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Characterisation of Metalloprotease-mediated EGFR Signal Transactivation after GPCR Stimulation
(2011)
In the context of metalloprotease-mediated transactivation of the epidermal growth factor receptor, different monoclonal antibodies against ADAM17 / TACE were characterized for their ability to block the sheddase. Activity of some of them was observed at doses between 2µg/mL and 10µg/mL. Kinetic analyses showed their activity starting at around 30 minutes. In cellular assays performed with the antibodies, especially upon treatment of cells with sphingosine-1-phosphate a reduction in proliferation was observed with some candidates. Moreover this study provides potential new roles for ß-Arrestins. Their involvement in the triple membrane-passing signal pathway of EGFR transactivation was shown. Furthermore, in overexpressing cellular model systems, an interaction between ADAM17 and ß-Arrestin1 could be observed. Detailed analysis discovered that phosphorylation of ß-Arrestin1 is crucial for this interaction. Additionally, the novel mechanism of UV-induced EGFR transactivation was extended to squamous cell carcinoma. The mechanism happens in a dose dependent manner and requires a metalloprotease to shed the proligand Amphiregulin. The involvement of both ADAM9 and ADAM17, being the metalloproteases responsible for this cleavage, was shown for SCC9 cells.
There is more and more evidence for the cancer stem cell hypothesis which believes that cancers are driven by a cellular subcomponent that has stem cell properties which is self-renewal, tumorigenicity and multilineage differentiation capacity. Cancer stem cells have been connected to the initiation of tumors and are even found to be responsible for relapses after apparently curative therapies have been undertaken. This hypothesis changes our conceptual approach of oncogenesis and shall have implications in breast cancer prevention, detection and treatment, especially in metastatic breast cancer for which no curative treatment exists. Given the specific stem cell features, novel therapeutic pathways can be targeted. Since the value of vaccinia virus as a vaccination virus against smallpox was discovered by E. Jenner at 18th century, it plays an important role in human medicine and molecular biology. After smallpox was successfully eradicated, vaccinia virus is mainly used as a viral vector in molecular biology and increasingly in cancer therapy. The outstanding capability to specifically target and destroy cancer cells makes it a perfect agent for oncolytic virotherapy. Furthermore, the virus can easily be modified by inserting genes which encode therapeutic or diagnostic proteins to be expressed when a tumor is infected. The emphasis in this study was the establishment of methods for the enrichment of human breast cancer stem-like cells from cancer cell lines and characterization of those cancer stem-like cells in vitro and in vivo. Furthermore, by using the Genelux Corporation vaccinia virus strain GLV-1h68, the isolated cancer stem-like cells can be targeted not only in vitro but also in vivo more efficiently. Side-population (SP) cells within cancers and cell lines are rare cell populations known to be enriched cancer stem-like cells. In this study, we used Hoechst 33342 staining and flow cytometry to identify SP cells from the human breast cancer cell lines MCF-7 and GI-101A as models for cancer stem-like cells. Considering the cytotoxicity of Hoechst dye and the restriction of instrument, we did not carry out further studies by this method. Utilizing in vitro and in vivo experimental systems, we showed that human breast cancer cell line GI-101A with aldehyde dehydrogenase activity (ALDH) have stemlike properties. Higher ALDH activity identifies the tumorigenic cell fraction which is capable of self-renewal and of generating tumors that could recapitulate the heterogeneity of the parental tumor. Furthermore, the cells with higher ALDH activity display significant resistance to chemotherapy and ionizing radiation, which proves their stem-like properties again. The cells which have higher ALDH activity also are more invasive compared to cells which have lower ALDH activity, which connects the cancer stem-like cells with cancer metastases. By analyzing the popular human breast cancer stem cells surface markers CD44, CD49f and CD24, it was discovered that the cells with higher ALDH activity have stronger CD44 and CD49f expression than in those cells with lower ALDH activity, which further confirms their stem-like properties. Finally, the cells with higher ALDH activity and lower ALDH activity were infected in vitro and used in virotherapy in a mouse xenograft model was performed. The results indicated that the vaccinia virus GLV-1h68 can replicate in cells with higher ALDH activity more efficiently than cells with lower ALDH activity. GLV-1h68 also can selectively target and eradicate the xenograft tumors which were derived from cells with higher ALDH activity. The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer invasion and metastases. EMT was induced in immortalized human mammary epithelial cells (HMLEs) and in GI-101A cells, which results in the acquisition of mesenchymal traits and in the expression of stem cell markers. Furthermore, the EMT-induced GI-101A cells showed resistance to chemotherapy and invasion capacity. CD44+/CD24- cells were enriched during the EMT induction. Following flow cytometry sorting by using CD44, CD24 and ESA surface marker, the sorted cells were tested in a mouse model regarding tumorigenicity. Unexpectedly, we found that CD44+/CD24+/ESA+ cells could initiate tumors more efficiently rather than CD44+/CD24-/ESA+ and other fractions in EMTinduced GI-101A cells. We also infected the CD44+/CD24+/ESA+ and CD44+/CD24- /ESA+ cells in vitro and performed virotherapy in a mouse xenograft model. The results indicated that the vaccinia virus GLV-1h68 is able to replicate in CD44+/CD24+/ESA+ cells more efficiently than in CD44+/CD24-/ESA+ cells. GLV-1h68 was also capable to selectively target and eradicate the xenograft tumors which derived from CD44+/CD24+/ESA+ cells. Moreover, CD44- cells have much lower tumorigenicity in the mouse model and CD44- cells derived-tumors are not responsive to vaccinia virotherapy. In summary, we have successfully established an in vitro and in vivo system for the identification, characterization and isolation of cancer stem-like cells from the human breast cancer cell line GI-101A by using the ALDEFLUOR assay. The vaccinia virus GLV-1h68 was able to efficiently target and eradicate the higher ALDH activity cells and tumors derived from those cells. Although contrary to the current assumption, CD44+/CD24+/ESA+ cells in the EMT-induced GI-101A cell line showed stem-like properties and GLV-1h68 was able to efficiently target and eradicate the CD44+/CD24+/ESA+ cells and tumors which derived from those cells. Finally, improved understanding of cancer stem cells may have tremendous relevance for how cancer should be treated. It is menacing that cancer stem cells are resistant to almost all anti-tumor approaches which have already been established for the treatment of metastatic diseases such as ionizing radiation, hormonal therapy, chemotherapy, and small molecular inhibitors. Therefore, it is promising that our results suggest that these cancer stem cells may be susceptible to treatment with oncolytic vaccinia virus.
Spir proteins are the founding members of the novel class of WH2-actin nucleators. A C-terminal modified FYVE zinc finger motif is necessary to target Spir proteins towards intracellular membranes. The function and regulation of the Spir actin organizers at vesicular membranes is almost unknown. Live cell imaging analyses performed in this study show that Spir-2 is localized at tubular vesicles. Cytoplasmic Spir-2-associated vesicles branch and form protrusions, which can make contacts to the microtubule network, where the Spir-2 vesicles stretch and slide along the microtubule filaments. The analysis of living HeLa cells expressing eGFP-tagged Spir-2, Spir-2-ΔKIND and Spir-2-ΔKW (lacking the 4 WH2 domains and the KIND domain) showed Spir-2-associated tubular structures which differ in their length and motility. Throughout the course of that study it could be shown that the tail domain of the actin motor protein myosin Vb, as a force-generating molecule, is colocalizing and co-immunoprecipitating with Spir-2-ΔKW. By using the tail domain of myosin Vb as a dominant negative mutant for myosin Vb-dependent vesicle transport processes it could be shown that Spir-2-ΔKW/MyoVb-cc-tail- associated vesicles exhibit an increased elongation. Moreover, using the microtubule depolymerizing drug nocodazole it could be shown that the elongation and the motility of Spir-2-ΔKW-associated vesicles depends on an intact microtubule cytoskeleton. Motility and morphological dynamics of Spir-2-associated vesicles is therefore dependent on actin, actin motorproteins and microtubule filaments. These results propose a model in which myosin/F-actin forces mediate vesicle branching, allowing the vesicles to move to and in between the microtubule filaments and thereby providing a new degree of freedom in vesicular motility. To determine the exact subcellular localization of Spir-2, colocalization studies were performed. It could be shown that Spir-2 shows a partial colocalization to Rab11a-positive compartments. Furthermore, Spir-2 exhibits an almost identical localization to Arf1 and the Arf1 small G protein but not Rab11a could be immunoprecipitated with Spir-2-ΔKW. This suggests, that Arf1 recruits Spir-2 to Arf1/Rab11a-positive membranes. Another important function of the Spir-2 C-terminus is the membrane targeting by the FYVE domain. By performing a protein-lipid overlay assay, it has been shown that purified GST- and 6xHis-tagged Spir-2-ΔKW bind phosphatidic acid suggesting a mechanism in which Spir-2 is recruited to phosphatidic acid-enriched membranes. To further elucidate the mechanism in which Spir-2 membrane-targeting could be regulated, interaction studies of C-terminal parts of Spir-2 revealed that the Spir-2 proteins interact directly.
This work delves into the recently developed ‘Whedo’-aquaculture-system in the rural community of Malanville (North Benin)and aims on providing a closer insight on this – for the area--recent system including the ecological but also the sociological and economical aspects in order to develop this extensive traditional fishery to a more productive semi-intensive aquaculture system. With the retreat of the flood ‘Whedos’ usually become infested with numerous hydato-and tenagophytes, while the presence and density of the free-floating macrophytes were positively related to the nutrient content of the ‘Whedo’. Extensive plant infestation also affects water quality through the decomposition of organic material and its accumulation in thick mud layers on the pond bottom as well as through the nocturnal oxygen consumption. Unfavourable water quality, especially low dissolved oxygen as well as high conductivity and nitrite levels, was identified to be the main factor determining which fish species were able to survive the harsh conditions prevailing in the ‘Whedos’ during the dry season. With the deteriorating water quality with advancing dry season, fish diversity decreased significantly leaving only species that are highly adapted to such unfavourable conditions. The most abundant species were Clarias gariepinus, Heterotis niloticus, Oreochromis niloticus L., Hemichromis c.f. letourneauxi, Polypterus senegalus and Epiplatys spilargyreius. Besides, the investigations also concentrated on the fish diversity of the rivers Niger and Sota with the results that for three species distribution gaps could be closed and for further three species their already known distribution could be expanded. But otherwise it could also be detected that some economically important species that were abundant in the past. In regard to the ‘Whedo’-management, the investigations showed that the owners lack most of the knowledge on appropriate management strategies, e.g. the feeding and stocking regime. The exploitation period depends on the extent of the previous annual flood and the location of the ‘Whedo’ within the floodplain, but the main season is from February to April. The biomass harvested on a hectare basis separated for each of the ‘Whedos’ averaged 17 tons/ha in 2008 and 8.6 tons/ha in 2009. However, 72 percent of the total biomass of Clarias only had an average weight of 40 grams. Therefore, two separated feeding trials were conducted and in total 6 supplementary feeds were tested on Clarias gariepinus. Groundnut cake, fish trash, rice bran, blood meal and azolla meal were used in different combination and rations to formulate the experimental diets. Diet containing 19 percent blood meal resulted in the best economical benefits showing that the use of high quality feed ingredients such as groundnut cake is not recommendable because local fish prices are too low to compensate the additional feeding costs. Instead of high quality feed farmers should focus on ingredients that are free of charge and easy to process. The supplementation based on 19 percent blood meal resulted in the doubling of the net profit compared to the income based on feeding only rice bran, thus provided higher additional income, enhancing the livelihood of the fish farmers. Concluding, the ‘Whedo’-aquaculture system is still in its infancy but nevertheless is an attractive system for the rural population because of existing knowledge of post-flood wetland fisheries as well as the low investment needed for its installation. Additionally, the local fish supply will increase and hence not only contribute to a better provision of protein-rich food and reduced pressure on the wild fish stocks but might also prevent fish prices to increase in a way that the poor won’t be able anymore to afford their most important source of animal protein. But fish farmers need more knowledge on appropriate management strategies and thus should be provided with technical support to guarantee a successful development and not to discourage the owners as a consequence of avoidable failures. Furthermore, the use of supplementary feed offers a cheap and effective means to increase the biomass production and thus enhance the extensive fishery to a semi-intensive aquaculture system.
Stroke, after myocardial infarction and cancer is the third most common cause of death worldwide and 1/6th of all human beings will suffer at least one stroke in their lives. Furthermore, it is the leading cause for adult disability with approximately one third of patients who survive for the next 6 months are dependent on others. Because of its huge socioeconomic burden absorbing 6% of all health care budgets and with the fact that life expectancy increases globally, one can assume that stroke is already, and will continue to be, the most challenging disease. Ischemic stroke accounts for approximately 80% of all strokes and results from a thrombotic or embolic occlusion of a major cerebral artery (most often the middle cerebral artery, MCA) or its branches Following acute ischemic stroke, the most worrisome outcome is the rapidly increasing intra-cranial pressure due to the formation of space-occupying vasogenic oedema which can have lethal consequences. Permeability changes at the Blood-Brain Barrier (BBB) usually accompanies the oedematous development and their time course can provide invaluable insight into the nature of the insult, activation of compensatory mechanisms followed by long term repair. Rodent models of focal cerebral ischemia have been developed and optimized to mimic human stroke conditions and serve as indispensable tools in the field of stroke research. The presented work constituting of three separate but complete works by themselves are sequential, where, the first part was dedicated to the establishment of non-invasive small animal imaging strategies on a 3 tesla clinical magnetic resonance scanner. This facilitated the longitudinal monitoring of pathological outcomes following stroke where identical animals can serve as its own control. Tissue relaxometric estimations were carried out initially to derive the transverse (T2), longitudinal (T1) and the transverse relaxation time due to magnetic susceptibility effects (T2*) at the cortical and striatal regions of the rodent brain. Statistically significant differences in T2*-values could be found between the cortex and striatal regions of the rodent brain. The derived tissue relaxation values were considered to modify the existing imaging protocols to facilitate the study of the rodent model of ischemic stroke. The modified sequence protocols adequately characterized all the clinically relevant sequels following acute ischemic stroke, like, the altered perfusion and diffusion characteristics. Subsequent to this, serial magnetic resonance imaging was performed to investigate the temporal and spatial relationship between the biphasic nature of BBB opening and, in parallel, the oedema formation after I/R injury in rats. T2-relaxometry for oedema assessment was performed at 1 h after ischemia, immediately following reperfusion, and at 4, 24 and 48 hours post reperfusion. Post-contrast T1-weighted imaging was performed at the last three time points to assess BBB integrity. The biphasic course of BBB opening with significant reduction in BBB permeability at 24 hours after reperfusion was associated with a progressive expansion of leaky BBB volume, accompanied by a peak ipsilateral oedema formation. At 48 hours, the reduction in T2-value indicated oedema resorption accompanied by a second phase of BBB opening. In addition, at 4 hours after reperfusion, oedema formation could also be detected at the contralateral striatum which persisted to varying degrees throughout the study, indicative of widespread effects of I/R injury. The observations of this study may indicate a dynamic temporal shift in the mechanisms responsible for biphasic BBB permeability changes, with non-linear relations to oedema formation. Two growth factor peptides namely pigment epithelium derived factor (PEDF) and epidermal growth factor (EGF) with widely different trophic properties were considered for their beneficial effects, if any, in the established rodent model of I/R injury and studied up to one week employing magnetic resonance imaging. Both the selected, trophic factors demonstrated significant neuroprotection as demonstrated by a reduction in infarct volume, even though PEDF was found to be the most potent one. PEDF also demonstrated significant attenuation of oedema formation in comparison to both the control and EGF groups, even though EGF could also demonstrate oedema suppression. In the present work, we noticed that interventions with macromolecule protein/peptides by itself could mediate remote oedema at distant sites even though the significance of such an observation is not clear at present. Susceptibility (T2*) weighted tissue relaxometric estimations were considered at the infarct region to detect any metabolic changes arising out of any neuroprotection and/or cellular proliferation / neurogenesis. PEDF group demonstrated a striking reduction of the T2*-values, which is indicative of an increased metabolic activity. Moreover, all the groups (Control, EGF and PEDF) demonstrated significantly elevated T2*-values at the contralateral striatum, which is indicative of widespread metabolic suppression usually associated with a variety of traumatic brain conditions. Moreover, as expected from the properties of PEDF, it demonstrated an extended BBB permeability suppression throughout the duration of the study. This study underlines the merits of considering non-invasive imaging strategies without which it was not possible to study the required parameters in a longitudinal fashion. All the observations are adequately supported by reasonably well defined mechanisms and needs to be further verified and confirmed by an immunohistochemical study. These results also need to be complemented by a functional study to evaluate the behavioural outcome of animals following these treatments. These studies are progressing at our laboratory and the results will be duly published afterwards.
The Ecology and Population structure of the invasive Yelllow Crazy Ant Anoplolepis gracilipes
(2011)
The invasive Yellow Crazy Ant Anoplolepis gracilipes is a widespread tropical ant species which is particularly common in anthropogenically disturbed habitats in South-East Asia and the Indopacific region. Its native range is unknown, and there is little information concerning its social structure as a potential mechanism facilitating invasion as well as its ecology in one of the putative native ranges, South-East Asia. Using mitochondrial DNA sequences, I demonstrated that the majority of the current Indopacific colonies were likely introduced from South-East Asian populations, which in turn may have been introduced much earlier from a yet unidentified native range. By conducting behavioral, genetic and chemical analyses, I found that A. gracilipes supercolonies contain closely related individuals, thus resembling enlarged versions of monogynous, polydomous colonies of other ant species. Furthermore, mutually aggressive A. gracilipes supercolonies were highly differentiated both genetically and chemically, suggesting limited or even absent gene flow between supercolonies. Intranidal mating and colony-budding are most likely the predominant, if not the exclusive mode of reproduction and dispersal strategy of A. gracilipes. Consequently, a positive feedback between genetic, chemical and behavioral traits may further enhance supercolony differentiation though genetic drift and neutral evolution. This potential scenario led to the hypothesis that absent gene flow between different A. gracilipes supercolonies may drive them towards different evolutionary pathways, possibly including speciation. Thus, I examined one potential way by which gene flow between supercolonies of an ant species without nuptial flights may be maintained, i.e. the immigration of sexuals into foreign supercolonies. The results suggest that this option of maintaining gene flow between different supercolonies is likely impaired by severe aggression of workers towards allocolonial sexuals. Moreover, breeding experiments involving males and queens from different supercolonies suggest that A. gracilipes supercolonies may already be on the verge of reproductive isolation, which might lead to the diversification of A. gracilipes into different species. Regarding the ecological consequences of its potential introduction to NE-Borneo, I could show that A. gracilipes supercolonies may affect the local ant fauna. The ant community within supercolonies was less diverse and differed in species composition from areas outside supercolonies. My data suggest that the ecological dominance of A. gracilipes within local ant communities was facilitated by monopolization of food sources within its supercolony territory, achieved by a combination of rapid recruitment, numerical dominance and pronounced interspecific aggression. A. gracilipes’ distribution is almost exclusively limited to anthropogenically altered habitat, such as residential and agricultural areas. The rate at which habitat conversion takes place in NE-Borneo will provide A. gracilipes with a rapidly increasing abundance of suitable habitats, thus potentially entailing significant population growth. An potentially increasing population size and ecological dominance, however, are not features that are limited to invasive alien species, but may also occur in native species that become ‘pests’ in an increasing abundance of anthropogenically altered habitat. Lastly, I detected several ant guests in supercolonies of A. gracilipes. I subsequently describe the relationship between one of them (the cricket Myrmecophilus pallidithorax) and its ant host. By conducting behavioral bioassays and analyses of cuticular hydrocarbon (CHC) profiles, I revealed that although M. pallidithorax is attacked and consumed by A. gracilipes whenever possible, it may evade aggression from its host by a combination of supreme agility and, possibly, chemical deception. This thesis adds to our general understanding of biological invasions by contributing species-specific data on a previously understudied invasive organism, the Yellow Crazy Ant Anoplolepis gracilipes. Introductions which may have occurred a long time ago may make it difficult to determine whether a given species is an introduced invader or a native pest species, as both may have pronounced ecological effects in native species communities. Furthermore, this thesis suggests that supercolonialism in invasive ants may not be an evolutionary dead end, but that it may possibly give rise to new species due to reproductive boundaries between supercolonies evoked by peculiar mating and dispersal strategies.
Molecular modelling and simulation are powerful methods in providing important in-formation on different biological systems to elucidate their structural and functional proper-ties, which cannot be determined in experiment. These methods are applied to analyse versa-tile biological systems: lipid membrane bilayers stabilized by an intercalated single wall carbon nanotube and retroviral proteins such as HIV protease and integrase. HIV-1 integrase has nuclear localization signals (NLS) which play a crucial role in nuclear import of viral preintegration complex (PIC). However, the detailed mechanisms of PIC formation and its nuclear transport are not known. Previously it was shown that NLSs bind to the cell transport machinery e.g. proteins of nuclear pore complex such as transportins. I investigated the interaction of this viral protein HIV-1 integrase with proteins of the nuclear pore complex such as transportin-SR2 (Shityakov et al., 2010). I showed that the transportin-SR2 in nuclear import is required due to its interaction with the HIV-1 integrase. I analyzed key domain interaction, and hydrogen bond formation in transportin-SR2. These results were discussed in comparison to other retroviral species such as foamy viruses to better understand this specific and efficient retroviral trafficking route. The retroviral nuclear import was next analyzed in experiments regarding the retroviral ability to infect nondividing cells. To accomplish the gene transfer task successfully, ret-roviruses must efficiently transduce different cell cultures at different phases of cell cycle. However, promising and safe foamy viral vectors used for gene transfer are unable to effi-ciently infect quiescent cells. This drawback was due to their inability to create a preintegra-tion complex (PIC) for nuclear import of retroviral DNA. On the contrary, the lentiviral vec-tors are not dependant on cell cycle. In the course of reverse transcription the polypurine tract (PPT) is believed to be crucial for PIC formation. In this thesis, I compared the transduction frequencies of PPT modified FV vectors with lentiviral vectors in nondividing and dividing alveolar basal epithelial cells from human adenocarcinoma (A549) by using molecular cloning, transfection and transduction techniques and several other methods. In contrast to lentiviral vectors, FV vectors were not able to effi-ciently transduce nondividing cell (Shityakov and Rethwilm, unpublished data). Despite the findings, which support the use of FV vectors as a safe and efficient alternative to lentiviral vectors, major limitation in terms of foamy-based retroviral vector gene transfer in quiescent cells still remains. Many attempts have been made recently to search for the potential molecules as pos-sible drug candidates to treat HIV infection for over decades now. These molecules can be retrieved from chemical libraries or can be designed on a computer screen and then synthe-sized in a laboratory. Most notably, one could use the computerized structure as a reference to determine the types of molecules that might block the enzyme. Such structure-based drug design strategies have the potential to save off years and millions of dollars compared to a more traditional trial-and-error drug development process. After the crystal structure of the HIV-encoded protease enzyme had been elucidated, computer-aided drug design played a pivotal role in the development of new compounds that inhibit this enzyme which is responsible for HIV maturation and infectivity. Promising repre-sentatives of these compounds have recently found their way to patients. Protease inhibitors show a powerful sustained suppression of HIV-1 replication, especially when used in combi-nation therapy regimens. However, these drugs are becoming less effective to more resistant HIV strains due to multiple mutations in the retroviral proteases. In computational drug design I used molecular modelling methods such as lead ex-pansion algorithm (Tripos®) to create a virtual library of compounds with different binding affinities to protease binding site. In addition, I heavily applied computer assisted combinato-rial chemistry approaches to design and optimize virtual libraries of protease inhibitors and performed in silico screening and pharmacophore-similarity scoring of these drug candidates. Further computational analyses revealed one unique compound with different protease bind-ing ability from the initial hit and its role for possible new class of protease inhibitors is dis-cussed (Shityakov and Dandekar, 2009). A number of atomistic models were developed to elucidate the nanotube behaviour in lipid bilayers. However, none of them provided useful information for CNT effect upon the lipid membrane bilayer for implementing all-atom models that will allow us to calculate the deviations of lipid molecules from CNT with atomistic precision. Unfortunately, the direct experimental investigation of nanotube behaviour in lipid bilayer remains quite a tricky prob-lem opening the door before the molecular simulation techniques. In this regard, more de-tailed multi-scale simulations are needed to clearly understand the stabilization characteristics of CNTs in hydrophobic environment. The phenomenon of an intercalated single-wall carbon nanotube in the center of lipid membrane was extensively studied and analyzed. The root mean square deviation and root mean square fluctuation functions were calculated in order to measure stability of lipid mem-branes. The results indicated that an intercalated carbon nanotube restrains the conformational freedom of adjacent lipids and hence has an impact on the membrane stabilization dynamics (Shityakov and Dandekar, 2011). On the other hand, different lipid membranes may have dissimilarities due to the differing abilities to create a bridge formation between the adherent lipid molecules. The results derived from this thesis will help to develop stable nanobiocom-posites for construction of novel biomaterials and delivery of various biomolecules for medi-cine and biology.