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The topicality of cyber defence has become unquestionable both domestically and internationally, which is also indicated by the rapid increase in the number of strategies, training programs, and professional forums focusing on this phenomenon. At the same time, the new connections between illegal activities and terrorism require governments, their competent bodies, agencies, and officials to regulate a phenomenon presented by only a few technologies which are understood by few people. Moreover, the defence against and societal resistance to the rapidly changing technologies must be further developed. The development of the cyber defence strategies of NATO and the European Union is a significant guideline for the development of the Hungarian specialist management system. KEYWORDS: cyber strategy, critical information infrastructure, terrorism, cyber security, NATO, EuropeanUnion, cyber-attack, hybrid warfare, force development, cyber defence, Hungarian Defence Forces

SummaryDual inhibition (2i) of Ras–MEK–ERK and GSK3β pathways enables the derivation of embryo stem cells (ESCs) from refractory mouse strains and, for permissive strains, allows ESC derivation with no external protein factor stimuli involvement. In addition, blocking of ERK signalling in 8-cell-stage mouse embryos leads to ablation of GATA4/6 expression in hypoblasts, suggesting fibroblast growth factor (FGF) dependence of hypoblast formation in the mouse. In human, bovine or porcine embryos, the hypoblast remains unaffected or displays slight-to-moderate reduction in cell number. In this study, we demonstrated that segregation of the hypoblast and the epiblast in rabbit embryos is FGF independent and 2i treatment elicits only a limited reinforcement in favour of OCT4-positive epiblast populations against the GATA4-/6-positive hypoblast population. It has been previously shown that TGFβ/Activin A inhibition overcomes the pervasive differentiation and inhomogeneity of rat iPSCs, rat ESCs and human iPSCs while prompting them to acquire naïve properties. However, TGFβ/Activin A inhibition, alone or together with Rho-associated, coiled-coil containing protein kinase (ROCK) inhibition, was not compatible with the viability of rabbit embryos according to the ultrastructural analysis of preimplantation rabbit embryos by electron microscopy. In rabbit models ovulation upon mating allows the precise timing of progression of the pregnancy. It produces several embryos of the desired stage in one pregnancy and a relatively short gestation period, making the rabbit embryo a suitable model to discover the cellular functions and mechanisms of maintenance of pluripotency in embryonic cells and the embryo-derived stem cells of other mammals.

Primordial germ cells (PGCs) are the precursors of germline cells that generate sperm and ova in adults. Thus, they are promising tools for gene editing and genetic preservation, especially in avian species. In this study, we established stable male and female PGC lines from 6Hungarian indigenous chicken breeds with derivation rates ranging from 37.5 to 50 percent. We characterized the PGCs for expression of the germ cell-specific markers during prolonged culture in vitro. An in vivo colonization test was performed on PGCs from four Hungarian chicken breeds and the colonization rates were between 76 and 100%. Cryopreserved PGCs of the donor breed (Partridge color Hungarian) were injected into Black Transylvanian Naked Neck host embryos to form chimeric progeny that, after backcrossing, would permit reconstitution of the donor breed. For 24 presumptive chimeras 13 were male and 11 were female. In the course of backcrossing, 340 chicks were hatched and 17 of them (5%) were pure Partridge colored. Based on the backcrossing 1 hen and 3 roosters of the 24 presumptive chimeras (16.6%) have proven to be germline chimeras. Therefore, it was proven that the original breed can be recovered from primordial germ cells which are stored in the gene bank. To our knowledge, our study is a first that applied feeder free culturing conditions for both male and female cell lines successfully and used multiple indigenous chicken breeds to create a gene bank representing a region (Carpathian Basin).

Background One of the most critical global problem nowadays is the increased environmental temperature. Agriculture is very susceptible to this adverse effect because the productivity of animals and poultry decreased. Although several studies reported the effects of heat-stress in chicken, the expression profile of heat-shock proteins and heat shock factors was not investigated in the gonads and germ cells of Transylvanian Naked Neck chickens. Methods In the first experiment, 24 hours after hatching 80 chicks were heat treated on 38.5oC ambient temperature with 60% humidity for 12 hours. After maturation, their primary productivity parameters, such as egg production, abnormalities in embryo development, sperm quantity, concentration, and motility were studied following two weeks of heat-stress on 30 °C room temperature. In the second experiment, the thermal manipulation of 60 chicks was the same but 15 treated and 15 control chicks were sacrificed immediately after the treatment. The other 15–15 chickens were raised to maturity. Expression levels for two heat-shock proteins and four heat shock factors were determined by real-time PCR in the gonads of heat-treated and heat-stressed chickens. Results We found that the heat-treated layers had significantly higher egg production than the control group in heat-stressed conditions. In cockerels, the sperm quality did not differ significantly between the heat-treated and heat-stressed group and the heat-stressed but not heat-treated group. We examined the expression pattern of HSPs and HSFs in the gonads. We found that the expression of HSP90 and HSF4 increased significantly (p HSF2 and HSF3 were significantly lower compared to the control. In case of adult heat-treated males, the HSP70, HSF1 and HSF3 expression levels showed a significant increase in both gonads, compared to the control expression levels (P HSP90 and HSF4 in heat-treated chickens.

Dual inhibition (2i) of Ras-MEK-ERK and GSK3β pathways enables the derivation of embryo stem cells (ESCs) from refractory mouse strains and, for permissive strains, allows ESC derivation with no external protein factor stimuli involvement. In addition, blocking of ERK signalling in 8-cell-stage mouse embryos leads to ablation of GATA4/6 expression in hypoblasts, suggesting fibroblast growth factor (FGF) dependence of hypoblast formation in the mouse. In human, bovine or porcine embryos, the hypoblast remains unaffected or displays slight-to-moderate reduction in cell number. In this study, we demonstrated that segregation of the hypoblast and the epiblast in rabbit embryos is FGF independent and 2i treatment elicits only a limited reinforcement in favour of OCT4-positive epiblast populations against the GATA4-/6-positive hypoblast population. It has been previously shown that TGFβ/Activin A inhibition overcomes the pervasive differentiation and inhomogeneity of rat iPSCs, rat ESCs and human iPSCs while prompting them to acquire naïve properties. However, TGFβ/Activin A inhibition, alone or together with Rho-associated, coiled-coil containing protein kinase (ROCK) inhibition, was not compatible with the viability of rabbit embryos according to the ultrastructural analysis of preimplantation rabbit embryos by electron microscopy. In rabbit models ovulation upon mating allows the precise timing of progression of the pregnancy. It produces several embryos of the desired stage in one pregnancy and a relatively short gestation period, making the rabbit embryo a suitable model to discover the cellular functions and mechanisms of maintenance of pluripotency in embryonic cells and the embryo-derived stem cells of other mammals.

The primordial germ cells (PGCs) are the precursors for both the oocytes and spermatogonia. Recently, a novel culture system was established for chicken PGCs, isolated from embryonic blood. The possibility of PGC long-term cultivation issues a new advance in germ cell preservation, biotechnology, and cell biology. We investigated the consequence of gga-miR-302b-5P (5P), gga-miR-302b-3P (3P) and dual inhibition (5P/3P) in two male and two female chicken PGC lines. In treated and control cell cultures, the cell number was calculated every four hours for three days by the XLS Imaging system. Comparing the cell number of control and treated lines on the first day, we found that male lines had a higher proliferation rate independently from the treatments. Compared to the untreated ones, the proliferation rate and the number of apoptotic cells were considerably reduced at gga-miR-302b-5P inhibition in all PGC lines on the third day of the cultivation. The control PGC lines showed a significantly higher proliferation rate than 3P inhibited lines on Day 3 in all PGC lines. Dual inhibition of gga-miR-302b mature miRNAs caused a slight reduction in proliferation rate, but the number of apoptotic cells increased dramatically. The information gathered by examining the factors affecting cell proliferation of PGCs can lead to new data in stem cell biology.

We studied the effect of different magnitudes (7000 PSI (48.26 MPa), 8000 PSI (55.16 MPa), and 9000 PSI (62.05 MPa)) of hydrostatic pressure on the ploidy of pikeperch larvae. Pressure shock was applied 5 min after the fertilization of eggs at a water temperature of 14.8 ± 1 °C. A 7000 PSI pressure shock was applied for 10 or 20 min, while 8000 and 9000 PSI treatments lasted for 10 min. Each treatment with its respective control was completed in triplicate, where different females’ eggs served as a replicate. In the treatment groups exposed to 7000 PSI for 10 min, only diploid and triploid larvae were identified, while 2n/3n mosaic individuals were found after a 20-min exposure to a 7000 PSI pressure shock. The application of 8000 or 9000 PSI pressure shocks resulted in only triploid and mosaic individuals. Among larvae from eggs treated with 8000 PSI, three mosaic individuals with 2n/3n karyotype were identified (4.0 ± 6.9%), while a single (2.0 ± 3.5%) 1n/3n mosaic individual was found in the 9000 PSI-treated group. To our knowledge, this is the first report that demonstrates the induction of a haplo-triploid karyotype by hydrostatic pressure shock in teleost fish. The dominance of triploid individuals with a reasonable survival rate (36.8 ± 26.1%) after 8000 PSI shock supports the suitability of the hydrostatic pressure treatment of freshly fertilized eggs for triploid induction in pikeperch.

In the last decade, avian gene preservation research has focused on the use of the early precursors of the reproductive cells, the primordial germ cells (PGCs). This is because avian PGCs have a unique migration route through the vascular system which offers easy accessibility. Furthermore, culturing of the cells in vitro, freezing/thawing, reintegration into a recipient embryo and the development of the germ cells can be carried out in well-defined laboratory circumstances. The efficient recovery of the donor genotype and the frequency of germline transmission from the surrogate host animals are still areas which need further development. Thus, the aim of the present study was to investigate an infertile interspecific hybrid (recipient) as an appropriate host for primordial germ cells from native poultry breeds. Guinea fowl × chicken hybrids were produced, the crossing was repeated inversely. The phenotype, the hatching time, the hatching rate, the sex ratio, the presence of own germ cells, the fertility and the phenotype of viable hybrids and the incidence of chromosomal abnormalities of dead hybrid embryos were described. 6.65% viable offspring was obtained with crossing of Guinea fowl females with domestic fowl males. Crossing of domestic fowl hens with Guinea fowl male resulted in lower fertility, 0.14% viable offspring. Based on the investigations, the observed offspring from the successful crossing were sterile male hybrids, thus an extreme form of Haldane’s rule was manifested. The sterile hybrid male embryos were tested by injecting fluorescently labeled chicken PGCs. The integration rate of labeled PGCs was measured in 7.5-day, 14.5-day and 18.5-day old embryonic gonads. 50%, 5.3% and 2.4% of the injected hybrid embryos survived and 40%, 5.3% and 2.4% of the examined gonads contained fluorescent labeled donor PGCs. Therefore, these sterile hybrid males may be suitable recipients for male PGCs and possibly for female PGCs although with lower efficiency. This research work shows that the sterility of hybrids can be used in gene conservation to be a universal host for PGCs of different avian species.

Chicken Primordial Germ cells (PGCs) are emerging pioneers in the field of applied embryology and stem cell technology. Now-a-days transgenic chickens are promising models to study human disease pathophysiology and drug designing. However, most of the molecular mechanism, which govern the stemness and pluripotency of chicken PGCs, not known in details. Recent studies have indicated the role of HSP70 in early embryonic development in many vertebrate species. Exposure of chicken to heat stress result in activation of heat shock factors which activate the transcription of HSP70. Exposure chicken eggs to acute heat stress effects HSP70 expression in PGCs and gonads. HSP70 helps in maintaining the integrity of chicken PGCs. A new emerging role of HSP70 in apoptosis has emerged. In our lab, we aim to characterize the expression of cHsp70 in chicken PGCs and gonads during embryonic development by subjecting the parents to acute levels of heat stress. Chickens whose parents subjected to heat stress showed varied expression of cHsp70 and also improved thermo tolerance. In the future we plan to study other factors and miRNAs, which is characterized as an emerging player in regulating heat shock protein response in chicken and also plays an important role in apoptosis.

Primordial germ cells (PGCs) were isolated from blood samples of chicken embryos. We established four PGC lines: two males (FS-ZZ-101, GFP-ZZ-4ZP) and two females (FS-ZW-111, GFP-ZW-5ZP). We could not detect a significant difference in the marker expression profile, but there was a remarkable difference between the proliferation rates of these PGC lines. We monitored the number of PGCs throughout a three-day period using a high-content screening cell imaging and analysing system (HCS). We compared three different initial cell concentrations in the wells: ~1000 cells (1×, ~4000 (4× and ~8000 (8×. For the GFPZW- 5ZP, FS-ZZ-101 and FS-ZW-111 PGC lines the lowest doubling time was observed at 4× concentration, while for GFP-ZZ-4ZP we found the lowest doubling time at 1× concentration. At 8× initial concentration, the growth rate was high during the first two days for all cell lines, but this was followed by the appearance of cell aggregates decreasing the cell growth rate. We could conclude that the difference in proliferation rate could mainly be attributed to genotypic variation in the established PGC lines, but external factors such as cell concentration and quality of the culture medium also affect the growth rate of PGCs.

Primordial germ cells (PGCs) are the precursors of adult germ cells, and among the embryonic stem-like cells in the bird embryo, only they can transmit the genetic information to the next generation. Despite the wide range of applications, very little is known about the mechanism that governs primordial germ cell self-renewal and differentiation. As a first step, we compared 12 newly established chicken PGC lines derived from two different chicken breeds, performing CCK-8 proliferation assay. All of the lines were derived from individual embryos. A significant difference was found among the lines. As microRNAs have been proved to play a key role in the maintenance of pluripotency and the cell cycle regulation of stem cells, we continued with a complex miRNA analysis. We could discover miRNAs expressing differently in PGC lines with high proliferation rate, compared to PGC lines with low proliferation rate. We found that gga-miR-2127 expresses differently in female and male cell lines. The microarray analysis also revealed high expression level of the gga-miR-302b-3p strand (member of the miR-302/367 cluster) in slowly proliferating PGC lines compared to the gga-miR-302b-5p strand. We confirmed that the inhibition of miR-302b-5p significantly increases the doubling time of the examined PGC lines. In conclusion, we found that gga-miR-181-5p, gga-miR-2127, and members of the gga-miR-302/367 cluster have a dominant role in the regulation of avian primordial germ cell proliferation.

Red-footed Falcons are gregarious trans-equatorial migrants, forming up to several thousand strong roost sites after the breeding season and before commencing migration. This pre-migration period is presumed to play a major role in defining the survival of long-range migrants. Here we investigate the autumn movements of 8 individuals caught and satellite-tagged within the Carpathian Basin. We found that birds may use multiple roost sites that can be separated by large distances. A single individual’s home range was 88 km2 (80% kernel home range) and was near concentrical to the roost site. Two individuals travelled to southern Ukraine soon after tag-deployment. Our results demonstrate that even a small number of satellite tagged birds show behavioural plasticity in terms of roost site selection indicating that post-breeding foraging habitat choice decisions may have substantial variability. The night localization points of birds marked out 2 and 5 yet unknown potential roost sites in Hungary and in the Ukraine, respectively. Using the data of an international weekly survey (2006-2011) carried out in the Carpathian Basin, we cross-referenced the departure dates of tagged individuals with the 6 year means of counted individuals. The tagged birds initiated migration with the first 25% percent of the surveyed population.

International audience; The conservation of genetic resources of avian species has become increasingly important over the past decade. The aim of the present study was to develop a genome preservation technique for the Hungarian goose Anser anser domestica. To this end, we developed a novel approach combining the simplicity of isolating a blastodermal cell suspension, which includes forming primordial germ cells (PGCs), with the efficiency of targeting future gonads by injecting these cells into the cardiac vein of the developing host embryo. First, we determined that the migratory period of PGCs in goose embryos was between 69 and 84 h of development. Then, we injected the blastodermal cell suspension into the bloodstream of recipient embryos at this stage of development and monitored donor cell transmission into the genital tract. In all, 249 embryos were injected; three were found to be chimeras in gonadal tissues, whereas only one was a chimera in other tissues. Based on these results, it is concluded that this method is suitable for producing chimeras in the domestic goose. The optimal time of cell injection was found to be between 74 and 76 h. The present study is the first report of the generation of chimeras in the domestic goose using intracardiac transplantation of embryonic cells.