A mouse model for improving cell survival of bisected cattle embryos

Selostus: Hiiren alkio mallina solukestävyyden parantamiseksi naudan alkioiden halkaisussa

Novel Roles of HCG/LH Signalling in the Mouse Mammary Gland and its Tumorigenesis.

Breast cancer is the most common cancer in women, and its development is intimately related to hormonal factors, but how hormones affect breast physiology and tumorigenesis is not sufficiently known. Pregnancy elicits long-term protection from breast cancer, but during the first ten years after pregnancy, breast cancer risk is increased. In previous studies, there has been conflicting data on the role of human chorionic gonadotropin (HCG) and the functionality of its receptor in extragonadal tissues. The aim of this study was to elucidate the role of chronically elevated HCG in mouse physiology. We have created a transgenic (TG) mouse model that overexpresses HCG. HCG is similar to lutenizing hormone (LH), but is secreted almost solely by the placenta during pregnancy. HCG and LH both bind to the LH receptor (LHR). In the current study, mammary gland tumors were observed in HCG TG mice. We elucidated the role of HCG in mammary gland signalling and the effects of LHR mediated signalling in mouse mammary gland gene expression. We also studied the effects of HCG in human breast epithelial cell cultures. Several endocrine disturbances were observed in HCGβ TG female mice, resulting in precocious puberty, infertility, obesity and pituitary and mammary gland tumors. The histology of the mammary gland tumors of HCGβ TG females resembled those observed in mouse models with activated Wnt/β-catenin signalling pathway. Wnts are involved in stem cell regulation and tumorigenesis, and are hormonally regulated in the mammary gland. We observed activated β-catenin signalling and elevated expression of Wnt5b and Wnt7b in TG tumors and mammary glands. Furthermore, we discovered that HCG directly regulates the expression of Wnt5b and Wnt7b in the mouse mammary gland. Pharmacological treatment with HCG also caused upregulation of several Wnt-pathway target genes in ovariectomized wild type (WT) mice in the presence of physiological concentrations of estradiol and progesterone. In addition, differential expression of several metabolic genes was observed, suggesting that HCG affects adipocyte function or glucose metabolism. When WT mice were transplanted with LHR deficient or wild type WT mammary epithelium, differential expression of several genes affecting the Wnt-signalling pathway was observed in microarray analysis. Diminished expression of several genes associated with LHR function in other tissues, such as the ovary, was observed in mammary glands deficient of epithelial LHR. In cultured human mammary epithelial cells HCG upregulated the expression of WNT5B, WNT7B similar to mouse, suggesting that the observations found are relevant in human physiology. These studies suggest that HCG/LHR signalling affects gene expression in non-gonadal tissues, and that Wnt-signalling is regulated by HCG/LH in human and mouse mammary glands.

Borrelia burgdorferi evades the effects of ceftriaxone treatment in a mouse model of Lyme borreliosis

Borrelia burgdorferi infektoitujen hiirten antibioottihoidon jälkeinen oireilu Lymen borrelioosi on puutiaisten välittämä monimuotoinen infektiotauti, jonka tunnetuin oire on ns. vaeltava ihottuma eli erythema migrans. Muita tavallisia ilmentymiä ovat erityisesti nivel- ja hermosto-oireet sekä harvemmin sydän- ja silmäoireet. Suurin osa potilaista paranee täysin terveeksi antibioottihoidon avulla, mutta jopa 10 % borrelioosiin sairastuneista oireilee suositusten mukaisesta hoidosta huolimatta. Pitkittyneen oireilun on ajateltu johtuvan mm. infektion laukaisemasta autoimmuunitaudista tai kroonisesta infektiosta, mutta teorioiden tueksi ei ole kyetty esittämään kiistattomia todisteita. Onkin todennäköistä, että antibioottihoidon jälkeisen oireilun takana on useampia mekanismeja eikä yksi teoria selitä kaikkien potilaiden oireilua. Tässä väitöskirjatyössä on tutkittu hoidonjälkeistä borrelioosia hiirimallin avulla. Varhaisvaiheessa (2 viikkoa infektoinnin jälkeen) annettu antibiootti vähensi hiirten nivelturvotusta ja esti B. burgdorferi – bakteerin kasvun kudoksista otetuista näytteissä. Hoidettu¬jen hiirten B. burgdorferi -spesifiset IgG-luokan vasta-aineet pysyivät kuitenkin koholla ja osasta kudosnäytteistä löytyi B. burgdorferi:n DNA:ta PCR-tutkimuksen avulla. Mikäli hiiret hoidettiin myöhäisessä vaiheessa (yli 18 viikkoa infektoinnista) tulokset olivat muuten samanlaiset, mutta keftriaksoni ei vaikuttanut nivelturvotukseen. Näin hiirissä oli aikaansaatu tilanne, joka on hyvin samankaltainen ihmisen hoitoresistentin borrelia-artriitin kanssa: oireet jatkuvat, mutta taudinaiheuttajaa ei saada esiin. Inflammaatiota vaimentavaa anti-TNF-alphaa on käytetty nivelreuman hoidossa menestyksekkäästi huonosti muuhun hoitoon reagoivilla potilailla ja siitä syystä sen ajateltiin voivan vaikuttaa suotuisasti myös B. burgdorferi -infektoitujen hiirten hoidonjälkeiseen nivelturvotukseen. Sillä ei kuitenkaan ollut vaikutusta nivelturvotukseen, mutta yllättäen hoidon jälkeen osa hiirten kudosnäytteistä osoittautui viljelypositiivisiksi. On siis ilmeistä, että hiirimallissamme osa B. burgdorferi spirokeetoista pystyy välttämään keftriaksonihoidon vaikutuksen joko hakeutumalla elimistössä kudokseen, jossa antibiootin pitoisuus ei nouse riittävän korkeaksi, tai ne kykenevät muuntautumaan metabolisesti inaktiiviin tilaan eikä mikrobilääke yhdessä immuunipuolustuksen kanssa onnistu tappamaan niitä. Jatkotutkimuksissa selvitimme B. burgdorferi - spirokeetan mahdollista piilopaikkaa tutkimalla antibioottihoidon jälkeen useita eri kudoksia PCR-menetelmällä. Tulosten perusteella spirokeetta näyttää suosivan nivelkudosta tai soluja, joita esiintyy nivelessä runsaasti. On kuitenkin edelleen epäselvää, missä muodossa B. burgdorferi –spirokeetat säilyvät kudoksessa antibioottihoidon jälkeen.

Designing and Implementing Mouse Emulation to Touchscreen Operated Mobile Web Browsers

Browsing the web has become one of the most important features in high end mobile phones and in the future more and more people will be using mobile phone for web browsing. Large touchscreens improve browsing experience but many web sites are designed to be used with a mouse. A touchscreen differs substantially from a mouse as a pointing device and therefore mouse emulation logic is required in the browsers to make more web sites usable. This Master's thesis lists the most significant cases where the differences of a mouse and a touchscreen affect web browsing. Five touchscreen mobile phones and their web browsers were evaluated to find out if and how these cases are handled in them. Also as a part of this thesis, a simple QtWebKit based mobile web browser with advanced mouse emulation model was implemented, aiming to solve all the problematic cases. The conclusion of this work is that it is feasible to emulate a mouse with a touchscreen and thus deliver good user experience in mobile web browsing. However, current highend touchscreen mobile phones have relatively underdeveloped mouse emulations in their web browsers and there is a lot to improve.

Chromatoid body mediated RNA regulation in mouse male germline

Male germ cell differentiation, spermatogenesis is an exceptional developmental process that produces a massive amount of genetically unique spermatozoa. The complexity of this process along with the technical limitations in the germline research has left many aspects of spermatogenesis poorly understood. Post-meiotic haploid round spermatids possess the most complex transcriptomes of the whole body. Correspondingly, efficient and accurate control mechanisms are necessary to deal with the huge diversity of transcribed RNAs in these cells. The high transcriptional activity in round spermatids is accompanied by the presence of an uncommonly large cytoplasmic ribonucleoprotein granule, called the chromatoid body (CB) that is conjectured to participate in the RNA post-transcriptional regulation. However, very little is known about the possible mechanisms of the CB function. The development of a procedure to isolate CBs from mouse testes was this study’s objective. Anti-MVH immunoprecipitation of cross-linked CBs from a fractionated testicular cell lysate was optimized to yield considerable quantities of pure and intact CBs from mice testes. This protocol produced reliable and reproducible data from the subsequent analysis of CB’s protein and RNA components. We found that the majority of the CB’s proteome consists of RNA-binding proteins that associate functionally with different pathways. We also demonstrated notable localization patterns of one of the CB transient components, SAM68 and showed that its ablation does not change the general composition or structure of the CB. CB-associated RNA analysis revealed a strong accumulation of PIWI-interacting RNAs (piRNAs), mRNAs and long non-coding RNAs (lncRNAs) in the CB. When the CB transcriptome and proteome analysis results were combined, the most pronounced molecular functions in the CB were related to piRNA pathway, RNA post-transcriptional processing and CB structural scaffolding. In addition, we demonstrated that the CB is a target for the main RNA flux from the nucleus throughout all steps of round spermatid development. Moreover, we provided preliminary evidence that those isolated CBs slice target RNAs in vitro in an ATPdependent manner. Altogether, these results make a strong suggestion that the CB functions involve RNA-related and RNA-mediated mechanisms. All the existing data supports the hypothesis that the CB coordinates the highly complex haploid transcriptome during the preparation of the male gametes for fertilization. Thereby, this study provides a fundamental basis for the future functional analyses of ribonucleoprotein granules and offers also important insights into the mechanisms governing male fertility.

Novel genes and regulatory systems in epididymal differentiation and sperm maturation of the mouse.

Mammalian spermatozoa gain their fertilizing ability during maturation in the epididymis. Proteins and lipids secreted into the epididymal lumen remodel the sperm membrane, thereby providing the structure necessary for progressive motility and oocyte interaction. In the current study, genetically modified mouse models were utilized to determine the role of novel genes and regulatory systems in the postnatal development and function of the epididymis. Ablation of the mouse β-defensin, Defb41, altered the flagellar movements of sperm and reduced the ability of sperm to bind to the oocyte in vitro. The Defb41-deficient iCre knock-in mouse model was furthermore utilized to generate Dicer1 conditional knock-out (cKO) mice. DICER1 is required for production of mature microRNAs in the regulation of gene expression by RNA interference. Dicer1 cKO gave rise to dedifferentiation of the epididymal epithelium and an altered expression of genes involved in lipid synthesis. As a consequence, the cholesterol:polyunsaturated fatty acid ratio of the Dicer1 cKO sperm membrane was increased, which resulted in membrane instability and infertility. In conclusion, the results of the Defb41 study further support the important role of β-defensin family members in sperm maturation. The regulatory role of Dicer1 was also shown to be required for epididymal development. In addition, the study is the first to show a clear connection between lipid homeostasis in the epididymis and sperm membrane integrity. Taken together, the results give important new evidence on the regulatory system guiding epididymal development and function