proving biological value (increasing the components ing experience in vegetable breeding, and has ac- with antioxidant effect), sensory characteristics, pest quired considerable practice in managing projects and diseases resistance, high temperature and supported by the Bulgarian Ministry of Science and drought tolerance by the use of conventional and Education, and Ministry of Agriculture and Forestry. biotechnological breeding methods; An international project on red pepper quality, de- Evaluation and exploitation of various veg- veloped by INCO-COPERNICUS 94, was success- etable germplasms, comprising local and introduced fully completed with participation of the pepper accessions, breeding lines, and cultivars; breeding team and Vegetable Fruit Quality Labora- Improvement of integrating systems for pest tory. There is an ongoing project, funded by the control (pest, diseases and weeds) in vegetables; International Atomic Energy Agency (IAEA) in Vienna, Investigations on bio-products as alterna- on the induction and study of tomato and pepper tive solutions to improve nutrient regime, plant growth mutants with high nutritive quality. The Breeding and plant qualit; Department is also responsible for cultivar mainte- The effect of environmental factors on nance and seed preservation (gene bank). quality and quantity of the yield in protected and The Technology-for-Growing Department open fields. comprises the following units: Agrotechnology, Plant Protection, Plant Nutrition, and Mechanization. The Tomato Program scientific staff includes 10 PhD officers, 6 of whom The tomato breeding program is directed are senior, and an additional 3 professors as associ- towards creation of cultivars for early production ate members. Great emphasis on investigations in and mid-season production for fresh consumption Plant Protection Unit is now being given to devel- and for processing. As a result of long-lasting opment of sustainable control systems and crop breeding program the team works with valuable

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Bulgarian Added Value to ERA

○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○ genetic materials derived from interspecific The aims of a joint program with the Cyto- hybridisation between Lycoprsicon esculentum and genetics Laboratory are to incorporate a male- wild species such as L. pimpinellifolium, L. chilense, L. sterile gene in valuable breeding lines and cultivars pennellii, and L. peruvianum. These species possess as well as to investigate the expression of domi- complex resistance to pathogens such as Cladospo- nance and recessiveness responsible for fertility and. rium fulvum, Fusarium oxysporum f.sp. lycopersici , sterility in hybrids and backcross progenies. Incom- Leveillula taurica, Corynebacterium michiganense, plete dominance and lethality were expressed in and Verticillium dahliae. Major emphasis is to cre- some of the homozygous genotypes. To prevent ate gene resources, combining high biological value flower abortion in winter and early spring, the Physi- with disease resistance to new races of economi- ology Laboratory licensed some substances, which cally important pathogens. As a result of crosses influence earliness and yield. between L. esculentum and L. chilense indetermi- A comprehensive system of soil and substrate nate tomato lines were created by the joint ef- analysis was elaborated at the Plant Nutrition Labo- forts of tomato breeders and the Vegetable Fruit ratory in order to diagnose soil and substrates nu- Quality Lab teams. Lines were divided into three trient status. Monitoring of nutrient needs of to- types according to concentration of b-carotene mato grown as soil and soilless culture is being based on total pigmentation in the fruits: 80-90%, carried out in order to recommend the optimal 50-60%, and about 30%. The fruits also possess norms for fertilization. Relationships are established comparatively high Vitamin C content reaching up between the nutrient levels and the quality of to 57 mg per 100 g fresh tissue. A number of production. In collaboration with Plant Protection gene sources with high level of lycopene (over 10 Unit the impact of vesicular mycorrhizae (VAM) mg) were created at the Tissue Culture Labora- fungi is being investigated on the growth, develop- tory as a result of in vitro callus treatment with ment, and productivity of tomato as well as the use sublethal doses of gamma rays Co60. In the same of biological agents against soil-borne pathogens laboratory effective procedures were developed and root-knot nematodes as an alternative to for micropropagation, calligenesis, organo-genesis, methylbromide as a treatment for soil disinfection and regeneration of valuable breeding lines, culti- in greenhouses. vars, and F1 tomato hybrids. Tomato hybrid forms Biomethods are being improved by searching and stable lines between L. esculentum and L. for new biological agents. Several entomopathogenic peruvianum and L. pennellii were created, as a fungi (Aschersonia sp., Verticillium lecanii), parasites result of embryo rescue technology. A system was (Encarsia formosa, Aphidius matricariae, A. ervi) and developed for in vitro selection of tomato forms predators (Phytoseiulus persimilis, Macrolophus costalis, tolerant to the fungal pathogen Alternaria solani M. nubilis, and Amblyseius sp., Orius niger) are used using its culture filtrates. In the last two years a as bioregulators to ameliorate the density of eco- successful method was developed for genetic nomically important pests on tomato: Trialeurodes transformation by Agrobacterium tumefaciens, vaporariorum, Myzus persicae, Aphis nasturtii, A. which resulted in the creation of transformed gossypii, Thrips tabaci, and Frankliniella occidentalis. tomato lines with heavy metal tolerance. At the The antagonistic activity of Trichoderma sp., Virus Immunity Laboratory, the most promising lines Gliocladium and Ånterobacter cloaceae is investi- with high biological value are transformed with gated. Procedures are elaborated for production tomato mosaic virus (ToMV) and cucumber mo- of soil-borne and aerogenic fungal pathogens. saic virus (CMV) resistance. A large part of the tomato breeding materials for fresh consumption Pepper Program and processing are assessed for their chemico- In the course of over 70 years great diversity technological and sensory properties at the Veg- of genetic materials has been created and main- etable Fruit Quality and Processing Laboratories. tained, mainly based on unique local forms typical Genetic diversity of the exploited materials of to- for the region of Bulgaria. They were improved by mato is characterized and identified at the Mo- methods of conventional breeding. Some of these lecular Genetics Lab using molecular markers. local cultivars, due to their valuable characteristics,

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are still in production and are also initial materials per cultivars. Breeding lines and hybrids resistant to for creation of new cultivars, responding to con- Verticillium dahliae and Phytophthora capsici have temporary requirements. At present the pepper been created thanks to a joint program with the breeding team is involved in a valuable collection Plant Protection Unit. An important aim of the of over 500 local and introduced accessions. breeding team is to create male sterile analogues Contemporary breeding program is focused of the most widespread commercial pepper culti- on the creation of green and red fruited types as vars in order to be used in a hybrid program. The well as pepper for grinding (paprika). The breeding requirements of new pepper cultivars to environ- program for „green pepper“ and „red pepper” em- mental factors are investigated with the phasizes the creation of lines and cultivars with Agrotecnology Unit in order to assess their suitabil- high biological value (mainly with ascorbic acid con- ity for different growing regions. tent over 120 mg per 100 g for green and over 200 mg for red one) as well as good sensory Cucumber Program properties. The desired characteristics for fresh con- The cucumber-breeding program deals with sumption are crispness, succulence, freshness and all types of cucumber species such as long Euro- non-pungency; for processing (for baking, canning, pean type, American slicers, mini and pickling cu-

and for juices) they are easy peeling, fleshy peri- cumbers. A great number of F1 hybrids of top per- carp and intensive red color. Attention is also paid formance have been created, most of them with to variable nuances of green (light yellow to dark predominantly gynoecious flowering habit, partheno- green) and red (light orange to dark red) colors carpic fruits with dark green to green color, smooth and to various shapes: oblate, edged, bell, blocky, to slightly rubbed surface, tolerant to Sphaerotheca

conical tapered, kapia, and slender type. fuliginea. Several slicer F1 hybrids types used as the The objectives of the paprika-breeding pro- seed parent combine CMV tolerance and predomi- gram are earliness and uniformity in ripeness as nantly gynoecious flowering habit combined with well as enhancement of quality and quantity of excellent taste and flavor of widespread monoe- total pigments and their long storage. Red pepper cious cultivars used as a pollen parent. lines with over 300 ASTA units are now established. As a result of collaboration between the Vi- One of the breeding directions is to search for or rus Immunity Laboratory and Plant Virology Torino create forms with stable pigment content after a cucumber virus was identified as a new putative frosting. tombusvirus species provisionally named cucumber At the Plant Tissue Culture Laboratory pro- Bulgarian latent virus (CBLV). Also, EMDV on cu- cedures are optimised for obtaining haploids in an- cumbers is announced for the first time in Bulgaria. ther culture, derived from local cultivars of Capsi- For creation of breeding materials with com- cum annuum. These procedures will be used for plete homozygosis in respect to some valuable char- species conservation, maintenance, and creation of acteristics, haploid cucumber plants were produced homozygous lines. Also effective procedures for with the joint efforts of the Department of Plant micropropagation, callusogenesis, organogenesis and Genetics, Breeding and Biotechnology at Warsaw regeneration of valuable forms, lines, cultivars and Agricultural University, Poland and the Cytogenetics

F1 hybrids are developed. Laboratory. Embryos were induced by gynogenesis The investigations at the Virus Immunity Labo- and cultured in vitro. The subsequent cytological ratory are focused on strain variability of the most analysis determined them as monohaploids. Several widespread viruses on pepper in Bulgaria: tobacco doubled haploids and genome chimeras were ob- mosaic virus (TMV) and cucumber mosaic virus tained by colchicine treatment. Interspecific hybrids (CMV). Recently pepper mild mottle virus (PMMV) were created in vivo between C. sativus and C. and eggplant mottle dwarf virus (EMDV) were melo var. agrestis subsp. sikimensis to increase ge- discovered in greenhouse pepper during a collabo- netic diversity. The Physiology Laboratory licenses rative program with the Plant Virology Institute in several complex substances which induce male flow- Torino, Italy. One of the aims of the team is to ers in gynoecious lines necessary for their repro- introduce TMV resistance in several traditional pep- duction as parents in hybrid programs.

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The intensive breeding program matches up in two directions: fresh consumption and process- with continuous new demands in term of disease ing. Most of the cultivars are bush type, with oval resistances. A new trend of the current plan is to or cylindrical, stringless, straight and smooth skinned combine the CMV resistance with resistance to pods, with dark green, light medium green or yellow Sphaerotheca fuliginea and Pseudoperonospora color, resistant to Pseudomonas phaseolicola and cubensis. The team disposes with lines, possessing bean common mosaic virus (BCMV). Recently some complex resistance to these three pathogens. cultivars were released for home gardeners with runner growth habit, flat pods and superb bean Cabbage Program flavor. Bulgaria appears to be a second center of Serious obstacles for bean production are origin for Brassica oleraceae var. capitata (heading virus diseases. A breeding program for virus resis- cabbage). Cultivars developed at the Institute pos- tance is carried out together with the Virus Immu- sess unique taste characteristics, appropriate for nity Laboratory to investigate virus variability and fresh consumption and processing. Two cultivars, create resistant gene resources. The team devel- ‘Kiose’ and ‘Balkan’, derived from a local accession, ops breeding lines and cultivars resistant to CMV, were widely used for years for fresh use and for temperature dependent necrotic strains of bean kraut. ‘Besapara’ is the only cultivar in Bulgaria for common mosaic virus (BCMV) as well as to bean early spring production by autumn planting. common mosaic necrosis virus (BCMNV). The col- The objectives of the breeding program are laboration program with the Vegetable Fruit Qual- to release early white cabbage cultivars for spring ity Laboratory is to release breeding lines combin- sowing and broccoli with high ascorbic acid content ing viral and bacterial resistance with good sensory and resistant to hollow stem. Collaboration pro- characteristics. grams with the Plant Tissue Culture and Cytoge- The Cytogenetics and Physiology Laborato- netics Laboratories strive to increase the genetic ries developed a procedure based on gamete se- diversity in heading cabbage using in vivo and in lection for obtaining high temperature-tolerant vitro muthagenesis mainly to separate male sterile genotypes in bean. Polyploids were induced in P. forms. Also procedures are optimised for obtaining vulgaris, P. coccineus, and P. acutifolius to obtain haploids in anther culture. fertile amphidiploids from interspecific hybrids in As a result of several years screening of Phaseolus. valuable gene sources from Brassica oleracea var. The collaboration program between the bean capitata, var. sabauda and var. italica, the breeding breeders and entomologists from the Plant Pathol- team has created several breeding lines resistant ogy Units aims to investigate the level of suscepti- to Peronospora parasitica. Current investigations are bility toward Acanthoscelides obtectus in different dealing with complex resistance towards three accessions and cultivars of P. vulgaris. A number of pathogens: P. parasitica, Alternaria brassicicola, and lines have been identified that possess pest toler- Xanthomonas campestris pv. campestris. The con- ance. The effectiveness of various phytopesticides ducted tests with entomologists from the Plant applied against bean weevil in open field is under Protection Unit revealed resistance to Breyicorine investigation. brassicae in white cabbage. Search for resistance to Mamestra brassicae, Pieris brassicae and Pieris Pea Program rapae is an aim of the team. A project with the Pea cultivars of Pisum sativum created at Agrotechnology Unit deals with introduction of or- Maritsa Institute are widespread in Bulgaria. They ganic farming systems appropriate for Bulgarian satisfied the needs for early, mid-early and late cultivars of white cabbages. production of peas mainly for canning and freez- ing. Most of the cultivars are wrinkle-seeded. The Bean Program new tendencies in the breeding program concern Phaseolus bean cultivars of the Maritsa Insti- the development of super early cultivars, uniform tute carry the genetic background of valuable lo- ripeness, multipoded, with increased number of peas; cal forms and accessions. They are orientated mainly resistance to the most spread viruses and toler-

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ance to high temperature and drought. Models for The current potato program place special sensory evaluation of various breeding materials, emphasis upon introducing cyst resistance to nema- adapted to various uses, are carried out at the todes (Globodera rostochiensis) in valuable and widely Vegetable Fruit Quality Laboratory. used cultivars. The Plant Tissue Culture Laboratory uses experimental mutagenesis to increase genetic Onion Program diversity, mainly to separate early forms. Serious dam- Since Bulgaria is considered to be a second- ages to potato production due to climate changes ary center of origin for onion, most of the cultivars impose a new trend in breeding program: creation created in the Institute possess valuable character- of breeding lines and cultivars tolerant to high tem- istics of local accessions and forms. They are distin- perature and drought. This program is a collabora- guished by good keeping quality due to firm, tightly tion of the Physiology and Cytogenetics Laborato- adhering skin and thin neck; by attractive yellow- ries. The potato team is currently working on the brown to light brown color; by variability in shape creation of specialized cultivars for boiled potatoes from globular to slightly flattened globe; for growing and puree, French fries and chips in order to re- by direct seeding or by sets. spond to the new requirements of the processing The Institute has significant achievements in industry in Bulgaria. To ameliorate and to increase hybrid breeding and hybrid seed production in on- the quality and quantity of yield from early pota- ion. The present breeding program is focused on toes, the Agrotechnology Unit is investigating the creating male-sterile lines and their maintainers, with application of nonwoven plant covers. strong pungent to sweet taste. Attention is paid to lines with white colored bulbs and high dry matter PROSPECTS FOR FUTURE RESEARCH content suitable for dried onion production as well Because of the forthcoming membership of as to gene resources, resistant to Botrytis sp. Bulgaria in the European Union there are several important research objectives for future research. Potato Program To transfer the recent achievements of Bulgaria is at the border area of optimal European knowledge in the field of plant genetics, conditions for potato growing. The most important fruit quality, integrated pest management (IPM) to limited factor for potato production is aphids as Bulgaria. This will be achieved by courses, conducted virus vectors causing degeneration of seed pota- by prominent European lecturers and by short- toes. In this respect the Institute is a pioneer in term visits of young PhD students and researchers elaborating a strategic breeding program in the of the Institute to European Institutes; 1960s for the creation of virus-resistant cultivars To develop Maritsa Institute as a promoter and technology for virus-free seed production. of Vegetable Crops Improvement Linkage (VCI In- Genes for virus resistance were transferred ner Linkage and VCI Balkan Linkage) in order to from different wild species such as Solanum acaule, integrate research and researchers in Bulgaria and S. demissum, S. sisimbrifolium, S. chacoense, S. in the Balkan region; stoloniferum to S. tuberosum materials. Valuable To create motivated young researchers, dis- breeding lines and cultivars (‘Iverze’ and ‘Nadejda seminate scientific information and results, and fa- 25’) were created with high productivity, earliness cilitate communication and collaboration between and good organoleptic properties. In order to in- the Balkan countries with similar scientific interests crease the genetic diversity and to create initial ma- in the field of vegetable science. terials with complex immunity to virus diseases and One of the contributions of the VCI Inner Link- late blight (Phytophthora infestans) an International age will be elaboration of strategy for Bulgarian Station was founded in the Rhodope Mountains with vegetable science that will be relevant to Europe. Bulgarian and German collaboration. A network for The Inner Linkage will be also exploited as a social virus-free seed production was elaborated and con- instrument for elaboration and diffusion of technol- sisted of tissue culture laboratory for virus-free ini- ogy and knowledge transfer program for farmers, tial materials, insect-proof greenhouses and highland seed producers and processors. The most important farms with low aphid infestation. achievement of VCI Balkan Linkage will be to bring in

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Maritsa Main Entrance House, built in 1960. It is surrounded by rare and valuable dendro species and comprises mainly administrative and library premises, some labs, and offices of researchers. The other Units are scattered in an Arboretrum, in smaller buildings.

Promising tomato breeding line for fresh Green pepper variety for fresh consumption. consumption.

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Pepper seed production.

Onion seed production.

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MOLECULAR MEDICINE CENTER – NOVEL CENTER FOR BIOMEDICAL RESEARCH IN BULGARIA

Albena Jordanova, PhD and Assoc. Prof. Ivo Kremensky, MD, PhD 2, Zdrave Str. 1431 Sofia, BULGARIA, Tel: + 359 2 9172 268, Fax: + 359 2 9172469, E-mail: [email protected], http://www.lmpbg.org

Molecular Medicine Center is a collaborative, University, and Ivo Kremensky, currently Associate multidisciplinary research center at Sofia Medical Professor at the Faculty and Head of the Labora- University established in 2005 with the support of tory. the Specific Support Action Program of the Euro- LMP was conceived as Bulgaria’s major diag- pean Union. The Center upgrades and promotes nostic, research and training center for biochemical the activities of the Laboratory of Molecular Pa- and molecular genetics. The World Health Organi- thology, the existing national facility for diagnosis zation (WHO) Fund and the United Nation Popula- and research in the field of human genetics, whose tions Fund (UNFPA) supported its establishment. Its 27-years tradition, expertise, and international links mission and future development have been care- provide solid foundations for future development. fully charted in consultation with WHO experts, and The major goal of the Molecular Medicine in compliance with international standards applied Center is enhancing the capacity for biomedical to the country’s population size, geography, health research in Bulgaria. This will be achieved through care system and infrastructure. promoting and developing the interaction between The Laboratory is functionally divided into six the Medical University’s departments and Bulgaria’s sections: Molecular Pathology, Biochemistry, Mass major diagnostic and research facility in the field neonatal screening (for PKU) and prenatal screen- of human genetics, the Laboratory of Molecular ing (for Down Syndrome); Prenatal Diagnosis of Pathology (LMP) and stimulation of genetic re- Inherited Disorders, Cytogenetic diagnosis and Hu- search in all fields of medicine, focusing on com- man Reproduction (with Sperm Bank), equipped mon genetic diseases of particular relevance to for biochemical and molecular genetics analyses. the country morbidity profile. LMP as the national centre for diagnosis and prevention of genetic diseases: BACKGROUND LMP conducts two national mass screening The Molecular Medicine Centre is built upon: programs - of newborns for PKU and galactosemia (i) the role of LMP as a national genetics center, (since 1977 more that 1 800 000 newborns have its existing capacity and know-how, established links been screened), and prenatal for Down syndrome with clinical medicine, and international reputation; and neural tube defects (since 2000). It offers a (ii) the research expertise of its members and the battery of metabolic and enzymological assays for role of Sofia Medical University as the country’s diagnosis of over 70 inborn errors of metabolism. It leading tertiary medical institution; (iii) the growing was among the first in Eastern Europe to intro- interest of Bulgarian clinical specialists from differ- duce molecular genetic testing. The Laboratory per- ent areas of medicine in the molecular factors forms molecular analyses for common aneuploidies, conferring common disorders. over 30 Mendelian disorders and other DNA-based The Laboratory of Molecular Pathology analyses, including forensic paternity testing. LMP LMP was founded in 1977 as a joint initiative has well-established procedures for transport, re- of the Faculty Departments of Pediatrics and Ob- ception, processing, and archiving of biological samples. stetrics and Gynecology, and the Ministry of Health. It is managing several national sample collections, The founding senior scientists were Luba Kalaydjieva, ranging in size from hundreds to many thousands, currently Professor of Molecular Genetics at the including Guthrie cards from newborns and a DNA University of Western Australia and Visiting Pro- bank of families with genetic disorders. The com- fessor at the Faculty of Medicine of Sofia Medical puterized register contains information on over

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20,000 families, including >2,000 with monogenic participate in the European Cystic Fibrosis Con- diseases. sortium and the Charcot-Marie-Tooth Disease Con- The Laboratory initiated and has been ac- sortium. Their studies of the common single-gene tively involved in designing and implementing of disorders have led to identification of a large num- the current National Program for Diagnosis and ber of novel disease mutations. This research has Prevention of Inherited Disorders and Congenital contributed to understanding the genetic land- Anomalies in Bulgaria, funded by the Ministry of scape of Europe, as well as to the study of geno- Health, and in the legislation and policy-making type-phenotype correlations. They have also made process in the field of healthcare, health insur- methodological contributions to the field by de- ance and other issues related to genetic testing. veloping highly sensitive mutation screening (SSCA) During the 27 years of its existence as a national protocols, now widely used by many international center LMP has gained a comprehensive over- laboratories. view of hereditary pathology, and knowledge of Gene discovery: the health care infrastructure of the country. Its The longstanding strong emphasis on close links to clinical medicine have shaped its neurogenetics, in close collaboration with the De- research profile and trained its staff in commu- partment of Neurology, has led to the identification nicating with the medical community. In its turn, and genetic characterization of four novel disor- LMP is known to the medical community coun- ders of the peripheral nervous system. This work is trywide as a reliable, long-standing partner in pa- now expanding into epilepsy and other tient care, and thus the first choice for scientific neurodegenerative disorders. partnership. Transition to complex disorders. The Laboratory staff members have helped The Laboratory of Molecular Pathology has in setting up parents’ and patients’ associations traditional links with the Departments of Pediatrics for Neuromuscular Disorders, Phenylketonuria, and Obstetrics due to its focus on Mendelian disor- Gaucher’s Disease, Cystic Fibrosis, Neurofibromato- ders. This is now changing, with an exponentially sis and are involved in the activities of these asso- growing interest of a wide range of clinical special- ciations, reporting on the advances of genetic re- ists in the genetic basis of common genetically com- search among patients. Major achievements of plex disorders. The MMC is involved in research LMP in studying genetic disorders and population into bipolar affective disorder in collaboration with structure of the Roma/Gypsies have been pos- the Department of Psychiatry. Research into com- sible due to the fruitful collaboration with the plex disorders is expanding into cardiovascular dis- non-profit organisations like the Roma Foundation orders, which are a major health problem and the and the „Health Problems of Ethnic Minorities” leading cause of death in Bulgaria, neurodegenerative Foundation- our partners in ongoing projects re- disorders, drug abuse, familial cancer, osteoporosis, lated to the Roma population. diabetes and genetic predisposition to complications MMC HAS INTERNATIONALLY RECOGNIZED of pregnancy and delivery. SCIENTIFIC PROFILE AND CONTRIBUTION TO Population genetics: EUROPEAN SCIENCE The key geographic location of Bulgaria makes Major objectives of the newly established it an important contributor to understanding the Molecular Medicine Centre are oriented towards population history of Europe. Publications of MMC research directly related to human health and well- staff members in population genetics are widely being. Its staff members are known to the Euro- cited in studies of European populations genetics. pean scientific community and respected for their We are regular contributors to the European fo- contributions to several areas of genetics. rensic database. The mutational basis of Mendelian disorders. MMC makes a unique contribution to genetic MMC researchers have a solid reputation in research and health care in Europe. the Medelian disorders research. They were one The Laboratory of Molecular Pathology un- of the founding members of the World Cystic der the supervision of Prof. Luba Kalaydjieva has Fibrosis Genetic Analysis Consortium and currently initiated pioneering genetic studies into Europe’s most

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retains young scientists and facilitates dissemination Management Committee. The Scientific Board de- of knowledge. termines the directions and guidelines of MMC re- search. The Project Management Committee en- MMC RESEARCH STRUCTURE sures the quality and timely implementation of the Research groups are the main structural unit deliverables and milestones outlined in the individual in MMC. They are formed, based on the specific research projects carried out in MMC. The Center demands from different Departments of Sofia is overseen by an Advisory Board, composed of Medical University and the current trends in ge- senior scientists affiliated with national and inter- netic research. These are multidisciplinary teams of national institutions outside the Medical Faculty. researchers from different departments of Sofia The research performed in MMC is supported Medical University and the LMP, working on specific by three main core facilities: Biobanking, Genomics collaborative projects with access to common tech- and Bioinformatics. The Biobanking core has the nical and managerial resources. The current re- capacity to provide a number of specialized high search topics in MMC are: quality services, such as genomic peripheral blood A. Inherited Peripheral Neuropaties (IPN) – lymphocyte, plasma and DNA isolation from large molecular defects and genotype-phenotype corre- numbers of samples, and cryopreservation for long- lations, mapping of novel loci and identification of term secure data storage and retrieval. The genes associated with IPN, clinical and genetic char- Genomics core offers an up-to-date technology acterization of Dominant Intermediate Charcot- platform for cost effective and mid- to high Marie-Tooth (DI CMT) disease, and functional throughput DNA/RNA analysis. The Bioinformatics genomics of DI CMT type C. core ensures access to various databases and analysis B. Muscular dystrophies – molecular defects software and organises the data produced by dif- and genotype-phenotype correlations. ferent individual projects into well-annotated da- C. Affective psychiatric disorders – large-scale tabases. This core makes the data available to the association studies, mapping of novel loci for bipo- Bulgarian scientific community through the devel- lar/unipolar affective disorders, case/control and opment of user interfaces/application program- trios studies. ming interfaces in combination with powerful analysis D. Familial Cancer – searching for genetic tools. The bioinformation actively trains researchers predispositions related to familial colorectal and and students in biomedical sciences in this novel endometrial cancer. biomedical discipline, so they can use these data E. Epilepsy syndromes – molecular defects efficiently. and genotype-phenotype correlations, mapping of novel loci associated with GEFS+ syndromes. MMC PERFORMS ACTIVE NETWORKING AND F. Genetic predispositions related to drug ad- DISSEMINATION OF KNOWLEDGE diction, autism, reproductive failures, dementias and MMC has established a local network within stroke. Sofia Medical University by maintaining monthly joint G. Ophthalmologic disorders – molecular de- seminars and joint supervision of PhD students and fects and genotype-phenotype correlations, map- postdocs affiliated to the different departments, ping of novel loci and identification of genes asso- who perform the molecular genetic part of their ciated with Retinitis pigmentosa and glaucoma. research in the MMC. H. Development of new DNA based methods MMC is subscribed to the most important for identification of aneuploidies. national and international journals in Biomedical .I Population genetics – molecular charac- science and serves as a platform for dissemination terization of the main ethnic groups of the Bulgar- of research achievements and project results among ian population, etc. a broad range of specialists. Annual open-door days for visits in the research facilities are orga- The Molecular Medicine Center is coordinated nized to attract the interest of young people (stu- by Assoc. Prof. Ivo Kremensky and two local gov- dents and pupils from high schools) and all inter- erning bodies: the Scientific Board and the Project ested in molecular medicine. Special Seminars are

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GENETIC SERVICE FOR CYSTIC FIBROSIS IN BULGARIA

Alexey Savov, PhD; Albena Jordanova, PhD and Assoc. Prof. Ivo Kremensky, MD, PhD Laboratory of Molecular Pathology, Sofia, Bulgaria

Laboratory of Molecular Pathology is the and construction of intragenic microsatellites of National Centre providing molecular diagnostics, pre- 262 CF alleles. Deletion delF508 accounted for 100% vention and genetic counseling of Cystic Fibrosis of Gypsy CF alleles, which thus differed significantly (CF) in Bulgaria. Since 1985 more than 350 CF from both Bulgarians and ethnic Turks. In Bulgar- families have been analyzed. The molecular screening ian and Turkish CF patients, 92% of the mutant approach includes a battery of highly informative alleles were identified, yielding a total of 25 differ- and sensitive methods for DNA analysis, such as ent mutations, of which only 7 occurred at fre- Heteroduplex analysis, Single Strand Conformation quencies higher than 1%. Genetic distances and Polymorphism Analysis (SSCA), direct DNA sequenc- population trees demonstrated that in the south- ing and haplotype analysis. eastern tip of Europe, the overall distribution of CF Our mutation detection strategy has allowed mutations and polymorphic haplotypes is very close detection of 92% of molecular defects in Bulgar- to that of Mediterranean populations, with a high ian CF patients. So far, we have identified 37 differ- frequency of N1303K and G542X, a large number ent mutations of which 13 were novel mutations of rare mutations and a prevalence of the 23 31 (Savov et al., 1994a, b; 1996). We were the first to 13 haplotype in association with deltaF508 demonstrate that double mutant alleles are not a (Angelicheva et al., 1997). rare finding in CF with serious implications for mo- Apart from the CF research, we provide pre- lecular diagnosis and genetic counselling (Savov et and postnatal diagnosis of the disease. So far, we al., 1999). have performed prenatal analyses in 139 CF fami- Our genotype-phenotype correlation studies lies and diagnosed 35 affected, 62 carriers and 42 revealed that Meconium ileus is a clinical complica- healthy fetuses. tion significantly associated with 1677delTA muta- tion and that R347P mutation causes CF charac- Reference list of articles related to Bulgarian terized by pancreatic insufficiency and pulmonary CF research: disease. We performed population genetics study 1. Angelicheva D., Calafell F., Savov A., Jordanova of CF in Bulgaria via comprehensive mutation analysis A., Kufardjieva A., Galeva I., Nedkova V., Ivanova T.,

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Yankova P., Konstantinova D., Genev E., Kalaydjieva L., tion. Hum Mutat. (1995); 6(3): 219-25. Cystic fibrosis mutations and associated haplotypes 5. Savov A., Jordanova A., Gavrilov D., Angelicheva in Bulgaria - a comparative population genetic study. D., Kalaydjieva L. G1244V: a novel missense muta- Hum Genet. (1997); 99(4): 513-20. tion in exon 20 of the CFTR gene in a Bulgarian 2. Savov A., Angelicheva D., Jordanova A., cystic fibrosis patient. Hum Mol Genet. (1994); 3(3): Kremensky I., Kalaydjieva L. 2176insC: a novel muta- 513-4. tion in exon 13 of the cystic fibrosis gene. Hum 6. Angelicheva D., Boteva K., Jordanova A., Savov Hered.(1996);46(3): 166-7. A., Kufardjieva A., Tolun A., Telatar M., Akarsubasi A., 3. Savov A., Angelicheva D., Balassopoulou A., Koprubasi F., Aydogdu S., et al. Cystic fibrosis pa- Jordanova A., Noussia-Arvanitakis S., Kalaydjieva L. tients from the Black Sea region: the 1677delTA Double mutant alleles: are they rare? Hum Mol mutation. Hum Mutat. (1994); 3(4): 353-7 Genet. (1995); 4(7): 1169-71. 7. Savov A., Mercier B., Kalaydjieva L., Ferec C. 4. Varon R., Stuhrmann M., Macek M. Jr., Identification of six novel mutations in the CFTR Kufardjieva A., Angelicheva D., Magdorf K., Jordanova gene of patients from Bulgaria by screening the A., Savov A., Wahn U., Macek M., et al. Pancreatic twenty seven exons and exon/intron boundaries insufficiency and pulmonary disease in German and using DGGE and direct DNA sequencing.Hum Mol Slavic cystic fibrosis patients with the R347P muta- Genet (1994); 3(6): 1034.

NEUROPSYCHIATRIC GENETICS RESEARCH GROUP

Radka Kaneva, PhD; Veselin Chorbov, Mina Angelova, MSc; Momchil Nikolov, MSc - Laboratory of Molecular Pathology, Tel: + 359-2-9172473, Fax: +359-2-9172469; E-mail: [email protected] Vihra Milanova, MD, PhD; Vessela Stoyanova, MD, PhD; Rositza Vladimirova, MD; Spiridon Alexiev, MD; Nikolina Djurova, MD, MSc - Department of Psychiatry, Sofia Medical University

Affective disorders are severe and common currently applied in order to identify potential psychiatric diseases with two main clinical forms: susceptibility genes. Bipolar and Unipolar Affective Disorders (BPAD The group of neuropsychiatric genetics works and UPAD). BPAD is characterized by major de- in the field of genetics and aetiology of affective pressive episodes alternating with phases of mania disorders and schizophrenia in the last 12 years. It (bipolar I disorder) or hypomania (bipolar II disor- consists of psychiatrists from First Psychiatric Clinic, der). UPAD is characterized by recurrent major Alexander University Hospital and Department of depressive episodes alone. Affective disorders are Psychiatry, Medical University –Sofia and molecular highly prevalent and associated with severe social biologists from the Laboratory of Molecular Pa- disability and considerable mortality both with ma- thology. The founding of the team was encour- jor economic impact. aged by Prof. A. Jablensky and Prof. L. Kalaydjieva The etiology of mood disorders is multi- to study the genetic factors contributing to the factorial involving genetic as well as environ- aetiology of major psychiatric disorders in the Bul- mental factors. Traditional methods such as family, garian population. The research group participated twin and adoption studies first have provided in many national and international research projects. cumulative evidence implicating genetic factors The clinical team lead by Assoc. Prof. Dr. Milanova in the vulnerability for affective disorders. Using works in the field of biological psychiatry and clini- these methods however, it is difficult to delin- cal psychopharmacology – treatment of bipolar eate which genetic variables are involved as well affective disorder, depression, and personality disor- as the exact mode of transmission of the dis- ders. The psychiatrists have been trained in using ease. Linkage and Association methodologies are various research diagnostic interviews.

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The Fogarty Research Foundation supported interview of (Cloninger, 1992). Psychosocial evalua- in 1992-1994 a research project on Foetal Neural tion is done in addition in patients in remission and Development and the aetiology of Schizophrenia. healthy controls. The interviews were translated The work was further extended by a collaborative from English to Bulgarian and tested among a pilot project with University of Western Australia, funded group of healthy controls. The interview has been by the Stanley Foundation 1995-1996 research given to all newly recruited patients and the par- award: Morphometric and Neural Connectivity Char- ents and sibs of affected probands from the trios. acteristics of Fetal Brains at Genetic risk for Bipo- After informed consent, venous blood is col- lar Disorder. lected from all patients, family members and healthy The collection of families with multiple af- controls. The DNA bank at present contains more fected members, suitable for linkage analysis was than 500 samples of large families with affective initiated in 1992 and supported by a project, disorder, small probands/parents trios and case/ funded by the Ministry of Education and Sci- controls collection. ence of Bulgaria. In 1994 the research team Under the BIOMED and BIOMED2 projects was invited to join the ongoing European initia- the Bulgarian case/control sample was included in tive under the BIOMED1 program of the EU: a large European database, with more than 600 European Collaborative Study of Affective Dis- patients with Bipolar Affective disorder, 600 pa- orders: Interaction between Genetic and Psy- tients with Unipolar Affective disorder (recurrent chosocial Vulnerability Factors (1991-1994). The depression) and 1200 healthy controls. coordinator of the project Prof. J. Mendlewicz, Candidate genes were selected, coding for Erasmus Hospital, Free University of Brussels, Bel- receptors of the major neurotransmitters (GABRA1, gium, formed a research consortium of 13 part- GABRA3, GABRA5, DRD2, HT2C, HT2A); their trans- ners from 8 European countries. The Bulgarian porters (dopamine and serotonin transporters) or contribution was extended with our participa- key enzymes such as MAOA, NOS1, PLA2A, COMT. tion in the second European project under Genotyping of selected polymorphisms was com- BIOMED2 program: European Collaborative Study pleted and association analysis was performed for on Molecular Genetics in Affective Disorders all selected markers in candidate genes in our (Genes in Affective Disorder). sample. The results from the European studies The patients included in the project were were published in more than 15 articles in interna- recruited from two Bulgarian Social Services for tional journals. Mental Health in Sofia and the Psychiatric Clinic After the completion of the two European of Alexander University Hospital, Sofia. The Bul- collaborative projects the research team carried garian association sample consists of 150 patients on with strengthening and expanding the clinical with affective disorder (Bipolar and unipolar) and team, recruiting more families with affective dis- 150 controls matched accordingly by sex, age and order. A unique collection of families, clinically ethnicity. well studied and suitable for genetic linkage Standardized validated diagnostic interviews analysis was gathered, including 7 small families (SCAN, DIP) criteria are performed by trained psy- of Bulgarian origin and 3 extended multigenera- chiatrists for all patients giving diagnosis based on tional Roma families. DSM-IV. The operational checklist for mental disor- In case of complex disorders, a complete ders (OPCRIT) and Family Interview for Genetic genome scan is needed to estimate the number Study (FIGS) are used in addition. The phenotype of contributing genes, as well as the magnitude data is stored in computerized clinical databases. of the gene effect for which we have no prior The BIOMED projects focused on psychoso- knowledge. A complete systematic genome scan cial parameters relevant to the phenotype of af- in these families was performed in the Max . . fective disorders evaluated with reliable instruments: Delbruck Center for Molecular Medicine in Ber- Social adjustment and self-esteem self reported lin. Statistical analysis of the results from the scale and Temperament and Character Inventory genome scan was performed in collaboration with

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the group of Prof. Wienker at the Institute of A multidisciplinary team was formed, includ- Medical Biometry, Informatics and Epidemiology ing psychiatric, neurological and genetic units, to ful- (IMBIE), University of Bonn. fil the aims of the project. The genetic and psychi- The findings of the large linkage study of atric teams have already been in close collabora- bipolar affective disorder (BPAD) that involved tion in previous studies on affective disorder. A genome-wide analysis of 52 families of Spanish, Roma leading neurologist, well experienced in the genetics and Bulgarian descent, and further fine mapping of of neuromuscular disorder in Gypsies, Assoc. Prof. the 1p34-p36, 4q28-q31, and 6q15-q24 regions con- Ivailo Tournev formed a team of neurologists and firm regions 4q31 and 6q24 as BPAD susceptibility neuropsychologists for detailed clinical study of epi- loci and 1p35-p36 is proposed as a new putative lepsy. Assoc. Prof. Vihra Milanova leads the psychi- locus requiring confirmation in replication stud- atric team and Dr. R. Kaneva is the leader of the ies. The results should facilitate the search of po- genetic team, as well as a coordinator of the project. tential susceptibility genes for BPAD in the candi- International collaboration on the project involves date regions, performing systematically linkage dis- leading experts in epidemiology of epilepsy (Prof. J equilibrium (LD) mapping. W Sander, Department of Clinical and Experimental To continue the work on the Gypsies, a ge- Epilepsy, UCL Institute of Neurology); psychiatry (Prof. netically isolated founder population, we have been A. Jablensky, Centre for Clinical Research in Neu- supported by a Research Grant from the NHMRC, ropsychiatry) and genetics of Roma population (Prof. Australia (2005-2007). The objectives of the project L. Kalaydjieva, WAIMR and UWA Centre for Medi- include the collection of additional large Roma fami- cal Research, University of Western Australia). lies with BP, as well as 150 probands/parents trios to serve for replication sample. The first step of Selected publications of the research team verification the positive genome scan data using a 2003-2005 denser genetic map and linkage analysis in the .1 Johannes Schumacher, Radka Kaneva, Rami existing expanded families is underway. The replica- Abou Jamra, Guillermo Orozco Diaz, Stephanie tion in large BP families from other Gypsy sub- Ohlraun, Vihra Milanova, Young-Ae Lee, Fabio Rivas, isolates (groups) and identification of a candidate Fermin Mayoral, Robert Fuerst, Antonia Flaquer, Chris- region harbouring a shared founder mutation will tine Windemuth, Eudoxia Gay, Sebastian Sanz, Maria be followed by fine mapping and linkage disequilib- Jose Gonzalez, Susana Gil, Francisco Cabaleiro, Fran- rium (LD) analysis. cisco del Rio, Fermin Perez, Jesus Haro, Christian Kostov, In parallel the Research team received a Vesselin Chorbov, Amelia Nikolova-Hill, Vessela grant by the National Science Fund at the Minis- Stoyanova, George Onchev, Ivo Kremensky, Konstantin . . try of Education and Science of Bulgaria under a Strauch, Thomas G. Schulze, Peter Nurnberg, Wolfgang new National Research Program „Genomics”. The Gaebel, Ansgar Klimke, Georg Auburger, Thomas F. research project is a natural follow up of our Wienker, Luba Kalaydjieva, Peter Propping, Sven Cichon, work on genetics of affective disorders. In addi- Assen Jablensky, Marcella Rietschel, and Markus M. tion to the main stream of work on fine map- Nothen.. . Genomewide Scan and Fine-Mapping Link- ping and looking for genes, contributing to the age Studies in Four European Samples with Bipolar aetiology of BPAD, we focus on the elucidation Affective Disorder Suggest a New Susceptibility Lo- of the relationship between affective disorders cus on Chromosome 1p35-p36 and Provides Fur- and epilepsy. The unique finding of a subset in ther Evidence of Loci on Chromosome 4q31 and the Roma families with BPAD with multiple cases 6q24. AJHG 2005 Dec 77(6): 1102-1111. of epilepsy in several generations and comorbid 2. Massat I., Souery D., Del-Favero J., Nothen idiopathic epilepsy and affective disorder serves M., Blackwood D., Muir W., Kaneva R,. Serretti A., as a starting point in the project. Other Roma Lorenzi C., Rietschel M., Milanova V., Papadimitriou families with affective disorders and epilepsy have GN., Dikeos D., Van Broekhoven C., Mendlewicz J. been recruited during fieldwork among Roma Association between COMT (Val158Met) functional groups in Bulgaria. polymorphism and early onset in patients with ma-

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○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○ jor depressive disorder in a European multicenter transporter 5HTTLPR polymorphism and affective genetic association study. Mol Psychiatry. 2005 disorders: no evidence of association in a large Eu- Jun;10(6):598-605. ropean multicenter study. Eur J Hum Genet. 2004 3. Kaneva R., Chorbov V., Milanova., Kostov C., May 12 (5): 377-82. Nickolov K., Chakarova C., Stoyanova V., Nikolova-Hill 6. Papadimitriou GN., Dikeos DG., Souery D., Del- A., Krastev S., Onchev G., Kremensky I., Kalaydjieva L., Favero J., Massat I., Avramopoulos D., Blairy S., Cichon Jablensky A. Linkage analysis in bipolar pedigrees S., Ivezic S., Kaneva R., Karadima G., Lilli R., Milanova V., adds support for a susceptibility locus on 21q22. Nothen M., Oruc L,. Rietschel M., Serretti A., Van Psychiatr Genet –2004 Jun;14(2):101-106. Broeckhoven C., Stefanis CN., Mendlewicz J. Genetic 4. Blairy S., Linotte S., Souery D., Papadimitriou association between the phospholipase A2 gene and GN,. Dikeos D., Lerer B, Kaneva R., Milanova V., unipolar affective disorder: a multicentre case-control Serretti A., Macciardi F., Mendlewicz J. Social adjust- study. Psychiatr Genet. 2003 Dec; 13(4):211-20. ment and self-esteem of bipolar patients: a multi- Oswald P., Souery D., Massat I., Del-Favero J., centric study. J Affect Disord. 2004 Apr;79(1-3):97- Linotte S., Papadimitriou G., Dikeos D., Kaneva R., 103. Milanova V., Oruc L., Ivezic S,. Serretti A,. Lilli R., Van 5. Mendlewicz J., Massat I., Souery D., Del-Favero Broeckhoven C., Mendlewicz J. Lack of association J., Oruc L., Nothen MM., Blackwood D., Muir W., between the 5HT2A receptor polymorphism (T102C) Battersby S., Lerer B., Segman RH., Kaneva R., Serretti and unipolar affective disorder in a multicentric A., Lilli R., Lorenzi C., Jakovljevic M., Ivezic S., Rietschel European study. Eur Neuropsychopharmacol. 2003 M., Milanova V., Van Broeckhoven C. Serotonin Oct;13(5):365-8.

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MADE IN BULGARIA WITH EUROPEAN SUPPORT

SELECTIVE SCREENING FOR DIAGNOSTICS OF INHERITED METABOLIC DISEASES IN BULGARIA

Maria Ivanova, MSc; Ivanka Sinigerska, MSc; Irfet Hasanova and Assoc. Prof. Ivo Kremensky, MD, PhD Laboratory of Molecular Pathology, Medical University, Sofia 1431, BULGARIA

Laboratory of Molecular Pathology offers ing program is the clinical symptoms and signs modern and widely applicable genetic services for constellation. To achieve this, the material for diagnosis and prophylactics of inherited metabolic analysis must be accompanied by a detailed clini- diseases (IMD). The highly effective methods per- cal and laboratory information. In case that a formed in the Laboratory of Molecular Pathology carrier status determination and prenatal diag- afford opportunities for reliable specialized genetic nosis are desired, it is mandatory that besides the analyses for other Balkan countries, which need detailed clinical, genealogical and laboratory in- genetic service. formation, a definitive clinical diagnosis is required The Laboratory of Molecular Pathology together with DNA samples from sufficient num- provides services for 1 specialised clinic, 5 medico– ber of family members. genetic counselling and more than 70 paediatric and 119 neonatal units in Bulgaria. The laboratory Analytical Approach and Methods performs: 1) Mass neonatal screening for PKU (up It is typical for the Bulgarian selective screen- to now 1 700 000 newborns were screened, 86 ing program that a comprehensive range of ana- patients with classical PKU were detected); 2) Se- lytical methods and techniques is used. lective biochemical screening for over 70 inborn The analytical programme includes three–stage disorders of metabolism – up to now 611 of 8592 systematic approach. Semiquantitative methods patients were precisely diagnosed; 3) Prenatal meta- (Thin–layer liquid chromatography–TLC, two–dimen- bolic and enzymatic diagnosis (up to now 43 pre- sional thin–layer liquid chromatography – 2DTLC) natal diagnoses for different metabolic disorders for urine metabolites assay are in use at the first were performed). stage. The second stage includes quantitative (high– performance liquid chromatography – HPLC, gas SELECTIVE SCREENING PROGRAM chromatography – mass spectrometry – GC/MS) In the Laboratory of Molecular Pathology methods and the third stage explores methods for about 400 patients are tested annually by selec- enzymatic activities assay in biological fluids and tis- tive screening program. sues (see Figure 1). Tandem mass spectrometry will It is obvious that the mass neonatal screen- be introduced into analytical approach soon. ing approach could ensure diagnostics and pro- All the methods and results obtained by them phylaxis in a restricted proportion of diseases (in are under strict internal and external laboratory Bulgaria – 2, in Europe – about 20). For all the control. remaining disorders, an alternative approach of The laboratory participates in European ex- selective screening is applicable. The basic crite- ternal quality control – ERNDIM Diagnostic Profi- rion for selection in a Bulgarian selective screen- ciency Testing.

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During the period of 25 years, 8592 pa- sonably good and completely comparable to the tients at high risk were included in the selective data of the leading European centres. screening program. In total 611 patients (7.1%) Significant progress in the diagnostics of in- were diagnosed in the basic groups of inherited herited metabolic disorders in Bulgaria has been metabolic diseases: Aminoacid disorders – 44; made since 1990. This is associated with the pro- Urea cycle defects–13; Organic acidurias –53; gressive introduction of DNA analysis. Thanks to Fatty acid oxidation disorders – 12; Peroxisomal DNA analysis it became possible to confirm the disorders –9; Disorders of the renal transport diagnoses of MCAD (A985G), galactokinase defi- – 61; Lactic acidurias – 5; Carbohydrate disor- ciency (P28T) and phenylketonuria – most com- ders–131; Glycoprotein degradation – 7; mon mutations in Bulgarian population. Mucopolysaccharidoses – 54; Ganglioside disor- PC network and specialised software are in ders –150. Our results can be assessed as rea- use for the selective screening program.

DETAILED CLINICAL PICTURE

PKU card URINE BLOOD

Galact ose Glucose AA-HPLC MCAD Su g ar s VLCFA-GC/ MS Ketones Ph en yl k et o n es Ketoacids Aminoacids MMA HPLC Su l f it e s

Aminoacids- TLC Oligosaccharides Suga r s- TLC MPS

ORGANIC ACIDS GC/ M S

EN ZYM ES

DIAGNOSIS OF INHERITED DISEASE

REGISTER – DNA ANALYSIS – DNA BANK

Figure 1. Analytical approach to diagnostics of inherited metabolic diseases – selective metabolic screening: All steps are chosen depending on the clinical picture and results from the previous steps of the analysis.

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DNA ANALYSIS FOR SOME OF THE NEUROMUSCULAR DISORDERS IN BULGARIA

Albena Todorova, PhD; Bilyana Georgieva, PhD; Tihomir Todorov - Laboratory of Molecular Pathology, University Hospital of Obstetrics and Gynecology, 2, Zdrave Str., Sofia 1431, Bulgaria

Application area: Diagnostics and prophylaxis, pre- This method permits deletions detection along natal diagnosis and carrier status determination for the whole gene sequence, as well as detection of some of the most severe, incurable neuromuscular duplications. Moreover, it is also possible to ana- disorders were successfully introduced in Bulgaria. lyze females and directly to determine their car- Duchenne/Becker muscular dystrophy rier status. (DMD/BMD) [OMIM # 310200; 300376] is the This modern diagnostic approach was intro- most common inherited neuromuscular disorder, duced in Bulgaria after the specialization of Dr. A. affecting 1:3500 live male births. The DNA diag- Todorova, as a fellow of the Alexander von nostics of this severe, progressive muscular dys- Humboldt Foundation in the Institute of Human . . trophy with onset early in childhood, invalidization Genetics, Munster (Dr. N. Bogdanova, prof. B. and death around the second decade of life, Dworniczak and prof. J. Horst). Very useful and was introduced in Bulgaria in 1990. It became profound collaboration was established after this possible after the enormous efforts of several specialization between both institutes. A number neurologists in the country, who performed pre- of Bulgarian patients and families benefit from cise clinical research and described in detail the this cooperation. clinical findings in hundreds of patients, affected Thanks to the above–mentioned professional by neuromuscular disorders. A group of patients trainings and the subsequently established collabo- with clinical diagnosis susceptible with DMD/BMD rations, nowadays in Bulgaria we can offer to all was selected and subjected to DNA analysis. Dr. DMD/BMD families the most recent and modern J. Bronzova introduced first trials for genetic diagnostic approaches for DNA tests. tests in Bulgaria thanks to a very fruitful col- Up to now 259 DMD/BMD families were laboration with the Sylvius laboratory, Leiden registered in our database. The performed screen- University Medical Center. ing for mutations in our patients revealed about Afterwards, several novel and modern meth- 70% deletions of one or more exons. Recently, 6 ods for DNA analysis of DMD/BMD were devel- duplications (dupl. 48–50, dupl. 8–11, dupl. 2–33, oped and introduced for routine analysis in our dupl. 13–40, dupl. 2–10 and dupl. 8–13) were also country by Dr. A. Todorova. This became possible found by MLPA (Figure 1). Moreover, 3 point muta- after the establishment of a very profound col- tions (exon 16 c.1975G>T, p. Glu659X; exon 41 laboration with the Laboratory of Human Genetics, c.6053delC and exon 54 c.7961A>G, p. Asp2654Gly) University of Padua (prof. G.A.Danieli) and with the and a small deletion of 16 bp in exon 44 were also Neuromuscular Center, University of Padua (Dr. described in our sample. Marina Fanin, prof. C. Angelini). Long–term special- Subsequently, the genetic analysis of some izations were performed in these leading laborato- forms of Limb–girdle muscular dystrophies ries in the field of diagnostics and prophylaxis of (LGMDs) became possible in Bulgaria. Several Eu- neuromuscular disorders. ropean studies showed that in Gypsy population Recently, the newest method for DNA analy- LGMD type 2C [OMIM # 253700] has been caused sis of the whole dystrophin gene, named multi- by a single mutation in the g–sarcoglycan gene, plex ligation–dependent probe amplifica–tion p.C283Y. An extensive field work in the Gypsy (MLPA) method, [www.mlpa.com] was applied also areas was performed in our country by Dr. I. to a number of DMD/BMD Bul–garian patients. Tournev and his team to search and clinically ex-

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V., Kremensky I. (2002) Screening for C283Y Muscular Dystrophy Type 1B. J Med Genet 40(10):115. Gamma–sarcoglycan Mutation in High Risk Group 14. Georgieva B., Todorova A., Tournev I., Mitev of Bulgarian Gypsies: Evidence for Geographical Lo- V., Kremensky I. (2004) C283Y Gamma– calization and Non–random Distribution among Gypsy sarcoglycan Gene Mutation in the Bulgarian Roma Subgroups. Community Genetics 5:217–2. (Gypsy) Population: Prevalence Study and Carrier 11. Todorova A., Constantinova D., Kremensky I. Screening in a High Risk Community. Clinical Ge- (2003) Dilated Cardiomyopathy and New 16bp netics 66(5): 467–472. Deletion in Exon 44 of the Dystrophin Gene: The 15. Todorova A., Kress W., Mueller C.R. 2005 Possible Role of Repeated Motifs in Mutation Gen- Novel Mutations in the Calpain 3 Gene in Ger- eration. Am J Med Genet 120A(1): 5–7. many. Clinical Genetics 67:356–358. 12. Kremensky I., Todorova A., Jordanova A., 16. Georgieva B., Todorova A., Tournev I., Plageras Savov A., Tournev I., Iankova S., Georgieva B., P., Mitev V., Kremensky I. (2005) 550delA Mutation Zaharova B., Kaneva R., Petkova R., Andonova S., in the Calpain 3 (CAPN3) Gene: DMD/BMD, SMA Ivanova M., Ivanova N., Rainova R., Kalaydjieva L. or LGMD2A – Clinically Misdiagnosed Cases. Am J (2003) Spectrum of Mutations of the Most Com- Med Genet 136A: 399–400. mon Genetic Disorders in Bulgaria. Journal for 17. Todorova A., Halliger–Keller B., Kress W., Quality of Life Research 1(2): 172–185. Tournev I., Dabauvalle MC., Plageras P., Kremensky I., 13. Todorova A., Halliger–Keller B., Walter M.C., Mueller CR (2006) Mutation Spectra of Lamin A/ Dabauvalle M–Ch., Lochmueller H., Mueller C.R. C (LMNA) Gene: Single Gene–multiple Disease. (2003) A Synonymous Codon Change in the LMNA Chapter in the book „Progress in Medical Genetics“, Gene Alters mRNA Splicing and Causes Limb–girdle Nova Science Publishers, Inc., in press.

Figure 1. MLPA analysis of the dystrophin gene. The duplication of exons 48-50 is presented in comparison to the control sample. Arrows point the duplicated exons.

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Figure 2. Geographic distribution of the mutation p.C283Y in the Bulgarian Gypsy population. Underlined numbers give the registered LGMD2C Gypsy patients per region. The percents provide the detected p.C283Y carrier frequency in each region. Arrows give the Gypsy influx on the Balkans.

Missense Ar g 118 Gl y Gly333Asp Arg49His Arg169Gly Asp753Asn

Nonsense

Glu323St op ( 3)

Large deletion exons 2- 8

Deletions 18 11- 12del TC 19 8 1- 8 4 del ATAG

Ex o ns

NS

NS I II IS1 III IS2 IV Protein domains

Figure 3. CAPN3 gene mutations detected in the Bulgarian sample of LGMD2A patients. The number of patients carrying the same mutation is given in parenthesis.

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METHODS AND TECHNOLOGIES FOR MANUFACTURE OF ENZYMES – ALPHA-AMYLASE, XYLANASE, CELLULASE

Contact Address: Mr. Vladislav Jivkov Applied Research and Communications Fund (ARC Fund), 5, Alexander Zhendov Str., 1113 Sofia, Bulgaria Phone:+359 2 986 7557, Fax:+359 2 9801833, Email: [email protected]

Application Area: Food – Agro Industry. logical problems were met during the production The technologies offered by the Bulgarian tests in Bulgaria. research institute are for the manufacture of 3 Innovative aspects: Innovative strains put in enzymes – alpha-amylase, xylanase and cellu- specific defined conditions, Content of the medium lase. Three different strains are developed, but used in the manufacturing process. technological equipment is the same. During the Main advantages: technological process these strains are put in Cheap source materials proper for the different specific conditions defined for each of European region are used: Alpha-amylase – par- them. Different source materials are used. The tially hydrolysed farina made of corn, potatoes or proposed technologies are not of the GMO type. rice; Xylanase - corn-cobs; Cellulase – wheat straw They are already realized in industrial conditions which leads to reduced price of the end products. in Bulgarian factories and prove to work. The About 50% of the price of the end prod- enzyme production can be set in different levels uct is formed by the source material. of purification of the end products. The enzyme These technologies use standard and al- products correspond to the European quality and ready existing equipment for the manufacture of safety standards. The equipment used is stan- bioproducts. dard for production of bioproducts. No techno- No waste is generated.

GRANULATED APPLE AND CITRUS PECTINS

Laboratory of Biologically Active Substances - Plovdiv 95, „Vasil Aprilov“ Blvd., 4002 Plovdiv Phone: +359 32/45 21 40, 32/44 19 29, Fax: 032/44 01 02 e-mail : [email protected]

Application area: Food industry, Human health. pressings.began long time ago. The pectin actively The pectic substances are acid polysaccha- affects some physiological processes in the hu- rides. They are well dissolved in water and give man constitution. It participates in metabolism of viscous solutions. They are widely applied in food lipids, bile acids and carbohydrates. It is recom- industry as hydrocolloids because of their gelling, mended for prophylaxis and treatment of ulcer emulsifying and stabilizing properties. Pectic sub- and gastritis, cardiovascular disease, binding and stances are found in all fruits and vegetables. They excreting of heavy metals and radioactive iso- are the most important soluble dietary fibres in topes. Pectin activates the immune system of a daily food of a man. Biological activity of pectic human constitution. substances increases when they are isolated from Combined accepting of granulated apple the plant raw material. For this reason industrial pectin with Aronia syrup – a unique product - production of pectin from apple and citrus PEKTARON, quite recently produced by the

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Laboratory’s manufacturing unit – strengthens excreting of heavy metals and radioactive isotopes. favourable biological effect of pectic substances. The product has immune stimulating effect and High content of stable anthocyanins in the berries keeps human health in good condition. of Aronia melanocarpa contributes to binding and

PHYTEX – NEW SOLUTIONS FOR EXTRACTION OF BOTANICALS

Prof. Lyubomir Boyadzhiev, DSc, Corr. Mem. Institute of Chemical Engineering, Acad. G. Bonchev Str., Bl. 103, 1113 Sofia, Bulgaria

Application area: Pharmaceutical, cosmetic, and food PHYTEX extraction systems are versatile, mul- industries. tipurpose equipment; they can operate as continu- World consumption of herb and spice ex- ous differential extractors (percolators) or in a tracts is continuously growing. Nowadays, more than multi-cycle cross flow regime, both offering com- two-thirds of the pharmaceuticals sold in the world plete extraction of the desired component(s). The market are completely or partially derived from extractors can be easily transformed into water- various plants. Considering this fact and the accu- steam distillation devices, suitable for production of mulated for more than 30 years experience in the natural aromas or essential oils. They can perform, field of extraction processes, the Institute of Chemical in a consecutive order, both processes with the same Engineering of Bulgarian Academy of Sciences equipment. launched an intensive research program oriented PHYTEX extractors operate at atmospheric towards development of efficient extraction pro- or reduced pressure. The latter mode is applied in cesses and value-added products. As a result PHYTEX case of thermo-sensitive products. Choosing the technology was born. appropriate vacuum level, one can remove most of PHYTEX extraction technology is a multi- the solvents residue from the enriched extract, op- purpose system, designed for extraction of vari- erating in the range of 30 to 50° C. ous pharmaceutical substances, flavors, colorants Using PHYTEX equipment, a large number of and other valuable components from wild or cul- extracts, oleoresins and essential oils are produced tivated botanicals. After the extraction process is from common or less known botanicals as: completed, retained solvent is removed from the Acacia, basil, bearberry, black elder, buckthorn, exhausted solid mass. Recovered solvent is re- caraway, celery, chamomile, common balm, dandelion, used in the process. The exhausted vegetable deadly nightshade, dill weed, eyebright, fennel, garlic, mass can be deposited with no harm for the geranium, ginger, hawthorn, hops, hemlock, horse- environment or even directly used for feeding chestnut, horse-radish, hyssop, juniper berry, marigold, the farm livestock. PHYTEX technology can be mustard, motherwort, nettle, nutmeg, parsley, pepper easily adopted as a water-steam distillation sys- mint, plantain, red paprika, periwinkle, puncture vine, tem, suitable for production of natural aromas or oregano, osier, savory, St. John’s wort, turmeric, vale- essential oils. rian, wild lettuce, wild thyme, etc.

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TRANSPLANTATION OF ANIMAL EMBRYOS IN BULGARIA

Assoc. Prof. D. Kacheva, PhD Institute of Biology and Immunology of Reproduction „Acad. K. Bratanov“ 1113 Sofia, 73 Tsarigradsko shosse Blvd.

Application areas: Reproduction biotechnologies in the village of R. Konare. During 1982 the first in animals. calf was born from transplantation. During the The investigations in the field of obtaining, same year the transfer of frozen cattle embryos storage and transplantation of preimplantation from which the calves transplants were born was embryos and manipulations with ova and embryos realized for the first time. Frozen calf embryos in vitro are part of the cellular and genetic engi- were transported from Czechoslovakia within the neering. These investigations in the reproductive bio- frames of mutual scientific collaboration with sci- technologies started at the beginning of the 80’s entists from the Institute of Physiology and Genet- of the last century. In 1974 initial studying started ics of Animals of the Czech Academy of Sciences. and already existed researches were enlarged on Successful experimental export of Bulgarian the initiative of the CMEA (Council for Mutual frozen sheep embryos was materialized in the In- Economic Assistance) in some scientific institutes of stitute of Genetics and Breeding of Animals at the all ex–socialist countries. According to Academician Polish Academy of Sciences, as well as in the Agrar- Bratanov’s proposal the preparations started for ian Faculty of the University in Zagreb, where the organizing of the staff in the Institute of Biology first lambs from frozen embryos in ex–Yugoslavia and Immunology of Reproduction for the work in were born. The successful interbred transfer of this field. In 1977 Assoc. Prof. Dr K. Vlahov, PhD; embryos from threatened with extinction ancient Assoc. Prof. Dr D. Kacheva, PhD (1978), later Assoc. native Bulgarian animal breeds were realized dur- Prof. Dr M. Petrov, PhD, research associate Dr Hr. ing next years. Experimental cryo bank was also Karaivanov, PhD (1980), Assoc. Prof. Dr E. Sapundjiev, created for frozen embryos from aborigine na- PhD were included in the membership of the sec- tional breeds of sheep, goats and cattle to store tion. The first experiments for the transplantation their genetic fund. of sheep and cow embryos were prepared and We used the embryo transfer also to obtain implemented with financial support of the Agricul- clinically healthy serum negative generation from tural Academy and personally of its President Acad. sheep, which were ill from viral pulmonary adeno- Tz. Hinkovski. In July 1979 in the agricultural experi- carcinoma through interbreed transfer of embryos. mental station of the Institute of Biology and Im- Experimental laboratory model was developed on munology of Reproduction – Sofia together with pulmonary adenocarcinoma in mice and the gen- scientists from Poland transplantation of fresh sheep esis of cancer of this zooanthroponosis was investi- embryos and deep frozen sheep embryos was re- gated. alized. The embryos were imported from Poland. In Experimentally, after transfer of allogen em- December lambs were born from fresh and fro- bryos between inbred mice which have difference zen embryos. These were the first animals–trans- of H–2 locus Major Histocompatibility Complex / plants that were born in our country. During the MHC / the immunity reaction of the recipients same year the team realized successful transplan- was modified and accelerated rejection of the vi- tation of sheep embryos in the Institute of Upland ral leukoma tumor was achieved, and vice versa, Stockbreeding and Agriculture (IUSA), Troyan, Bul- increase of metilholanthrene sarcoma growth was garia. By this action they started a long useful reached. collaboration, almost a quarter of a century, be- During all these years there was scientific tween IBIR and IUSA. Successful transplantation of improvement of the elements of the embryo trans- cow embryos was realized in productive conditions fer technology in relation to induction of superovu-

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EQUAL IN EUROPEAN RESEARCH AREA

BULGARIAN VIPs:

Acad. ATANAS ATANASSOV, DSc

Governors of the International Center of Genetic Engineering and Biotechnology (I.C.G.E.B.), Trieste, Italy; Member of Steering Committee of UNEP Multi–year programme for Capacity Building and Regional Co–operation on Safety in Biotechnol- ogy in Central and Eastern Europe (CEE) (1995) and a FACE Program of EC (1995); Chairman of Steering Committee on Biosafety for CEE (2002 – 2004); Executive Secretary of Bulgarian Coun- cil for Biosafety Use of Genetically Modified Higher Plants, Ministry of Agriculture and Forestry (1996 – 2005); National coordinator for Plant Biotech- nology in Bulgaria. Coordinator of Plant Biotech- nology for Balkan countries (1995); Expert to FAO’s Plant Biotechnology Program, Rome, Italy and IAEA, Vienna, Austria; National Correspondent of the In- Dr. Biology (1977); DSc. Agronomy (1989); ternational Plant Tissue Culture Association, Euro- Corresponding member (1997) and member (2003) pean Association for Research and Plant Breeding of the Bulgarian Academy of Sciences; Member – EUCARPIA, International Society for Molecular of Agricultural Academy of the Ukraine (1993) Plant–Microbe Interaction (IS–MPMI), Japan Soci- and Agricultural Academy of Russia (2003). Doc- ety of Plant Physiology, ESNA – European Society tor Honoris Causa of the University of Agricul- for New Methods in Agricultural Research; Chair- tural Sciences and Veterinary Medicine, Kluj–Napoca, man of the National Committee of Bioethics, Min- Romania (2001). Director of the Central Labora- istry of Education and Sciences, Bulgaria (2002); tory of Genetic Engineering (since 1985), renamed Chairman of Bulgarian Association of Seed and Institute of Genetic Engineering (1989) and Planting Material (since 2001); President of AgroBioInstitute (2000) – Center of Excellence AgroBioTech Park (since 2003); President and in Plant Biotechnology (2000), National Center for Chairman of the Board of Directors of the Black Agricultural Sciences, Sofia, Bulgaria; Member of Sea Biotechnology Association (since 2004). the Board of Governors of Agricultural Academy; Acad. A. Atanassov specializes in tissue cul- Professor and Director of the Bulgarian part of ture at the Institute of Plant Physiology, Moscow, the International Center on Plant Science, De USSR (1969, supervisor: R. G. Butenko, one of the Montford University, Leicester, UK (1994); National most famous scientists in the field of plant cell and coordinator and a representative of the Bulgarian tissue cultures). He passed training on pollen and Affiliated Center and Member of the Board of protoplasts regeneration (1975) in the laboratories

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Professor ANGEL SIMEONOV GALABOV, MD, DSc, Corr. Mem.

Born in 1939 in Sredetz, Stara Zagora district, Bulgaria. Graduated in Medicine at the Medical University of Sofia (1962). Defended his PhD thesis in Biology (1968) [Study of the Inhibiting Activity of N’,N’–anhydrobis (b–hydroxyethyl) on the Repli- cation of Paramyxovirus parainfluenzae 1 (Sendai)] and his DSc in Virology (1978) [N, N’–Disubstituted Thiourea and Abitylguanid – Specific Viral Inhibi- tors]. Academic ranks: Associate Professor (1974), Professor (1988). Postdoctoral Fellowship at the Institut Pasteur, Paris with the Nobel Prize winner Andre Lvoff (1968/69) and with Acad. Vadim Agol (Moscow State University) (1975). antialphavirus substance, described in scientific lit- Angel S. Galabov is currently the most distin- erature; guished virology scientist in Bulgaria and is widely benzodioxan derivatives (BMM–8, BMM– acclaimed worldwide. His work and publications 10) – alpha– and flavivirus manifesting a protec- comprise 162 scientific studies, including a mono- tive in vivo effect; graph and a monograph section (published by Aca- aporphinoidal alkaloid oxoglaucine– with a demic Press). 95 of his works have been published pronounced inhibitory effect upon enteroviruses in renowned international editions. He has been replication. cited in over 550 references. These researches were successful due to the Prof. Galabov’s main scientific contributions methodology developed by Angel S. Galabov – in are connected with antiviral substances – experi- creating new methods and testing–patterns of an- mental chemotherapy of viral infections and bio- tiviral substances, including a screening system for logical response modifiers. viral replication inhibitors. In close collaboration with almost all leading Prof. Galabov reveals the molecular mecha- groups of Bulgarian chemists–synthetics and chemis- nism of antiviral activity of the most perspective try specialists on chemistry of natural substances, An- new antivirals proved by him and determines the gel Galabov has discovered and identified a number target of the virus inhibitors–ligands of viral pro- of highly active viral replication inhibitors. These include: teins: for thiourea PTU–23 – the protein in en- N,N’–disubstituted thiourea, active against teroviruses regulating the replication cycle, for enteroviruses and other picornaviruses, among which tetrahydro–2 [1H]pyrimidinone mopyridone – the the compound PTU–23 is considered prominent matrix protein M1 of influenza A virus, for biguanide with its antienteroviral effect and in vivo activity abityilguanide – a capsid protein of adenoviruses. and is regarded as a „classic“ enteroviral inhibitor; In his investigations together with chemists derivatives of tetrahydro–2[1H]pyrimidinone from Bulgaria and the Ukraine, Prof. Galabov and other cyclic urea, effective against influenza, succeeded in clarifying the chemical structure– toga– and flaviviruses. The most effective among antiviral activity relationship in active classes of them is mopyridone, which is highly active in experi- compounds (N,N’–disubstituted thioureas, mental infections with influenza A and B viruses tetrahydro–2[1H]pyrimidinones), which serve as and has a synergistic effect in combination with a base for directed synthesis of new highly ac- rimantadine. Its effect upon alphaviruses (which tive compounds. cause several diseases spread by mosquitoes in the Angel S. Galabov has been working in the tropics) determines it as the most effective field of drug–resistance, stressing mainly on en-

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for antiviral activity and virucidal activity by em- nizer and first President (1999–2001). Currently ploying the screening systems developed by him. he is a member of the Board of BSM and Chair- Angel S. Galabov has read more than 140 man of the Programme Committee of BSM. Angel reports at international scientific forums, 24 of them S. Galabov is initiator of the most significant activity being plenary or following an invitation. He has of BSM – the Microbiologia Balkanica forums, which presided at meetings and sections at 26 interna- take place every two years and have great success tional scientific events. He was invited to conduct in integrating the efforts of all microbiology scien- seminars in 17 foreign universities and scientific in- tists in Southeastern Europe. He had two mandates stitutes. as a Member of the International Research Coun- Prof. Galabov is a member of the interna- cil of the International Society for Antiviral Re- tional editorial boards of the following scientific search, ISAR (1992–96) and won high approval for journals: „Acta virologica“ (the first European spe- his work, including his effort in incorporating in ISAR cialized journal on virology), „Future Microbiology“, scientists from Central and Eastern Europe. He „Problems in Russian Virology“, „Balkan Journal of was also a member of the Permanent Organiza- Medical Genetics“, „Interferon & Biotechnology“, two tional Committee of the International Antiviral Sym- foreign national scientific journals, and the Bulgar- posia; he is also a member of six other interna- ian „Biotechnology and Biotechnological Equipment“ tional scientific organizations. and „Bulgarian Medicine“. An active member of the Union of Scientists In his area of research he has been supervi- of Bulgaria, since 2001 Prof. Galabov is the Presi- sor of 19 PhD students, 11 of them having de- dent of the Bulgarian Society for Microbiology and fended their theses, and of a number of other a member of the Board of the Union of Scientists young specialists from Bulgaria and abroad. More of Bulgaria. He represents Bulgaria in the interna- that 10 PhD theses of specialists from other Bul- tional scientific organizations FEMS (Federation of garian and foreign scientific groups are based on European Microbiological Societies) and IUMS (In- his research. ternational Union of Microbiological Societies). He is Angel S. Galabov has founded two laborato- an organizer of many scientific activities in microbi- ries specialized in antiviral substances – at the Medi- ology and virology in Bulgaria, the most important cal Academy, Sofia, and at the Institute of Microbi- and perfectly organized being the 10th Congress ology (BAS). The importance of these laboratories of Microbiologists in Bulgaria (2002) and „Virology is highly appreciated by scientists all over the world Days in Bulgaria“ (2004). Angel S. Galabov is one and they are acknowledged as leading centers for of the co–founders of the Bulgarian National Acad- research of antiviral substances in Central and East- emy of Medicine (1995) and a member of its Aca- ern Europe, and one of the most significant centres demic Board. in Europe in general. He has been a long–standing member of This activity of vast scope and high quality the Specialized Scientific Council of Virology and characterizes the creation of a Bulgarian school actually of Microbiology, and Virology and Immu- connected with the research of antiviral substances. nology with the High Attestation Commission, a Angel S. Galabov has been a longstanding member of scientific boards of a number of insti- expert on viral infections (since 1983) of the World tutes, member of the expert council at the Ministry Health Organization. of Health in Bulgaria, President of the National His scientific research correlates with a wide Sub–committee for poliomyelitis eradication, and of range of activities, which involve initiating and orga- the Committee of Biological Defense, etc. nizing scientific forums and working in scientific He has been involved to a great extent as a organizations. He initiated and actively took part in reviewer in scientific boards of the High Attesta- the functioning of the first symposium chain on tion Commission, as an expert in the Bulgarian Na- antiviral substances in Europe (since 1974). He also tional Fund for Scientific Research, etc. initiated the foundation of the Balkan Society for Prof. Galabov is a member of the General Microbiology (BSM) in 1998 and was its main orga- Assembly of the Bulgarian Academy of Sciences

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(BAS) since its foundation in 1993 and member of are organized at the Institut Pasteur Atelier in the Council for National Security and Safety of the Institute of Microbiology. the Executive Council of BAS, and other expert Angel S. Galabov has a long experience as a committees in BAS. teacher in virology: for post–graduate qualification Since 1995 he is the Director (third man- of students in the former Institute for Postgradu- date since 2003) of the Institute of Microbiology ate Training of Physicians and the Medical Academy, (BAS). He has contributed to a very great extent at the Faculty of Veterinary Medicine of the For- to the institute’s recognition as a national re- estry and Technology University in Sofia (founder of search centre in microbiology and as a compe- the Department of Microbiology and endorsing vi- tent scientific institution in Southeastern Europe. rology as a separate discipline, creator of the teach- On his initiative the Institute of Microbiology was ing syllabus and of the virology course), at the Fac- associated to the Institut Pasteur, Paris ulty of Biology at Sofia University as a teacher in (26.06.2004). The institute became the eighth specialized courses in medical virology and antiviral European institute in this family of associated agents (master degree courses and as diploma works members. The Institute of Microbiology of the supervisor), as initiator and founder of the first course Bulgarian Academy of Sciences has thus attained of lectures in virology for students of medicine (cur- high recognition for its level of scientific research rently an optional course) in the Medical Faculty of and intensive activity. Angel S. Galabov has be- the Medical University, Sofia, lecturer of pre–qualifi- come a regular member of the Board of Direc- cation courses for pharmaceutics at the Faculty of tors of the Institut Pasteur and associated insti- Pharmaceutics of the Medical University, Sofia. tutes. Meanwhile he was also chosen as coordi- Prof. Galabov is also famous for his active nator for one of the research directions of the work connected with popularizing scientific knowl- institutes in Europe. edge via printed works and electronic media (ap- In the crucial environment of the recent proximately 250 interviews and publications, including economic realities Prof. Galabov’s aim was to in- a book and a radio lecture course) – on the newest corporate the Institute of Microbiology in the in- achievements in the field of virology, on current viral ternational market of scientific products – by infections and how to fight them. He is also involved strictly observing project financing as a base for in popularizing the achievements of his institute and scientific and research work, by developing inter- of the Bulgarian Academy of Sciences. national scientific cooperation, and the Institute In 2004 Angel S. Galabov was elected a Cor- has been rated as being among the most active responding Member of the Bulgarian Academy of regarding programmes of the EU and NATO (an Sciences. award given to Prof. Galabov by the Ministry of In 2005 the Romanian Academy of Medical Education and Science). The increasing number of Sciences elected him a „Member of Honour“ for publications in international scientific journals should his contribution as a scientist in virology and for his also be mentioned, as well as the excellent per- achievements related to scientific cooperation in sonnel policy with special attention paid to young the field of microbiology in the region of South- scientists, the increase in the Institute’s revenue eastern Europe. (first place among the biological institutes of BAS), The Russian Academy of Medical Sciences and the developing teaching activity. A very suc- awarded him with the Medal „D. I. Ivanovsky“ for cessful tendency is the international courses for his contribution in the development of virology and young specialists from Southeastern Europe, which international cooperation in this scientific field.

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Associate Professor IVO KREMENSKY, MD, PhD

Assoc. Prof. Ivo Kremensky is one of the most Kremensky is internationally recognized in the field eminent Bulgarian scientists in the field of Biochem- of inborn errors of metabolism, genetics of neuro- istry and Molecular Genetics of Human Diseases. As muscular and psychiatric disorders and population an expert in genetic screening programs, he has com- genetics. The Medical Faculty, Sofia Medical Univer- prehensive overview on the hereditary pathology in sity, awarded Assoc. Prof. Kremensky the „Acad. Dr. the Bulgarian population, and knowledge of the health D. Orahovatz“ prize for research excellence in 2001. care infrastructure of the country. He is well recog- Over the past 10 years Assoc. Prof. Kremensky nized in all aspects of organization and implementa- published more than 130 papers in international tion of large–scale diagnostics and prevention pro- peer reviewed journals, including Nature Genetics grams based on genetic analysis. Assoc. Prof. and the American Journal of Human Genetics. He Kremensky is actively involved in the legislation and has a long teaching carrier at Sofia Medical Uni- policy–making process in the field of healthcare in versity and is involved in the undergraduate and Bulgaria and other issues, related to genetic testing. postgraduate medical and biology education with Assoc. Prof. Kremensky has made major con- lectures and tutorials in molecular pathology. Assoc. tributions to the introduction of two national mass Prof. Kremensky is the author of the first full– screening programs of newborns for phenylketo- time course in Molecular Medicine at Sofia Medical nuria and galactosemia (since 1977), to Biochemical University. He has supervised more than 24 MSc screening for Down syndrome and neural tube de- and 15 PhD students. fects in the second trimester of pregnancy (since Assoc. Prof. Kremensky is a member of the 2000), and the DNA prenatal diagnosis of aneuploidias governing administrative and scientific body of the (since 2001). One of his major achievements is the Medical Faculty, Sofia Medical University, the Na- design and implementation of the first two National tional Council on Genetics at the Ministry of Health, Programs for Diagnosis and Prevention of Inherited chairman of the Scientific Panel on Medicine and Disorders and Congenital Anomalies in Bulgaria member of the Scientific Panel on Genomics at the (2000–2005 and 2006–2010). He has been princi- Ministry of Education and Science. He is a member pal investigator or collaborator in 17 genetic studies, of the Panel on Genetics and Molecular Biology, 12 of which were international collaborations. Biochemistry and Biophysics of the Higher Accredi- Assoc. Prof. Kremensky, together with Prof. tation Commission of Bulgaria. Luba Kalaydjieva, founded in 1977 the Laboratory Assoc. Prof. Kremensky is an expert member of Molecular Pathology, Sofia Medical University and of the International Federation of Clinical Chemistry is currently the Head of Laboratories at the Uni- and participates in a number of prestigious interna- versity Hospital of Obstetrics and Gynaecology tional organizations, as International Society for Neo- „Maichin Dom“. The Laboratory of Molecular Pa- natal Screening, International Society of Inherited thology is the major diagnostic, research and train- Metabolic Diseases, American Society of Human Ge- ing centre for biochemical and molecular genetics netics, International Working group on Nanotechnology in Bulgaria. The research team of Assoc. Prof. and European Down’s Syndrome Screening Group.

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AWARDS

SCIENTISTS AWARDED IN THE CONTEST FOR HIGH SCIENTIFIC ACHIEVEMENTS IN 2005

A DIPLOMA FOR HIGH SCIENTIFIC ACHIEVE- tant scientific and applied contributions to theoreti- MENTS AND CASH PRIZE cal calculations and experimental research of para- metric, nonlinear and nonlinear–technical oscillating A. YOUNG SCIENTISTS systems are reflected in the work. Chief assistant MARIA PETKOVA Senior research associate DORA TOKMAKOVA from the Clinic of Cardiology at KARAGYOZOVA from the Institute of Mechanics the University Multiprofile Hospital for Active Treat- at BAS for 11 scientific publications abroad in the ment (UMHAT) of Medical University in Plovdiv for field of shell deformation at intensive dynamic load three publications in prestigious medical journals causing great plastic deformations as well as char- and participation in a monograph in the field of acterization of cell materials at great plastic de- chronical cardiovascular insufficiency. formations. The main contributions of the works are con- Methods for investigation of complex rod nected with risk evaluation at patients with cardio- systems with cell hexagonal structure at biaxial vascular insufficiency and possibilities for influenc- power action are created and different states at ing the prognosis. These accents are among the pipes with ring–shaped and orthogonal vertical sec- present day problems of modern cardiovascular tion are defined. The conclusions made can be ap- practice. plied at designing tubular mechanisms and con- Senior research associate NIKOLAI structions from aluminium and steel alloys at im- MARINOV NIKOLOV from the Institute of pulse excitations. Mathematics and Informatics at the Bulgarian Acad- Senior research associate I grade, DSc. ILIYA emy of Sciences (BAS) for 12 publications in the DIMITROV HRISTOV from the Institute for Soil Sci- field of multidimensional complex analysis – 11 in ence and Agroecology „N. Pushkarov“ for his mono- foreign journals and 1 in the Reports of the Bul- graph „Estimation of Water Status of Agroecosystems garian Academy of Sciences during the last three and Formation of Water Reserves in Soils“. Publishing years. House „Publish SaiAT–ECO“, Sofia, 2004. The investigations are connected with the The author for the first time in this country border behavior of invariant metrics. This is a very and abroad presents a new scientific basis for evalu- important topic having application in problems of ation of water status of the system „soil – complex analysis for continuation of holomorphic agroculture – ground layer of the atmosphere“ images. The theorem on localization of the kernel for the whole vegetation period and its efficiency. and the Bergman metric round the holomorphic A new integral index and method for determining peak point is proved. it are introduced for energy levels of soil moisture. Senior research associate NADYA B. SCIENTISTS OVER 35 YEARS OLD MLADENOVA ANTONOVA from the Institute of Prof. VLADIMIR DAMGOV from the Mechanics and Biomechanics at BAS for 8 scientific Space Research Institute at BAS for the mono- publications in prestigious international editions in graph „Nonlinear and Parametric Phenomena: Theory the field of haemo– and biorheology, as well as and Applications in Radiophysical and Mechanical for investigation of dependences between surface Systems“. World Scientific: New Jersey, London, forces by means of experimental observation of Singapore, 2004. parameters of thin liquid films from pulmonary Capital work in the field of nonlinear phe- surfactants. nomena with application in microelectromechanical Important haemorheological dependences are systems and nanotechnologies. Original and impor- found with patients with cerebrovascular disorders

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of ischemical origin and hyperviscosity syndrome, as applicable for solving problems in the field of mo- well as the impact of high–molecular polymeric lecular immunology are developed. additives upon haemorheological and haemodynamical parameters with animals. B. SCIENTISTS OVER 35 YEARS OLD Associate professor HRISTO SLAVOV Associate professor ALEXANDAR KYUCHUKOV from Veliko Tarnovo University „St. OMARCHEVSKI from the Faculty of Theology at Cyrill and Methodius“ for his two monographs Sofia University „St. Kliment Ohridski“ for his works „Methods of Teaching Bulgarian Language in El- „Religious Policy of the Emperor Justinian I (527 – ementary School in Conditions of Bilinguism“, 565)“. Publishing House of Sofia University „St. „Prosveta“ Publishing House, 2004 and „Written Kliment Ohridski“, 2004 and „In Search of Truth“ Speech in Bulgarian of Romany Schoolchildren in III (Publication of the Faculty of Theology, 2003). – V Grades“, Balkan Foundation „Diversity“, 2002. The works investigate problems not studied The problems treated in the book are very in detail in the national theology. A thorough analy- topical not only for our country, but also for the sis of very contradictory in their contents conclu- rest of the European countries. In the light of sions about Justinian I and other persons as well as integration processes of ethnic minorities, preserv- of new and up to now not investigated by Bulgar- ing their ethnic culture and specific features, the ian science facts and documents is done. offered methods of teaching Bulgarian language Associate professor DIMITAR DIMITROV and developing writing skills have exceptional ap- POPOV from Shumen University „Bishop K. plied value for our system of education. Preslavski“ for the monograph „ Phonostylistics of the Discourse“, University Publishing House, Shumen, HONORARY DIPLOMA FOR ACHIEVEMENTS IN 2004. SCIENCE This is the first monographic research on phonostylistics in Bulgarian linguistics and the first A. DOCTORANDS serious attempt to bring phonostylistics forward as Dr. VENTSISLAVA YANKOVA PETROVA an independent subject within the sphere of lin- – part–time assistant at Biological faculty of Sofia guistics. The work contains serious theoretical treat- University „St. Kliment Ohridski“ for defended in ments connected with the sound qualities, pronun- 2004 dissertation „Investigation of Mechanisms for ciation and pronunciational qualities of Bulgarian Detoxication of Reactive Oxygen Species in speech. Mitochondries of Yeast“ and three articles in for- Associate professor NIKOLAI STANEV eign publications connected with the dissertation. PRODANOV from Veliko Tarnovo University „St. On the basis of conducted original experi- Cyrill and Methodius“ for his monograph „Prob- ments a number of dependences are proved in the lems of Historiographic Textology. On the Material dissertation and a new model for in vivo detoxica- of Bulgarian Historiography of VII –XX Centuries“. tion of active oxygen generated in cell mitochondries „Faber“ Publishing House, V. Tarnovo, 2003. is offered. The work fills in the gap in the Bulgarian Research associate KLIMENT BORISOV historiographic research. For the first time the study PETROV from the Institute of Molecular Biology of the most important historiographic conclusion – at BAS for defended in 2004 dissertation „Ex- historically written text – is put on a broad basis. pression of Fragments of Antibodies and Strate- Senior research associate RAYA ZAIMOVA gies for Their Stabilization“ and two articles in from the Institute of Balkan Studies at BAS for foreign journals. the monograph „The Way to the „Other“ Europe. This is an important and topical problem in From French–Ottoman Cultural Intercourse in the the field of albumen engineering. Systems for ex- XVI–XBVIII Centuries.“ „Queen MAB“ Publishing pression of catalytic antibody, for determining opti- House, 2004. mal conditions for cultivation, creation of a new The work contributes to clarification of cul- pseudo–fragment and other vanguard approaches tural intercourse and acquaintance of separate parts

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WINNERS IN THE SECOND ANNUAL COMPETITION „INNOVATIVE BULGARIAN ENTERPRISE OF THE YEAR 2005“ ANNOUNCED

The Second National Innovation Forum which 2. Turnover: relative share of new products/ ARC Fund organized jointly with the World Bank processes in the total sales of the company. office in Bulgaria, DG Enterprise of the European 3. Exports: relative share of the new prod- Commission, the Ministry of Economy, the Austrian ucts/processes placed on foreign markets in the Embassy in Sofia, EPIQ Electronic Assembly and the overall export of the company. Center for the Study of Democracy on October 25, 4. Prospects: opportunities for further devel- 2005 featured the awards ceremony for the Sec- opment of the new products/processes. ond National Contest recognizing the most Innova- 5. Intellectual Property Rights protection of tive Bulgarian Enterprise of the Year 2005. the new products/processes: intangible assets ac- quired (patents, IPR certificates, licenses, etc.) The main objectives of this contest were: 6. R&D expenses: relative share of R&D ex- to encourage the innovation activities of penses in the total amount of revenues. Bulgarian enterprises; and 7. R&D personnel: relative share of employ- to promote the companies participating in ees involved in R&D in the overall personnel (this the contest and the results achieved by them in criterion is only valid for large enterprises). the field of innovation. 8. Number of new products/processes: num- ber of new products/processes placed on the mar- Innovative companies from all sectors of the ket in the last 3 years. Bulgarian economy were eligible to participate in 9. Connectivity/Networking: company’s link- the contest. ages/cooperation with universities; scientific, research A company was considered to be innovative and engineering organizations, participation in inter- if it had developed and marketed new or advanced national programs, etc. products (goods or services) and/or processes over 10. Level of production and management in the last three years. Innovation was defined in the enterprise: implemented certification and qual- broader terms as application of new approaches ity control systems, preservation of the environ- or technologies improving the economic performance ment, product safety; utilization of computers, soft- and competitiveness of the respective company. ware products, network devices, online services of- The innovation performance of participating fered, e–commerce, etc. companies was evaluated by an expert panel against a set of criteria and data collected on the basis of The ranking of companies per their final evalu- a special application form – an entry questionnaire ation was ultimately thoroughly analyzed by the that companies participating in the contest com- expert panel. pleted and submitted to ARC Fund. Awards Evaluation In order to ensure comparability with the The ranking of participating companies was accepted EU enterprise categories, two groups of performed on the basis of a set of criteria combin- companies have been defined: ing quantitative and qualitative indicators, measur- innovative small enterprises (of up to 50 able against a scale of 1–to–5. employees); Evaluation criteria: innovative medium–sized or large enter- .1 Technical level of the new products/pro- prises (of over 50 employees). cesses: assessment of the basic technical parameters of the new products/processes developed by the The awards ceremony took place during the contestant as compared to the respective analogue National Innovation Forum on October 25, 2005 at of new products/processes of competing companies. the Sheraton Sofia Hotel Balkan. Mr. Plamen

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Vachkov, Chairman of the newly established Agency and database development, application and sys- for IT and Communications, handed in the awards. tem development for a variety of hardware/ The Daisy Technology Ltd. company was software platforms and complex solutions. The presented with the Award for Innovative Small company participated in the contest with the Enterprise of 2005. The SD ELTA – R company following products: a system for visual modeling won the contest in the category for medium– and automation of business procedures in the sized and large enterprise. They were fol- Workflow.Cubes company, and with an innova- lowed by five other SMEs also recognized as tive software solution that provides profession- innovative, which received Certificates for Inno- als and amateurs in the field of interior design vative Enterprise of the Year 2005: Trapen SP, with the opportunity to prepare proficient inte- Coattex Ltd., Antipodes Ltd., Orgachim Co. rior solutions – 3D Spacer. and Optix Co. SD ELTA – R was ranked first in this The Daisy Technology Ltd. was ranked category with its product for development of first for the following new products it had in- digital telecommunication network in lowly troduced to the Bulgarian market: mp3 players, populated regions. ELTA R is a company with Digital Multimedia Cameras. Trapen SP partici- experience in development, manufacture, engi- pated in the contest with its microprocessor sys- neering and trade in products in the communi- tem for automation and optimization of differ- cation industry. Orgachim Ltd. was awarded ent kinds of mills for grinding of cement, raw for the DEKO professional products for con- materials, ores, etc., in the cement, dressing and struction and interior design. Optix Co. oper- power industries and a number of related ates in the field of designing, manufacturing branches. Coattex Ltd. is specialized in the field and testing quality of optical components and of technologies and techniques for powder poly- systems, encompassing the capabilities required mer coatings, metallic coatings, enameling and to undertake opto–mechanical and opto–elec- talcking and was nominated for its product for tronic assemblies and devices meeting the needs electrostatic powder coating machine APEN® – of the medicine, military and commercial mar- 30 G 1(2). Antipodes Ltd. works in the field of kets. The company participated in the contest system architecture and software design, web with 13 innovative products.

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RECENT PUBLICATIONS OF BULGARIAN SCIENTISTS

ARTICLES

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Title: Prospects for Biological Control of Teasels, Dipsacus Spp., a New Target in the United States. Authors: Rector, Brian G.1 [email protected]; Harizanova, Vili2, Sforza, Rene 1; Widmer, Tim1; Wiedenmann, Robert N.3 Source: Biological Control; Vol. 36, 1 (2006 Jan.), 1-14 Author Affiliations: 1USDA-ARS, European Biological Control Laboratory, Montpellier, France;2Department of Entomology, Agricultural University, 12 Mendeleev Str., 4000 Plovdiv, Bulgaria; 3Department of Entomology, University of Arkansas, Fayetteville, AR 72701, USA

ISSN: 1049-9644

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Title: Nonlinear Robust Temperature–Humidity Control in Livestock Buildings.

Soldatos, A.G.1, [email protected]; K.G.2 [email protected]; Daskalov, P.I.3 Authors: [email protected];Pasgianos, G.D.1 [email protected]; Sigrimis, N.A.2 [email protected]

Source: Computers & Electronics in Agriculture; Vol. 49, 3 (2005 Dec), 357-376. 1National Technical University of Athens, Department of Electrical and Computer

Author Affiliations: Engineering, Division of Signals, Systems and Robotics, Zographou, 15773 Athens, Greece; 2Agricultural University of Athens, Department of Agricultural Engineering, Laboratory of Agricultural Machinery and Automation, era Odos 75, Botanikos, 11855 Athens, Greece;3Department of Automatics, University of Rousse, 8, Studentska Street, 7017 Rousse, Bulgaria

ISSN: 0168-1699

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Title: In Vitro Regeneration and Genetic Transformation of Common Bean (Phaseolus vulgaris L.)—Problems and Progress.

Authors: Veltcheva, M.1 [email protected]; Svetleva, D.1; Petkova, Sp.1; Perl, A.2

Source: Scientia Horticulturae; Vol. 107, 1 (2005 Dec), 2-10

Author Affiliations: 1Department of Genetics, Agricultural University, 12, Mendeleev Str., Plovdiv 4000, Bulgaria;2Department of Fruit Tree Breeding and Molecular Genetics, Agricultural Research Organization, the Volcani Center, P.O. Box 6, Bet-Dagan 50250, Israel

ISSN: 0304-4238

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Title: Parasitism of Cameraria Ohridella (Lepidoptera, Gracillariidae) in Natural and Artificial Horse Chestnut Stands in the Balkans.

Authors: Grabenweger, G.1 [email protected]; Avtzis, N.2; Girardoz, S.3; Hrasovec, B.4; Tomov, R.5; Kenis, M.3

Source: Agricultural & Forest Entomology; Vol. 7, 4, (2005 Nov.), 291-296, 1 chart, 1 diagram

Author Affiliations: 1Institute of Plant Protection, Department of Applied Plant Sciences and Plant Biotechnology, University of Natural Resources and Applied Life Sciences, A-1190 Vienna, Austria;2Technological Educational Institution of Kavala, Department of Forestry, Proastio, 66100 Drama, Greece;3CABI Bioscience Switzerland Centre, Rue des Grillons, 2800 Delémont, Switzerland;4Faculty of Forestry, University of Zagreb, Svetosimunska 25, 41001 Zagreb, Croatia;5Faculty of Agronomy, University of Forestry, 10, Kliment Ohridski Blvd., 1756 Sofia, Bulgaria

ISSN: 1461-9555

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Title: Reviving Pre-Socialist Cooperative Traditions: The Case of Water Syndicates in Bulgaria.

Authors: Theesfeld, Insa1; Boevsky, Ivan2;

Source: Sociologia Ruralis; Vol. 45, 3 (2005 Jul.), 171-186,

Author Affiliations: 1Humboldt University of Berlin; 2Institute of Agricultural Economics, Bulgaria

ISSN: 0038-0199

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Title: Growth Control of Listeria Innocua 2030c during Processing and Storage of Cold-Smoked Salmon Trout by Carnobacterium Divergens V41 Culture and Supernatant.

Authors: Vaz-Velho, M.1,2 [email protected];Todorov, S.3,4; Ribeiro, J.1; Gibbs, P.1,5

Source: Food Control; Vol. 16, 6, (2005 Jul.), 540-548,

Author Affiliations: 1Escola Superior de Biotecnologia, Universidade Catolica Portuguesa, R. Dr. Antonio Bernardino de Almeida, 4200-072 Porto, Portugal; 2Escola Superior de Tecnologia e Gestao, Instituto Politecnico de Viana do Castelo, Av. Atlantico, 4900 Viana do Castelo, Portugal;3 Ecole Nationale d’Ingenieurs des Techniques des Industries Agricoles et Alimentaires (ENITIAA), Nantes, France; 4Faculty of Biology, Department of Microbiology, University of Sofia, 8, Dragan Tzankov Blvd., 1423 Sofia, Bulgaria; 5Leatherhead Food Research Association, Surrey, UK

ISSN: 0956-7135

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Title: Molecular Farming in Plants: An Approach of Agricultural Biotechnology.

Authors: Kamenarova, Kunka1; Abumhadi, Nabil1, Gecheff, Kostadin2; Atanassov, Atanas1

Source: Journal of Cell & Molecular Biology; Vol. 4, 2, 2005, 77-86,

Author Affiliations: 1AgroBioInstitute, 8, Dragan Tzankov Blvd., 1164 Sofia, Bulgaria; 2Bulgarian Academy of Science, 1113 Sofia, Bulgaria

ISSN: 1303-3646

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Title: Immunomodulatory Effects of C3bgp on the Antibody Response to Hemocyanin in Outbred Rabbits and the F1 Generation of Breeding with Siblings.

Authors: Stanilova, Spaska A.1 [email protected]; Miteva, Lyuba D.1; Tanchev, Svetlin G.2

Source: Veterinary Immunology & Immunopathology; Vol. 106, 1/2 (2005 Jun.), 15-21,

Author Affiliations: 1Department of Molecular Biology, Immunology and Genetics, Faculty of Medicine, Thracian University, 11, Armeiska Str., 6000 Stara Zagora, Bulgaria; 2Department of Genetics, Breeding and Reproduction, Faculty of Agriculture, Thracian University, Students’ Campus, 6000 Stara Zagora, Bulgaria

ISSN: 0165-2427

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EVENTS

SECOND NATIONAL INNOVATION FORUM TOOK PLACE IN SOFIA ON OCTOBER 25, 2005

ARC Fund organized the Second National In- nor of Stara Zagora, EPIQ Electronic Assembly Com- novation Forum at the Sheraton Sofia Hotel Balkan pany, Ludogorie–91 Ltd. and Point L Bulgaria Ltd. on October 25th, 2005. The event was organized in Dr. Ognyan Shentov, Chairman of ARC Fund, cooperation with the Ministry of Economy and the welcomed the participants and underlined a num- World Bank Sofia Mission, with the support of DG ber of key factors for achieving qualitative eco- Enterprise of the European Commission, the Aus- nomic growth amongst which he underscored the trian Embassy in Sofia, the EPIQ Electronic Assem- need for strong political will at the highest level, a bly Company and the Center for the Study of new long–term political vision and intensive pub- Democracy. The Forum was held under the pa- lic–private partnerships. tronage of the President of the Republic, Mr. Georgi Mr. Oscar de Bruyn Kops, Country Manager Parvanov. of the World Bank’s Bulgaria Office, stressed upon The main objective of the Forum was to Bulgaria’s challenge to sustain high growth level continue the dialogue and networking among the and productivity despite the unfavorable demo- stakeholders in the country’s national and regional graphic situation in the country. Mr. de Bruyn Kops innovation systems and serve as a platform for also emphasized crucial needs for human capital exchange of ideas and cooperation between the improvement, labor market performance optimiza- government, business community and research or- tion and decrease in unemployment. He highlighted ganizations. It additionally sought to promote the the support, which the World Bank provides for RIS (i.e. Regional Innovation Strategies) process in reforms in Bulgaria, in particular structural reforms, all planning regions of Bulgaria and the on–going strengthening of institutional capacity and social RIS BRIDGE initiative for the South West region of sustainability. Bulgaria. By presenting ARC Fund’s annual report In his keynote address President Georgi Innovation.bg the Forum also served as an open Parvanov recognized the importance of the second mechanism for benchmarking the local innovation National Innovation Forum as an evidence for an knowledge and expertise with the European and emerging and necessary tradition. The President international best practice. suggested a possible and successful approach for The forum’s agenda featured presentations bridging the gap between SMEs and the pool of by representatives of the European Commission, innovative information. This should be sought in the DG Enterprise, ARC Fund, State Agency for IT and application of the popular formula „business trains Communications, Bulgarian Small and Medium En- business“, as well as in the active cooperation among terprises Promotion Agency, National Science Fund, NGOs, professional associations, academic commu- University of National and World Economy, Bulgar- nity and the partners from the EU and USA. ian Academy of Sciences, Center for the Study of The forum continued with the award cer- Democracy, Coventry University Enterprises Ltd., UK, emony announcing the winners in the second na- Cambridge University, UK, URENIO Research Unit at tional Contest for Innovative Enterprise of the year the Aristotle University, Greece, the Regional Gover- 2005. The contest was open to participants from

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all industrial sectors with innovative ideas, products policy, as well as an overview of the new initia- and services. Awards were given in two main cat- tives and different policies in the area of innova- egories according to the companies’ size: for small tion were outlined by Prof. Milanka Slavova from innovative enterprise (of less than 50 employees), the University of National and World Economy. and medium–sized and large enterprise (over 50 She drew the audience’s attention to the fact employees). The chairman of the jury, Prof. Marin that the only strategic plan, which includes con- Petrov, announced the winners in the contest and crete measures for promotion of the innovation, Mr. Plamen Vachkov, Chairman of the Agency for is the National Plan for Regional Development. IT and Communications, handed in the awards. Ms. Zoya Damyanova, Program Director of ARC The first thematic session devoted to Fund, extended this issue by presenting the role Innovation.bg: the State and Perspectives of of the RIS initiatives for the development of the the National Innovation System was opened by regional innovation systems and successful ab- Mr. Yannis Tsilibaris, Deputy Head of the Support sorption of the EU Structural Funds in the fu- for Innovation Unit at the European Commission’s ture. She announced ARC Fund’s initiative of cre- DG Enterprise, who gave an overview of the Com- ating a National Network of RIS regions by sign- mission policy on raising European competitiveness ing a Memorandum of Cooperation, which was and innovation for overcoming the persistent gap welcomed by the coordinators of all the other of some 30 % in GDP per capita between the EU RIS projects in Bulgaria. and the USA, for addressing the disparities in eco- Within the Second Thematic Session on Re- nomic development among European regions and gional Innovation Strategies Dr. Clive Winters, Pro- the variations in the regional innovation perfor- gram Director at the Coventry University Enter- mance. Mr. Tsilibaris introduced the audience to prises Ltd, UK, shared with the audience the expe- the Competitiveness and Innovation Framework rience of the West Midland Region in the RIS Program (CIP) and its objective to promote coordi- process and made an overview of pilot and emerg- nation between regions in terms of Regional Inno- ing projects suggesting mechanisms for changing vation Strategies. Mr. Plamen Vachkov, Chairman the innovation landscape in the region. The shar- of the Agency for IT and Communications at the ing of RIS experiences was continued by Prof. Council of Ministers listed the necessary factors Nicos Komninos, Director of URENIO – Research for the development of innovation in the ICT sec- Unit at the Aristotle University of Thessaloniki, tor as one of the most important sectors for the Greece, who presented some successful RIS initia- Bulgarian economy. The need for more flexible tives that could serve as models for wiring up a economy was stressed upon as a necessary means successful innovation system in Bulgaria. The ex- of deploying the potential Bulgaria has in the ICT perience from the implementation of the pilot RIS sector – 15,000 IT specialists, which is 12% of the in the South Central Region of Bulgaria, along total number of IT specialists in Eastern Europe, with some conclusions, lessons learned, and Ms. Maria 500 software companies and 110 Cisco academies. Neikova, Regional Governor of the Stara Zagora However, the investments in R&D remain highly in- Region, presented recommendations. She appealed sufficient – they are four times smaller than those to the coordinators of the current RIS initiatives in the EU countries. to clearly formulate at the very beginning the Mr. Ruslan Stefanov, Project Coordinator expected results and measures to be undertaken at the Center for the Study of Democracy, pre- so as to achieve synergy. Ms. Daniela Tchonkova, sented the objective and main findings of the RIS BRIDGE Project Coordinator at ARC Fund, who Innovation.bg Report, which is intended to fa- presented the new RIS BRIDGE initiative for the cilitate the process of shaping a new long–term South West Planning Region, concluded the ses- vision for innovation. The approach that under- sion. She put strong emphasis on the specifics of pinned this report is based on the consolidation the RIS BRIDGE initiative – in contrast to the of all aspects of the innovation policy in a single standard RIS concept – which takes account of document. The key aspects of the EU innovation the intra–regional disparities and applies a differ-

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FP6 PROJECT „RESEARCHERS IN EUROPEAN KNOWLEDGE SOCIETY“ COMPLETED SUCCESSFULLY.

Conference with international participation Survey on the state–of–the–art in the „The Researcher – a Factor for the Development researcher’s profession, conducted during the REKS of the Knowledge Society“ which took place on project with results reported at the final conference. 28–29 November 2005 in Sofia finalized the set TV broadcasting– six integrated TV trans- of activities of the „Researchers in European missions within the TV production ‘Hi Tech’ on the Knowledge Society“ (REKS) project in Bulgaria national Nova Televisia– one every month during http://www–it.fmi.uni–sofia.bg/REKS/index.html. the ‘Researchers in Europe Initiative’. The REKS project addressed the overall stra- Dissemination– on–line and off–line tegic objective of the FP6 Human Resources and channels for dissemination and reaching a wider Mobility activity: „to provide broad support for the audience in Bulgaria were applied. development of abundant and dynamic world–class Participants in the REKS Project were as human resources in the European research system“. follows: Co–ordinator– Sofia University „St. Kliment It focused on public recognition of researchers and Ohridski“; University of Rousse „Angel Kantchev“; the development of researchers’ careers both at Technical University of Varna; Trakia University of national and regional levels in Bulgaria, and paid due Stara Zagora and Paisij Hilendarski University of attention to participation of women in research. Plovdiv. Regional centres of the Bulgarian Network The project formulated its main goal to of Research Mobility Centres (RMC) also had con- raising the awareness of Bulgarian society on the tribution to the fulfillment of the project objec- importance of researchers for building a knowl- tives This network was established as Bulgarian edge economy and society in Bulgaria as a part of part of the ERA–MORE – The European Network the European ones. of RMC (http://europa.eu.int/eracareers) in the The main goal of REKS was detailed into the framework of the EC Communication ‘A Mobility following project objectives: Strategy for the European Research Area’. – facilitation of the dialogue between dif- The outcomes of the project can be summa- ferent stakeholders in the economy and the soci- rized as follows: ety on the importance of researchers and identifi- increased awareness of the general public cation of the respective policy needs; and businesses on the importance of research of – improvement of the appreciation of research- human resources for building the knowledge ers in society, and by young people in particular; economy and society; – dissemination of information regarding prac- increased awareness on Bulgarian and Eu- tical tools supporting the researcher’s career in rope–wide initiatives related to researchers; Europe and in Bulgaria. increased awareness on career and job Beyond the stated objectives the project con- opportunities for young scientists in Europe; tributed to raising the awareness of Bulgarian soci- contribution to research of human capac- ety on the researchers’ mobility initiatives, and fa- ity building in the country; cilitated the more active utilization of the available minimizing weaknesses in research labour tools supporting the researcher’s career in Europe. force and thus supporting the knowledge–based The project implementation included the society in Bulgaria as a part of the European one; following groups of activities: enhanced participation of Bulgarian re- Awareness–raising rally– four regional searchers in the Marie Curie Actions, and other EU seminars, carried out in the towns of Rousse, Varna, programs related to human capacity building. Stara Zagora and Plovdiv dedicated to the The project activities had a substantial im- researcher’s profession. pact on Regional and National levels, and the project National conference– organized in Sofia results had a clear European added value. at the end of the project.

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