Kunstlike ja rakuvälise maatriksi valkudega kaetud pindade osa rakkude kasvu ja diferentseerumise reguleerimises

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Loomade kõik rakud puutuvad oma elu jooksul kokku neid ümbritseva rakuvälise maatriksiga. See on rakkude poolt sekreteeritud vaheaine, mis peamiselt koosneb valkudest ja polüsahhariididest. Koes annab rakuväline maatriks rakkudele mehhaanilise toe ja võimaldab üles ehitada keerukaid organeid. Lisaks mõjutab rakuväline maatriks rakkude kasvu, migreerumist ja diferentseerumist, kuivõrd maatriksi komponentidelt lähtuvad neid protsesse toetavad signaalid. Ühed enimuuritud rakuvälise maatriksi valgud on kollageenid ja laminiinid. Käesolevas doktoritöös uuriti rakk-maatriks kontaktide mõju rakkude kasvule ja diferentseerumisele. Esmalt uuriti kuidas rakkude kasvupinna topograafilised omadused mõjutavad inimese naha fibroblastide kasvu. Kasvupinnasena kasutati ränil põhinevat materjali (n.ö tehisklaasi), mis andis võimaluse tekitada pindu, mis oli kaetud erineva suurusega ümarate struktuuridega. Nende struktuuride keskmine diameeter oli 200 nm, 500nm, 1 µm ja 10 µm. Selgus, et mida suuremad olid struktuurid (diameeter 500 nm, 1 µm ja 10 µm), seda aeglasem oli rakkude kasv ning sellega kaasnes rakkude nn „vananemine”. Meie teine uuring näitas, et želatiini ja glükoosi elektrospinnimise teel on võimalik tekitada kasvupindasid, mis sobivad inimese naha fibroblastide kasvatamiseks. Lisaks ilmnes, et sellised kasvupinnad tõstavad rakuvälise maatriksi valkude, laminiinide, ekspressiooni taset fibroblastides. Kolmandas uuringus käsitlesime laminiinide paiknemist ja sekreteerimist inimese trombotsüütidest. Vastupidiselt varemarvatule selgus, et laminiinid ei paikne trombotsüütide α-graanulites. Kui trombotsüüte aktiveeriti trombiiniga, siis väljusid laminiinid rakkudest mikrovesiikulite, mitte aga eksosoomide koosseisus. Doktoritöö neljandas uurimuses iseloomustati laminiinide ekspressiooni inimese embrüonaalsetes tüvirakkudes. Selgus, et retinoolhappega mõjutatud tüvirakkude varajase diferentseerumise käigus suureneb neis laminiini 511 hulk, aga laminiini 521 hulk väheneb. Lisaks leiti, et inimese embrüonaalsetes tüvirakkudes ekspresseerub lisaks varemkirjeldatule veel mitmeid teisi laminiinide isovorme. Veel enam, selgus, et mitmed erinevad laminiinide isovormid ekspresseeruvad ka sellistes inimese embrüonaalsetes tüvirakkudes, mis ei ole läinud diferentseerumise teele.All animal cells interact with extracellular matrix (ECM) either continuously or during important phases of their life-time. ECM provides mechanical support for tissues and is thus necessary for building up organs with complex structures. The interactions between cells and ECM regulate cell growth, migration and differentiation. The major components of ECM are proteins and polysaccharides that are secreted by various cell types. Collagens and laminins are examples of widely-studied ECM proteins. The research presented in the current thesis was designed to study the ways in which cells interact with the surrounding extracellular matrix and how these bidirectional interactions affect cell growth and differentiation. Firstly, we studied how topographical properties of silica-based surfaces affect the growth of human dermal fibroblasts. Surfaces with four different sizes of round structural elements were produced (200 nm, 500nm, 1 µm ja 10 µm). It was found that increase in the size of these structural elements coincided with the decrease in cell growth and induced cell senescence. The second study demonstrated that electrospun gelatin-based scaffolds that contain glucose are suitable for growth of primary dermal fibroblasts. These scaffolds induced expression of ECM proteins such as laminins in fibroblasts. The third study investigated the localization and secretion of laminins in human platelets. Our investigation revealed that platelets do not store laminins in typical alpha granules as was shown previously, and that these proteins are secreted via microvesicles but not via exosomes when platelets become activated. The fourth study of this thesis characterized the expression of laminins in human embryonic stem cells. We observed that the relative amount of laminin-511 increases while the amount of laminin-521 decreases during early differentiation induced by retinoic acid. Additionally, we found that human embryonic stem cells express a wider range of different laminin chains than previously described, and that the laminin repertoire was independent of their differentiation status

Influence of rivet to sheet edge distance on fatigue strength of self-piercing riveted aluminium joints

Self-piercing riveting (SPR) is one of the main joining methods for lightweight aluminium automotive body structures due to its advantages. In order to further optimise the structure design and reduce the weight but without compromising strength, reduction of redundant materials in the joint flange area can be considered. For this reason, the influence of rivet to sheet edge distance on the fatigue strengths of self-piercing riveted joints was studied. Five edge distances, 5 mm, 6 mm, 8 mm, 11.5 mm and 14.5 mm, were considered. The results showed that the SPR joints studied in this research had high fatigue resistance and all specimens failed in sheet material along joint buttons or next to rivet heads. For lap shear fatigue tests, specimens failed in the bottom sheet at low load amplitudes and in the top sheet at high load amplitudes except for specimens with very short edge distance of 5 and 6 mm; whereas, for coach-peel fatigue tests, all specimens failed in the top sheet. For both lap shear and coach-peel fatigue tests, specimens with an edge distance of 11.5 mm had the best fatigue resistance. It was found that for coach-peel fatigue, length of crack developing path before specimens lost their strengths was the main factor that determined the fatigue life of different specimens; for lap shear fatigue, the level of stress concentration and subsequent crack initiation time was the main factor that determined the fatigue life

Correlation of eigenstates in the critical regime of quantum Hall systems

We extend the multifractal analysis of the statistics of critical wave functions in quantum Hall systems by calculating numerically the correlations of local amplitudes corresponding to eigenstates at two different energies. Our results confirm multifractal scaling relations which are different from those occurring in conventional critical phenomena. The critical exponent corresponding to the typical amplitude, $\alpha_0\approx 2.28$, gives an almost complete characterization of the critical behavior of eigenstates, including correlations. Our results support the interpretation of the local density of states being an order parameter of the Anderson transition.Comment: 17 pages, 9 Postscript figure

MoBPS - Modular Breeding Program Simulator

The R-package MoBPS provides a computationally efficient and flexible framework to simulate complex breeding programs and compare their economic and genetic impact. Simulations are performed on the base of individuals. MoBPS utilizes a highly efficient implementation with bit-wise data storage and matrix multiplications from the associated R-package miraculix allowing to handle large scale populations. Individual haplotypes are not stored but instead automatically derived based on points of recombination and mutations. The modular structure of MoBPS allows to combine rather coarse simulations, as needed to generate founder populations, with a very detailed modeling of todays’ complex breeding programs, making use of all available biotechnologies. MoBPS provides pre-implemented functions for common breeding practices such as optimum genetic contributions and single-step GBLUP but also allows the user to replace certain steps with personalized and/or self-written solutions

Utilising FGF2, IGF2 and FSH in serum-free protocol for long-term in vitro cultivation of primary human granulosa cells

Human granulosa cells acquired as leftover from IVF treatment can be used to study infertility problems and area valuable tool in the research of follicle maturation and ovulation. There is a need for more defined and long-term culture protocols for studying the response of granulosa cells upon treatment with selected hormones/chemicals. In the current study, we tested the effect of adding FGF2, IGF2 and FSH into defined basal medium inorder tofind culture conditions that would support proliferation of cumulus and mural granulosa cells alongwith the expression of common granulosa cell type markers such asFSHR,AMHR2,LHRandCYP19A1. We foundthat FGF2, IGF2 together with FSH helped to retain granulosa cell marker expression while supporting cellsurvival at least for two weeks of culture. The defined serum-free culture conditions for long-term culturing willbe valuable in providing new standards in the research of human granulosa cells

Target prediction and validation of microRNAs expressed from FSHR and aromatase genes in human ovarian granulosa cells

MicroRNAs (miRNAs) are known post-transcriptional regulators of various biological processes including ovarian follicle development. We have previously identified miRNAs from human preovulatory ovarian granulosa cells that are expressed from the intronic regions of two key genes in normal follicular development: FSH receptor (FSHR) and CYP19A1, the latter encoding the aromatase enzyme. The present study aims to identify the target genes regulated by these miRNAs: hsa-miR-548ba and hsa-miR-7973, respectively. The miRNAs of interest were transfected into KGN cell line and the gene expression changes were analyzed by Affymetrix microarray. Potential miRNA-regulated genes were further filtered by bioinformatic target prediction algorithms and validated for direct miRNA:mRNA binding by luciferase reporter assay. LIFR, PTEN, NEO1 and SP110 were confirmed as targets for hsa-miR-548ba. Hsa-miR-7973 target genes ADAM19, PXDN and FMNL3 also passed all verification steps. Additionally, the expression pattern of the miRNAs was studied in human primary cumulus granulosa cell culture in relation to the expression of their host genes and FSH stimulation. Based on our findings we propose the involvement of hsa-miR-548ba in the regulation of follicle growth and activation via LIFR and PTEN. Hsa-miR-7973 may be implicated in the modulation of extracellular matrix and cell-cell interactions by regulating the expression of its identified targets.Peer reviewe

Assessment of the Potential of CDK2 Inhibitor NU6140 to Influence the Expression of Pluripotency Markers NANOG, OCT4, and SOX2 in 2102Ep and H9 Cells

As cyclin-dependent kinases (CDKs) regulate cell cycle progression and RNA transcription, CDKs are attractive targets for creating cancer cell treatments. In this study we investigated the effects of the small molecular agent NU6140 (inhibits CDK2 and cyclin A interaction) on human embryonic stem (hES) cells and embryonal carcinoma-derived (hEC) cells via the expression of transcription factors responsible for pluripotency. A multiparameter flow cytometric method was used to follow changes in the expression of NANOG, OCT4, and SOX2 together in single cells. Both hES and hEC cells responded to NU6140 treatment by induced apoptosis and a decreased expression of NANOG, OCT4, and SOX2 in surviving cells. A higher sensitivity to NU6140 application in hES than hEC cells was detected. NU6140 treatment arrested hES and hEC cells in the G2 phase and inhibited entry into the M phase as evidenced by no significant increase in histone 3 phosphorylation. When embryoid bodies (EBs) formed from NU6104 treated hES cells were compared to EBs from untreated hES cells differences in ectodermal, endodermal, and mesodermal lineages were found. The results of this study highlight the importance of CDK2 activity in maintaining pluripotency of hES and hEC cells and in differentiation of hES cells