Interakcje pomiędzy BCR/ABL a RNAi

Chronic myeloid leukemia (CML) is a malignant disease of progenitor myeloid cells caused by chromosomal translocation that results in the forming of diminutive Philadephia chromosome that harbors BCR/ABL fusion oncogene. The product of this oncogene, a tyrosine kinase, alters several important regulatory pathways related to cell growth and differentiation thus leading to cancer transformation. Major form of CML therapy is based on tyrosine kinase inhibitors, first of all imatinib (IM). Some patients develop resistance to IM in the course of treatment. In the process of leukemogenesis the activity of miRNAs – one of groups of RNAs involved in RNA interference (RNAi) – is altered. Signatures of altered miRNAs activity may serve as a prognostic factor in the development and therapy of several diseases. Moreover, other group of RNAs involved in RNAi – siRNA – might be valuable addition to array of specific therapeutics targeted the BCR/ABL kinase

FLUKA simulations of the target thickness dependence of Cu-Kβ\beta/Kα\alpha intensity ratios

The numerical Monte-Carlo simulations of Cu-K$\alpha$ and Cu-K$\beta$ fluorescence lines induced by monoenergetic Am-241 radiation in copper have been presented. The simulations included modeling the K$\beta$/K$\alpha$ intensity ratios for various thicknesses of copper. The results obtained using the FLUKA code were compared to available experimental and theoretical values. A clear relationship was observed between the simulated K$\beta$/K$\alpha$ intensity ratios and the sample thickness: as the thickness increased, the K$\beta$/K$\alpha$ ratio also increased until it reached saturation

Protocol for Studying Embryonic Mammary Gland Branching Morphogenesis Ex Vivo

Mammary gland development starts during embryogenesis, and the process continues after birth. During development, the mammary gland undergoes massive morphological and physiological alterations including growth, invasion, and branching morphogenesis providing an ideal model for stem cell and cancer biology studies. Great efforts have been made in understanding mammary gland development during puberty and adulthood; however, the process during embryogenesis is still elusive. One reason is that the tools to study tissue dynamics during development are limited, which is partially due to the lack of an ex vivo culture method. Here we describe an updated organ culture protocol of the murine embryonic mammary gland. This powerful tool allows monitoring of growth and branching morphogenesis of mammary gland ex vivo by live imaging. In addition, we introduce a novel method for culturing intact, stroma-free mammary rudiments from late gestation mouse embryos in 3D in Matrigel. This approach can be used to identify the direct stromal cues for branching morphogenesis.Non peer reviewe

Ectodysplasin/NF-kappa B Promotes Mammary Cell Fate via Wnt/beta-catenin Pathway

Mammary gland development commences during embryogenesis with the establishment of a species typical number of mammary primordia on each flank of the embryo. It is thought that mammary cell fate can only be induced along the mammary line, a narrow region of the ventro-lateral skin running from the axilla to the groin. Ectodysplasin (Eda) is a tumor necrosis factor family ligand that regulates morphogenesis of several ectodermal appendages. We have previously shown that transgenic overexpression of Eda (K14-Eda mice) induces formation of supernumerary mammary placodes along the mammary line. Here, we investigate in more detail the role of Eda and its downstream mediator transcription factor NF-kappa B in mammary cell fate specification. We report that K14-Eda mice harbor accessory mammary glands also in the neck region indicating wider epidermal cell plasticity that previously appreciated. We show that even though NF-kappa B is not required for formation of endogenous mammary placodes, it is indispensable for the ability of Eda to induce supernumerary placodes. A genome-wide profiling of Eda-induced genes in mammary buds identified several Wnt pathway components as potential transcriptional targets of Eda. Using an ex vivo culture system, we show that suppression of canonical Wnt signalling leads to a dose-dependent inhibition of supernumerary placodes in K14-Eda tissue explants.Peer reviewe

Ectodysplasin target gene Fgf20 regulates mammary bud growth and ductal invasion and branching during puberty

Mammary gland development begins with the appearance of epithelial placodes that invaginate, sprout, and branch to form small arborized trees by birth. The second phase of ductal growth and branching is driven by the highly invasive structures called terminal end buds (TEBs) that form at ductal tips at the onset of puberty. Ectodysplasin (Eda), a tumor necrosis factor-like ligand, is essential for the development of skin appendages including the breast. In mice, Eda regulates mammary placode formation and branching morphogenesis, but the underlying molecular mechanisms are poorly understood. Fibroblast growth factor (Fgf) receptors have a recognized role in mammary ductal development and stem cell maintenance, but the ligands involved are ill-defined. Here we report that Fgf20 is expressed in embryonic mammary glands and is regulated by the Eda pathway. Fgf20 deficiency does not impede mammary gland induction, but compromises mammary bud growth, as well as TEB formation, ductal outgrowth and branching during puberty. We further show that loss of Fgf20 delays formation of Eda-induced supernumerary mammary buds and normalizes the embryonic and postnatal hyperbranching phenotype of Eda overexpressing mice. These findings identify a hitherto unknown function for Fgf20 in mammary budding and branching morphogenesis.Peer reviewe

Molecular and cellular basis of early development of the mammary gland

The mammary gland, a specialized organ providing nutrition for mammalian offspring, commences during embryogenesis with the formation of an epithelial thickening known as, placode. Subsequently, the placode enlarges creating a hillock and invaginates into underlying mesenchyme, marking the transition to a bulb stage. By the time of birth, the primary rudimentary ductal tree forms through branching morphogenesis. The development of the organ continues postnatally, and its full maturation is accomplished during pregnancy and lactation. Signaling pathways involved in mammogenesis are well characterized. Among them ectodysplasin (Eda) is key regulator of ectodermal appendages formation in all vertebrates. Eda belongs to tumor necrosis factor family and mediates signaling through transcription factor NF-κB. In humans, mutations in Eda or other components of this signaling pathway lead to hypohidrotic ectodermal dysplasia (HED), characterized by sparse hair, missing teeth, and defects in several exocrine glands including the breast. Mice overexpressing Eda (K14-Eda) exhibit supernumerary mammary glands and a hyperbranching phenotype. Despite growing knowledge on molecular signaling regulating mammogenesis, cellular mechanisms are still poorly characterized. The aim of this thesis work was to uncover how Eda regulates mammary gland development and to identify the key cellular mechanisms driving mammary bud formation. In this thesis work, molecular and cellular mechanisms of early mammary gland development were studied. The transcriptional target genes of Eda were identified, among them members of Wnt, Fgf, TNF, Tgfβ, chemokine and Hh signaling pathways. It was shown that in K14-Eda mice supernumerary glands also formed in the neck region, whereas formation of ectopic mammary placodes on the flank was mediated through Eda-dependent Wnt pathway upregulation. Using the Fgf20 null mouse model the role of Eda target gene, Fgf20 in mammogenesis was studied. Results showed that while Fgf20 is dispensable for mammary placode formation, its depletion led to the bud size defect. Cellular mechanisms driving early mammary primordium development were elucidated using 3D and 4D confocal microscopy. It was shown that early mammary rudiment growth is predominantly mediated through migration-driven cell influx, with cell hypertrophy and cell proliferation making minor contributions in this process. Additionally, cell migration in the mammary placode stage was documented by live imaging for the first time. The invagination of the mammary bud was shown to be mediated through contractile rim of epidermal keratinocytes adjacent to the bud, named ring cells. Conditional deletion of Myh9, encoding heavy chain of non-muscle myosin IIA, led to decreased actomyosin network and a compromised ring cell shape indicative of their impaired function. In addition, abnormal mammary bud shape and defective invagination was observed suggesting an essential role of NMIIA in this process.Maitorauhanen on pitkälle erikoistunut avoeritteinen rauhanen, joka tuottaa ravintoa nisäkkäiden imeväisille. Maitorauhasen kehitys alkaa, kun sikiön ektodermaaliseen epiteelikudokseen muodostuu paksuuntuma eli plakodi (”placode”). Plakodi suurenee kumpareeksi (”hillock”) ja tunkeutuu alla olevaan mesenkyymiin alkaen muistuttaa ensin muodoltaan silmua (”bud”) ja sitten hehkulamppua (”bulb”). Tämän jälkeen maitorauhanen alkaa haaroittua ja syntymään mennessä muodostuu alkeellinen rauhaspuun rakenne. Maitorauhasen kehitys jatkuu syntymän jälkeen ja saavuttaa täyden toimintavalmiutensa raskauden ja imetyksen aikana. Maitorauhasen kehitystä ohjaavat signaalinvälitysketjut on hyvin karakterisoitu. Niihin lukeutuu ektodysplasiini (Eda), joka on ektodermaalisten elinten kehityksen keskeinen säätelytekijä selkärankaisissa. Eda kuuluu tuumorinekroositekijäperheeseen (TNF) ja sen käynnistämä signaalinvälitysketju säätelee NF-κB transkriptiotekijän kohdegeenien ilmentymistä. Mutaatiot Eda:ssa tai muissa signaalivälitysketjun osasissa aiheuttaa geneettisen sairauden nimeltä hypohidroottinen ektodermaalinen dysplasia (HED), joka ilmenee heikkona hiusten ja karvojen kasvuna, puuttuvina hampaina ja puutteina rauhasten toiminnassa, rintarauhanen mukaan luettuna. Eda:n yli-ilmentäminen hiirissä (K14-Eda) johtaa ylimääräisten maitorauhasten muodostumiseen, sekä liialliseen kasvuun ja haaroittumiseen. Vaikka tietous maitorauhasen kehityksen molekyylisäätelystä on lisääntynyt, ovat sen solumekanismit verrattain heikosti ymmärretty. Tämän väitöskirjatutkimuksen tavoitteena oli selvittää miten Eda signaalipolku säätelee maitorauhasen kehitystä ja tunnistaa maitorauhasen varhaiskehitystä ohjaavat solubiologiset mekanismit. Tässä väitöskirjassa tutkittiin maitorauhasen varhaiskehityksen molekyyli- ja solumekanismeja. Eda:n kohdegeeneiksi tunnistettiin Wnt, Fgf, TNF, Transformoiva kasvutekijä β, kemokiini ja Hedgehog -signaalinvälitysketjujen osasia. K14-Eda hiirimallissa havaittiin, että kaulalle muodostui ylimääräisiä maitorauhasplakodeja ja osoitettiin ylimääräisten plakodien syntyvän Wnt signaalinvälitysketjun aktiivisuuden seurauksena. Eräs löydetyistä Eda:n kohdegeeneistä oli Fgf20, jonka roolia tutkittiin tarkemmin poistogeenisessä hiirilinjassa. Fgf20 ei ollut välttämätön plakodien muodostukselle, mutta maitorauhasten silmut ovat normaalia pienempiä. Tutkin myös maitorauhasen muodostumisen solumekanismeja kolmi- ja neliulotteisella (so. elävän kudoksen aikaviivekuvantamisella) konfokaalimikroskopialla. Näytin ensi kertaa eläviä soluja kuvantamalla, että maitorauhasaihion varhainen kasvu on seurausta solujen liikkeestä. Solujen koon kasvulla ja solujakautumisella sen sijaan oli pienempi vaikutus. Maitorauhassilmun työntyminen alla olevaan mesenkyymiin oli riippuvainen sitä reunustavista ektodermin epiteelisoluista (keratinosyyteistä), jotka muodostivat supistuvan kehän sen ympärille. Supistuvan kehän merkitys kävi selväksi tutkimalla hiirten sikiöitä, joiden ei-lihasperäisen myosiini-IIA:n (NMIIA) raskasta ketjua koodaava Myh9 -geeni oli inaktivoitu ektodermissä. Myh9:n puute johti kehäsolujen aktiini-myosiini-verkoston heikkenemiseen ja niiden muotojen pyöristymiseen. Lisäksi havaitsin maitorauhassilmun muotoutumisen ja mesenkyymiin työntymisen olevan puutteellista, mikä osoitti NMIIA:n tärkeän roolin varhaisessa maitorauhasen kehityksessä

Surface prospection of soil samples composition of elements originate from “Meteoryt Morasko” reserve

The research was focused on strewnfield in “Meteorite Morasko” reserve. The main goal of the project was to find correlation between chemical composition of soil and concentration of cosmogenic material which was discovered in the region. After field prospection the sampling was made for three reference regions in the reserve. ICP-MS method was used to determine the elemental composition of soil samples. Investigation has been carried out to indicate the chemical components which iron meteorite matrix is built of like Fe, Ni and Co. Results of the study gave information about distribution and correlation of chemical components in the reserve

Mesenchyme instructs growth while epithelium directs branching in the mouse mammary gland

The mammary gland is a unique organ that undergoes dynamic alterations throughout a female’s reproductive life, making it an ideal model for developmental, stem cell and cancer biology research. Mammary gland development begins in utero and proceeds via a quiescent bud stage before the initial outgrowth and subsequent branching morphogenesis. How mammary epithelial cells transit from quiescence to an actively proliferating and branching tissue during embryogenesis and, importantly, how the branch pattern is determined remain largely unknown. Here, we provide evidence indicating that epithelial cell proliferation and onset of branching are independent processes, yet partially coordinated by the Eda signaling pathway. Through heterotypic and heterochronic epithelial-mesenchymal recombination experiments between mouse mammary and salivary gland tissues and ex vivo live imaging, we demonstrate that unlike previously concluded, the mode of branching is an intrinsic property of the mammary epithelium whereas the pace of growth and the density of ductal tree are determined by the mesenchyme. Transcriptomic profiling and ex vivo and in vivo functional studies in mice disclose that mesenchymal Wnt/ß-catenin signaling, and in particular IGF-1 downstream of it critically regulate mammary gland growth. These results underscore the general need to carefully deconstruct the different developmental processes producing branched organs

<i>Edar</i> expression and NF-κB reporter expression co-localize in the mammary epithelium.

<p>(A) <i>Edar</i> transcripts were detected by in situ hybridization with a ³⁵S-UTP-labeled probe in mammary buds at E12.5 and E13.5. (B) NF-κB reporter was initially expressed throughout the mammary bud in control embryos, but became localized to the basal layer of the epithelium around E13.5. (C) In <i>K14-Eda</i> embryos, reporter activity stayed high throughout the mammary bud at E12.5 and E13.5. mb4 = mammary bud number 4. (D) The supernumerary buds exhibited mosaic expression of the reporter which was less pronounced in the in the neck (left) than in mammary primordia forming between buds 3 and 4 (right). (Scale bar: 100 μm.)</p

NF-κB is required for the formation of Eda induced supernumerary mammary placodes.

<p>(A-C) Expression of <i>Tbx3</i> (A), <i>Wnt10b</i> (B) and <i>PTHrP</i> (C) was detected in the mammary buds of the WT, <i>K14-Eda</i>, <i>IκBαΔN</i> and compound <i>IκBαΔN</i>; <i>K14-Eda</i> embryos at E13.5. Arrows highlight the supernumerary mammary placodes in <i>K14-Eda</i> embryos. Note that no localized expression was visible between the 3^rd and the 4^th mammary bud in the compound <i>IκBαΔN</i>; <i>K14-Eda</i> embryos. (D) Scanning electron microscopy images of the mammary forming region confirmed absence of accessory mammary primordia in compound mutants. (Scale bar: 500 μm.)</p