<p>Hyaluronan (HA), a structural component in the extracellular matrix (ECM), has been recognized as a signaling molecule. It is important during various biological activities such as embryogenesis, angiogenesis, wound healing and tumor progression. Increased amount of hyaluronan during embryonic development is necessary for cell migration and differentiation, but the increased production of hyaluronan by tumor cells or tissue fibroblasts is correlated to poor prognosis for tumor progression and chronic inflammation, respectively. Therefore, understanding the mechanisms regulating HA-enriched matrices and the roles of HA in the biological functions is of fundamental biological importance.</p><p>Four novel findings are described in this thesis: (1) HA fragments (HA12) and the known angiogenic factor FGF-2 promote endothelial cell differentiation by induction of common but also distinct sets of genes, particularly, upregulation of the chemokine <i>CXCL1/GRO1</i> gene is necessary for HA12-induced angiogenesis and this effect is dependent on CD44 activation. (2) High concentrations of hyaluronan suppress PDGF-BB-induced fibroblasts migration and PDGFRβ tyrosine phosphorylation upon activation of hyaluronan receptor CD44, probably by recruiting a CD44-associated phosphatase to the PDGFRβ. (3) PDGF-BB stimulates <i>HAS2</i> transcriptional activity and HA synthesis through upregulation of MAP kinase and PI3 kinase signaling pathways in human dermal fibroblasts. (4) Specific suppression of <i>HAS2</i> gene in the invasive breast cancer cell line Hs578T by RNA interference (RNAi) leads to a less aggressive phenotype of breast tumor cells. This suppressive effect can be reversed by exogenously added hyaluronan.</p><p>In conclusion, binding of hyaluronan to CD44 plays an important role in cell signaling, inflammation and tumor progression. Further studies are required to elucidate the molecular mechanisms through which hyaluronan levels are regulated under physiological or pathological conditions, and to explore compounds involved in hyaluronan accumulation and activity as targets for therapies of chronic inflammation and tumors.</p>

This thesis describes recombinant proteins that have been generated by rational and combinatorial protein engineering strategies for use in subunit vaccine delivery and tumor targeting. In a first series of studies, recombinant methods for incorporating immunogens into an adjuvant formulation, e.g. immunostimulating complexes (iscoms), were evaluated. Protein immunogens, which are not typically immunogenic in themselves, are normally administered with an adjuvant to improve their immunogenicity. To accomplish iscom incorporation of a Toxoplasma gondii surface antigen through hydrophobic interaction, lipids were added either in vivo via E. coli expression, or in vitro via interaction of an introduced hexahistidyl (His6) peptide and a chelating lipid. The possibility of exploiting the strong interaction between biotin and streptavidin was also explored, in order to couple a Neospora caninum surface antigen to iscom matrix, i.e. iscom particles without any antigen. Subsequent analyses confirmed that the immunogens were successfully incorporated into iscoms by the investigated strategies. In addition, immunization of mice with the recombinant Neospora antigen NcSRS2, associated with iscoms through the biotin-streptavidin interaction, induced specific antibodies to native NcSRS2 and reduced clinical symptoms following challenge infection. The systems described in this thesis might offer convenient and efficient methods for incorporating recombinant immunogens into adjuvant formulations that might be considered for the generation of future recombinant subunit vaccines. In a second series of studies, Affibody® (affibody) ligands directed to the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu), which is known to be overexpressed in ∼ 20-30% of breast cancers, were isolated by phage display in vitro selection from a combinatorial protein library based on the 58 amino acid residue staphylococcal protein A-derived Z domain. Biosensor analyses demonstrated that one of the variants from the phage selection, denoted His6-ZHER2/neu:4, selectively bound with nanomolar affinity (KD ≈ 50 nM) to the extracellular domain of HER2/neu (HER2-ECD) at a different site than the monoclonal antibody trastuzumab. In order to exploit avidity effects, a bivalent affibody ligand was constructed by head-to-tail dimerization, resulting in a 15.6 kDa affibody ligand, termed His6-(ZHER2/neu:4)2, that was shown to have an improved apparent affinity to HER2-ECD (KD ≈ 3 nM) compared to the monovalent affibody. Moreover, radiolabeled monovalent and bivalent affibody ligands showed specific binding in vitro to native HER2/neu molecules expressed in human cancer cells. Biodistribution studies in mice carrying SKOV-3 xenografted tumors revealed that significant amounts of radioactivity were specifically targeted to the tumors in vivo, and the tumors could easily be visualized with a gamma camera. These results suggest that affibody ligands would be interesting candidates for specific tumor targeting in clinical applications, such as in vivo imaging and radiotherapy.

Conventional cancer treatment based on radiotherapy or chemotherapy affects all dividing cells. By directing the therapy specifically to the tumor cells, normal cells can be spared. Tumor targeting molecules carrying a cytotoxic moiety is then an attractive approach. In this thesis, an affibody molecule with high affinity for the protein HER-2, that is strongly associated with aggressive forms of breast cancer, was selected. After radiolabeling with 125I, the affibody molecule, in monovalent and bivalent form, was tested in vitro in HER-2 overexpressing tumor cells and in transplanted tumors in mice. It was shown that the HER-2 targeting affibody molecule bound its target in a specific manner, both in vitro and in vivo. The small size of the affibody molecule resulted in fast clearance through the kidneys. An impressive tumor-to-blood ratio of 10 eight hours post injection was achieved and the tumors could easily be visualized in a gamma camera. The biologic effects of the bivalent affibody molecule and a monovalent affinity maturated version was measured and compared with the effects of the monoclonal antibody trastuzumab. It was found that although all molecules target the same protein, the effects differed greatly. The affibody molecule was also labeled with the alpha-emitting radionuclide 211At. Specific decrease in survival was seen in HER-2 overexpressing cells receiving the 211At labeled affibody molecule. The sensitivity to the treatment differed between cell lines, probably as a result of differences between the cell lines in internalization and nuclear size. The 211At labeled affibody molecules were also tested in vivo, where stability of the 211At label was a problem. To circumvent this problem, more stable conjugation chemistry was tested, as well as strategies to prevent uptake of released 211At by normal organs. This thesis describes the selection and optimization of affibody molecules for medical use for the first time.

Hyaluronan (HA), a structural component in the extracellular matrix (ECM), has been recognized as a signaling molecule. It is important during various biological activities such as embryogenesis, angiogenesis, wound healing and tumor progression. Increased amount of hyaluronan during embryonic development is necessary for cell migration and differentiation, but the increased production of hyaluronan by tumor cells or tissue fibroblasts is correlated to poor prognosis for tumor progression and chronic inflammation, respectively. Therefore, understanding the mechanisms regulating HA-enriched matrices and the roles of HA in the biological functions is of fundamental biological importance. Four novel findings are described in this thesis: (1) HA fragments (HA12) and the known angiogenic factor FGF-2 promote endothelial cell differentiation by induction of common but also distinct sets of genes, particularly, upregulation of the chemokine CXCL1/GRO1 gene is necessary for HA12-induced angiogenesis and this effect is dependent on CD44 activation. (2) High concentrations of hyaluronan suppress PDGF-BB-induced fibroblasts migration and PDGFRβ tyrosine phosphorylation upon activation of hyaluronan receptor CD44, probably by recruiting a CD44-associated phosphatase to the PDGFRβ. (3) PDGF-BB stimulates HAS2 transcriptional activity and HA synthesis through upregulation of MAP kinase and PI3 kinase signaling pathways in human dermal fibroblasts. (4) Specific suppression of HAS2 gene in the invasive breast cancer cell line Hs578T by RNA interference (RNAi) leads to a less aggressive phenotype of breast tumor cells. This suppressive effect can be reversed by exogenously added hyaluronan. In conclusion, binding of hyaluronan to CD44 plays an important role in cell signaling, inflammation and tumor progression. Further studies are required to elucidate the molecular mechanisms through which hyaluronan levels are regulated under physiological or pathological conditions, and to explore compounds involved in hyaluronan accumulation and activity as targets for therapies of chronic inflammation and tumors.

Poly(A) polymerase (PAP) is the enzyme catalyzing the synthesis of the adenine tail to the 3’-end of mRNA. This A-tail is present on the majority of the primary RNA transcripts of protein-coding genes, and is important for mRNA stability, export to the cytoplasm and translation. Therefore, PAP is a key regulator of eukaryotic gene expression. This thesis describes the heterogeneity of PAP and the functional significance of multiple isoforms of PAP. PAP exists in many different isoforms generated by three different mechanisms, gene duplication, alternative mRNA processing and post-translational modification. In HeLa cell extracts three different forms of PAP being 90, 100 and 106 kDa in size have been detected, where the 106 kDa isoform is a phosphorylated version of the 100 kDa species. It is shown that the N-terminal region of PAP contains a region required for catalysis, while the C-terminal end is important for the interaction with the cleavage and polyadenylation specificity factor (CPSF). Interestingly, it was found that also the extreme N-terminal end is important for the interaction with CPSF. This region is post-translationally modified by phosphorylation. Five alternatively spliced forms of PAP mRNAs are encoded by the PAPOLA gene while one unique species is encoded by the PAPOLG gene. The analysis showed that the exact structure of the alternatively spliced C-terminal end of PAP played an important role for catalytic efficiency. Thus, the C-terminal end contains a region important for modulating the catalytic efficiency of PAP. Aminoglycoside antibiotics inhibit PAP activity, most likely by displacement of catalytically important divalent metal ions. Data shows that different aminoglycosides inhibit PAP activity by different mechanisms suggesting that the binding sites for the different aminoglycosides do not completely overlap. It is concluded that aminoglycosides interfere with enzymes important for housekeeping functions in mammalian cell, which may explain some of the toxic side effects caused by aminoglycoside antibiotics in clinical practice.

Šiame magistro darbe nagrinėjama mokslo laisvė kaip teisės kategorija bei atskleidžiami mokslo laisvės ypatumai ir ribos biomedicinoje. Kadangi mokslo laisvės turinys iki šiol yra neaiškus, susidarė prielaidos atsirasti teisiniams ir praktiniams neaiškumams mokslo laisvės reglamentavimo srityje. Esminės problemos, susijusios su mokslo laisvės kaip teisės kategorijos neapibrėžtumu yra mokslo laisvės ribų teisinio reglamentavimo, mokslo laisvės biomedicinoje tapatinimo su teise (laisve) ir mokslo laisvės biomedicinoje tapatinimo su esminiu principu problemos. Todėl šio darbo tikslas – išanalizuoti ir įvertinti mokslo laisvės kaip teisės kategorijos ypatumus biomedicinos kontekste. Magistriniame darbe analizuojama mokslo laisvės teisinio reglamentavimo tarptautiniuose ir nacionaliniuose teisės aktuose raida, nagrinėjama mokslininko teisė (laisvė) atlikti mokslinį tyrimą ir tiriamojo asmens teisė naudotis mokslo pažangos vaisiais bei įvertinami mokslo laisvės ypatumai ir ribos biomedicinoje. / The thesis analyses freedom of science as a category of law and its peculiarities in biomedicine. Freedom of science as a legal category is not adequately regulated and cannot sufficiently provide the legal basis for protecting society against adverse effects resulting from biomedical and technological development. The main problems associated with the regulation of the freedom of science are the problem of regulating the limits of the freedom of science, the problem associated with the conception of the freedom of science as a human right (freedom), and the problem concerned with the conception of the freedom of science as a fundamental principle. Therefore the aim of the thesis was to analyze and evaluate the characteristics of the freedom of science in biomedicine. The thesis analyses the right (freedom) of the scientist to perform research and the right of the investigated subject to enjoy the benefits of scientific progress and its applications, as well as the peculiarities and limitations of the freedom of science in biomedicine.

The thesis was designed to gain insight into how Alzheimer’s disease influences selfhood from first-personal accounts of illness. The focus of the study was narrowed further by concentrating on the autobiographies of individuals diagnosed with Early-Onset Alzheimer’s disease (EOAD). The purpose of this thesis was to analyze the autobiographies of individuals with EOAD with the aim of understanding their selfhood. In this thesis I argue that, Alzheimer’s disease may influence a change in self, however, the self is not lost entirely. This thesis draws on the philosophical conception of narrated self as it allows for one perpetually constructed self, whereby a change in self does not necessarily mean the self is lost entirely. Through an interpretive analysis of six autobiographical accounts of Alzheimer’s, this thesis demonstrates that Alzheimer’s disease influences a loss of sense of self but that autobiography enables individuals with Alzheimer’s to (re)construct self.

The purpose of this writing is to analyses the doctrine of informed consent. Person exercises his autonomy by making informed and reasonable decisions. The writing attempts to disclose a theoretical and practical role of the informed consent institute and its influence on physician’s civil liability. This work consists of five parts. First of all is given ethical importance of informed consent doctrine. In the following parts of the work it is analyzed national and international regulations on informed consent. There is short Lithuanian case law on informed consent overview. At the end it is concluded that there are some gaps in Lithuanian law concerned with informed consent.

This thesis proposes the relevance of epigenetic research to feminist studies and gender studies, and vice versa, and asks how epigenetics speaks to the so-called sex-gender distinction. It also discusses what epigenetics could potentially tell us about ourselves, and our place in a world where we are all creatures of both nature and nurture. The author proposes that with its promise of insight into the relationship of the body to environment and experience over time, epigenetics could be an inextricable link between nature and nurture. Combining a modified version of diffractive analysis, and gender/sex as an analytical device, the author engages with epigenetic research and its representation in popular science and in the public imaginary. After discussing the striations of feminist discourse on permeable bodies, the author proposes epigenetics as another layer in the strata, placing epigenetics within feminist and gender studies literature and discourse. Noting that as research gains ground the way in which the public imagines and describes epigenetics gives shape to its materialization and development, this thesis asserts the urgent need for social sciences, and in particular feminist and gender studies, to engage in critical discourse

This thesis proposes the relevance of epigenetic research to feminist studies and gender studies, and vice versa, and asks how epigenetics speaks to the so-called sex-gender distinction. It also discusses what epigenetics could potentially tell us about ourselves, and our place in a world where we are all creatures of both nature and nurture. The author proposes that with its promise of insight into the relationship of the body to environment and experience over time, epigenetics could be an inextricable link between nature and nurture. Combining a modified version of diffractive analysis, and gender/sex as an analytical device, the author engages with epigenetic research and its representation in popular science and in the public imaginary. After discussing the striations of feminist discourse on permeable bodies, the author proposes epigenetics as another layer in the strata, placing epigenetics within feminist and gender studies literature and discourse. Noting that as research gains ground the way in which the public imagines and describes epigenetics gives shape to its materialization and development, this thesis asserts the urgent need for social sciences, and in particular feminist and gender studies, to engage in critical discourse with epigenetic research as it is carried out and as it is translated to the wider public.