15. The Role of Mechanical Load in Treating Osteoporosis: Designing a Mechanical Osteoblast Stimulation Model to Investigate Curative Applications

Coordinator: Basdra Efthimia, Institution: National & Kapodistrian University of Athens

Osteoporosis is the most common metabolic bone disease with severe social and economic implications and high rates of morbidity and mortality. It is characterised by reduction of the bone mass, disruption of bone microarchitecture, decreased tolerance and increased fracture frequency. Bone loss is due to excessive activity of osteoclasts (bone reduction cells) and reduced activity of osteoblasts (bone production cells). Osteoblastic activity can be promoted by osteoblast mechanical stimulation.
The aim of this study was to design an in vitro osteoblast mechanotransduction model in order to investigate the molecular mechanisms implicated in osteoporosis. The findings indicate that, under the application of mechanical force for 6 and 12 hours in pro-osteoblastic cells, the activation of transcriptional factor NF-κB/cRel and its binding to cellular DNA become apparent, indicating that NF-κB is possible to regulate the expression of osteospecific genes (such as Runx2) after mechanical stimulation.
The involvement of the inflammatory factor NF-κB in the regulation of osteospecific genes’ expression and the molecular mechanism underlying this relationship, opens new perspectives for understanding the physiology of osteoblastic differentiation and, consequently, of bone reconstruction after physical stimulation such as mechanical stress, the dysfunction of which is responsible for a number of bone pathophysiologies, such as osteoporosis, fracture healing, restoration of bone deficits etc, further underlining the fundamental importance of regular exercise in their prevention and treatment.
Final Report

16. The Creation and Analysis of a New Genetic Model of Osteoporosis in Transgenic Mice Overexpressing the RANKL Cytokine

Coordinator: Douni Helen, Institution: Alexander Fleming Biomedical Sciences Research Center

Osteoporosis is a chronic disease that primarily affects menopausal women and is characterised by low bone mass and increased risk of fractures. RANKL is the main mediator in bone resorption by inducing osteoclast generation. Recently, it was discovered that pharmaceutical inhibition of RANKL effectively prevents bone loss and fractures in osteoporotic patients, making RANKL the most specialised target against osteoporosis.
The research group has recently generated mice that over-produce human RANKL using genetic engineering. The study analyses new genetic models of osteoporosis resulting from the overexpression of human RANKL. Specifically, we characterised two models of osteoporosis in detail, one with low expression of RANKL and mild osteoporosis and one with highly elevated levels of RANKL and characteristics of severe osteoporosis, such as trabecular bone loss, cortical porosity and reduced bone strength.
The administering of a commercial anti-osteoporotic drug in these models showed that bone loss was prevented, making them ideal models for the evaluation of new inhibitors against human RANKL in the preclinical level. The development of these first animal models of osteoporosis induced by overexpression of human RANKL will pave the way for the discovery of highly-specific novel drugs.
Final Report

17. The Genetic Differentiation of the Mitochondrial Genome and Its Co-evolution with the Nuclear Genome as a Speciation Mechanism in the Terrestrial Snails of Greece

Coordinator: Parmakelis Aristeidis, Institutions: National & Kapodistrian University of Athens / University of Patras

Mitochondria are factories producing cellular energy and are necessary for cell life, as well as, sometimes, cell death. Mitochondria have their own DNA and it evolves, like the rest of the genetic material. Indeed, since it is believed that its evolution occurs at a steady rate and without adaptive leaps, mitochondrial DNA is widely used to measure evolutionary time. For example, such measurements support humanity’s African origin. Is the evolution of mtDNA, however, truly neutral, or is it influenced by the evolution of the remaining nuclear genetic material? Coevolution appears to be a plausible interpretation, since energy production requires the close interaction of nuclear and mitochondrial genes. Indeed, it has recently been suggested that the absence of such an effective interaction may lead to reproductive barriers and, therefore, to the appearance of new species.
The co-evolution hypothesis was tested in a specific and precise manner within the project. For this purpose, populations from three genera of land-snails were used and one nuclear and one mitochondrial gene engaging in close interaction to produce energy were molecularly analysed.
The findings of this study strongly support the mito-nuclear co-evolution as a likely speciation mechanism for the land-snails of Greece. Furthermore, the results indicate that the most likely diferrentiation level that this mechanism identifies is among con-specific populations that, even though are currently isolated, maintained some form of communication during their recent past.
Finally, this research study contributes to the understanding of complex evolutionary mechanisms and adds new data regarding the adaptability of the cell’s bioenergy mechanisms.
Final Report

18. What Lies Behind a Giant: the Case of the Skyros Lizard

Coordinator: Pafilis Panagiotis, Institutions: University of Michigan / National & Kapodistrian University of Athens / Imperial College

Skyros island is well known for the famous pony, a typical case of dwarfism. At the same Archipelago though, there is an impressive case of gigantism, the endemic lizard of Skyros (Podarcis gaigeae), present only in the islet Mesa Diavates. This population is characterised by high density and cannibalism while a numerous seagull colony nests in the islet. According to the “island rule”, all these features favour gigantism.
This study tried to clarify the underlying factors of gigantism and its implications to the overall biology of lizards. It failed to detect another incident of gigantism after sampling all islets of the Archipelago. This finding certifies the uniqueness of the Diavates population, though all insular lizards had larger body size than their Skyros conspecifics while an intermediate case was found at Lakonissi. High food availability is the main factor diversifying Diavates from the rest of the islets, supported by breeding seagulls that provide nutrients. Thanks to seabird contribution to the energy flow and the particular substrate of the islet, vegetation is lush and has switched to more nitrophilous species, fueling an augmented primary productivity.
Minimal predation pressure has increased lizard population densities, which experience stronger intraspecific competition, expressed even as cannibalism. Under these circumstances large body size turns to be an advantage for both juveniles (since they may survive from cannibalism) and male adults (that have access to an extra food resource through cannibalism but also possess higher social status, territoriality and reproductive success).
The impact of gigantism on reproductive output was strong. Contrary to classical life-history strategies that predict for clutches of either many but smaller eggs or few but larger eggs, in this case females from Diavates lay many and larger eggs. No effect was found regarding thermoregulation. The occurrence of gigantism and unusual reproductive investment in these small island populations is probably best explained by the occurrence of two underlying factors: (i.) the existence of striking cannibalistic behaviours in the form of attacks to the tail and intense intraspecific predation on juveniles, and (ii.) substantial marine subsidies by resident seabird colonies.
Final Report

19. Development of a Diagnostic Drug for Alzheimer’s Disease

Coordinator: Pelekanou Maria, Institution: National Centre of Scientific Research "Demokritos"

The study focuses on the in vivo biological evaluation of a technetium-99m complex (Bz-Tc), as a potential radiodiagnostic agent for Alzheimer’s disease. To date, there exists no specific clinical diagnostic examination for the early and accurate diagnosis of the disease, and the search for a specific diagnostic procedure for Alzheimer’s is a major research target worldwide.
The Bz-Tc complex carries in its structure a benzothiazole derivative moiety that renders it capable of binding to the amyloid plaques that characterise Alzheimer’s disease. Within this project, biodistribution studies of the radioactive Bz-Tc in healthy mice revealed that 0.2% of the injected radioactivity was localised at the brain area, a sufficient quantity for radioimaging applications. In experiments conducted with transgenic mice/models for the disease, a considerably higher dose of radioactivity was measured in the brain area (1.5%-3.2%) of the transgenic mouse compared to the normal. Finally, toxicity experiments performed in mice after administration of a high dose of a non-radioactive analogue of Bz-Tc followed by body weight measurements, behavioural examination and histopathological examination of liver and kidneys did not reveal any appreciable degree of toxicity of the complex.
In conclusion, the in vivo results are encouraging and prompt for the continuation of the biological evaluation Bz-Tc as a radiodiagnostic agent for Alzheimer’s disease.
Final Report

20. Automatic Detection of Heart Murmurs in Children’s Phonocardiograms and Development of Paediatric Auscultation Training Software

Coordinator: Stilianou Ioannis, Institutions: Foundation for Research &Technology (FORTH) / University of Crete

Cardiac auscultation is a first line diagnostic tool, as it can detect abnormal sounds (murmurs) associated with underlying congenital or acquired heart malformations. The appropriate differentiation of abnormal murmurs from innocent (without underlying pathology) murmurs is of paramount importance when screening otherwise healthy populations with a high incidence of innocent murmurs, such as children, for heart disease.
Despite the fact that cardiac auscultation is the cheapest and simplest screening tool, general paediatricians present rather suboptimal clinical skills; they would fail to detect 20% of heart defects with abnormal murmurs (sensitivity 80%), while they would inappropriately refer more than 30% of children with innocent murmurs (specificity 65%).
A computer-based automated heart murmur classification system was developed for paediatrics, having a 95% sensitivity and 97% specificity to differentiate innocent from abnormal systolic murmurs in childhood, a performance comparable to that of experienced paediatric cardiologists. The system is based on an appropriately selected and representative sample of paediatric heart sounds having a definitive echocardiographic diagnosis. The system aims to support paediatricians and primary health care physicians, to establish appropriate referral patterns of children with murmurs and to correctly refer only those children with abnormal murmurs, while preventing referrals of children with innocent murmurs.
Final Report

21. The Genetic and Molecular Role of the AIF (Apoptosis Inducing Factor) Proteins in Activating the Innate Immune System of Plants

Coordinator: Tsitsigiannis Dimitrios, Institution: Agricultural University of Athens

Plants, like all organisms, have developed an innate immune system that allows them to defeat infections from various pathogens. The first line of defense is to identify the invasive microorganisms and to produce various molecules in order to destroy them. The second line of defense is the programmed death of infected cells, thereby limiting the spread of infection.
Programmed cell death (PCD) is a process that normally takes place during development and defense of multicellular organisms and it may be linked to serious diseases like cancer and degenerative diseases. Research in recent years has demonstrated the existence of common biochemical pathways of PCD among plant, animal and microbial cells but, unlike other organisms, this process in plants has not been thoroughly explored.
The aim of this study was to investigate a family of genes identified in the genetic model plant Arabidopsis thaliana. These genes have similarities with the mammalian AIF gene (Apoptosis Inducing Factor), a phylogenetically old, 57 kDa flavoprotein, which shares similarity to bacterial, fungus and plant oxidoreductases. AIF is associated with diseases related to increased apoptotic events such as infection with HIV, neurodegenerations and heart attacks. The present study showed for the first time that the At-AIF genes play an important role in the activation of the plant immune system and their resistance or susceptibility to a number of important crop pathogens.
Final Report