Functions of human papillomavirus E5 oncogene in epithelial cells and in the onset of cervical cancer

Human papillomaviruses (HPVs) are the causal agents of cervical cancer, which is the second most common cancer among women worldwide. Cellular transformation and carcinogenesis depend on the activities of viral E5, E6 and E7 proteins. Alterations in cell-cell contacts and in communication between epithelial cells take place during cervical carcinogenesis, leading to changes in cell morphology, increased cell motility and finally invasion. The aim of this thesis was to study genome-wide effects of the HPV type 16 (HPV-16) E5 protein on the expression of host cell messenger RNAs (mRNAs) and microRNAs by applying microarray technology. The results showed that the HPV-16 E5 protein alters several cellular pathways involved in cellular adhesion, motility and proliferation as well as in the extracellular matrix. The E5 protein was observed to enhance wound healing of epithelial cell monolayers by increasing cell motility in vivo. HPV-16 E5-induced alterations in the expression of cellular microRNAs and their target genes seem to favour increased proliferation and tumorigenesis. E5 was also shown to affect the expression of adherens junction proteins in HaCaT epithelial keratinocytes. In addition, a study of a membrane cytoskeletal cross-linker protein, ezrin, revealed that when activated, it localizes to adherens junctions. The results suggest that ezrin distribution to forming adherens junctions is due to Rac1 activity in epithelial cells. These studies reveal for the first time the holistic effects of HPV-16 E5 protein in promoting precancerous events in epithelial cells. The results contribute to identifyinging novel markers for cervical precancerous stages and to predicting disease behaviour.Papilloomaviruksen (HPV) aiheuttama kohdunkaulan syöpä on maailmanlaajuisesti naisten toiseksi yleisin syöpä. Epiteelimuutoksia ja syöpiä todetaan yhä nuoremmilla naisilla, ja HPV-infektiot nuorilla naisilla ovat hyvin tavallisia. Tutkimuksemme pyrki selvittämään ihmisen papilloomaviruksen vaikutuksia solujen väliseen kiinnittymiseen ja kommunikaatioon kohdunkaulan syövän synnyssä. Papilloomaviruksella on kolme proteiinia, E5, E6 ja E7, joiden vuorovaikutus solun proteiinien kanssa ovat merkittäviä syövän syntyyn vaikuttavia tekijöitä. Keskeisiä tapahtumia syövän ja epiteelimuutosten kehittymisessä ovat epiteelin erilaistumisen, solujen välisen kommunikaation ja solujen tarttumiskyvyn häiriintyminen. Nämä johtavat solumorfologian muutoksiin, soluliikkuvuuden lisääntymiseen ja yhdessä muiden tapahtumien kanssa lopulta invaasioon. Tämän väitöskirjatutkimuksen tavoitteena oli tutkia laaja-alaisesti mikrosirujen avulla ihmisen papilloomavirus tyyppi 16:n (HPV-16) E5-proteiinin vaikutuksia isäntäsolun lähetti-RNA:iden sekä mikro-RNA:iden ilmentymiseen. Tutkimuksen tavoitteena oli tarkastella solu- ja kudostasolla niiden proteiinien ilmentymistä ja sijoittumista solussa, joiden ilmentymisen havaittiin muuttuneen HPV-16 E5-proteiinin vaikutuksesta sekä tutkia tarkemmin eri soluproteiinien ilmentymisen muutoksia ajan funktiona. Tutkimuksessa selvitettiin myös E5-onkogeenin merkitystä epiteelisolujen kiinnittymiseen ja liikkuvuuteen. Tulokset osoittivat, että E5-proteiini muuttaa mm. useiden solukiinnittymiseen ja solunsisäiseen signaalinkuljetukseen osallistuvien geenien ilmentymistä sekä RNA- että proteiinitasolla. Näitä geenejä pystyttiin ryhmittelemään ja yhdistämään tiettyihin solunsisäisiin signalointireitteihin. E5-proteiinin vaikutus solujen liikkuvuuden lisääntymiseen havaittiin myös eläviä soluja kuvantamalla. Tulokset osoittavat myös, että E5-proteiini pääasiassa vähentää isäntäsolun mikro-RNA:iden ilmentymistä. Havaitut muutokset mikro-RNA:iden ja näiden kohdeproteiinien ilmentymisessä näyttävät edistävän syöpään johtavia tapahtumia solussa. Väitöskirjatyössä selvitettiin myös pieniin GTPaaseihin kuuluvien Rho-proteiinien merkitystä epiteelisolun tukirangan säätelyproteiinin, esriinin, säätelyssä. Rac1-aktiivisuuden osoitettiin olevan tarpeen esriinin sijoittumiseksi epiteelisolujen vyöliitoksiin. Työssä pyrittiin myös mallintamaan HPV-16 E5-proteiinin kokonaisvaltaisia vaikutuksia pahanlaatuisten muutosten käynnistämisessä epiteelisolussa. Tutkimus antaa uusia mahdollisuuksia löytää muun muassa merkkiaineita kohdunkaulan syövän esiasteiden toteamiseen ja taudin käyttäytymisen ennustamiseen

Identifying the value of sustainability in competition policy – an expedition to Article 101(3) TFEU

European Union was established to bring about peace into a violent Europe. Currently we are facing a problem, the climate crisis, that has no precedence and demands immediate actions. The European Union has made its stand on the importance of saving the world from a disaster by adopting measures to enable the Green transit. To become the world’s first climate-neutral continent by 2050, the EU requires all EU policies to take environmental values into account. One of these policies, that is currently seeking a way to coordinate multiple values is competition policy. This thesis has focused on pinpointing, what is the value of sustainability in the European Union’s competition policy. Competition policy aims at maximizing the consumer welfare and the question is, can sustainability constitute an element of that welfare. Co-operation between companies can be fruitful from the point of view of sustainability but at the same time, it can be prohibited. This thesis has focused on finding out, whether companies can co-operate when there are sustainability gains to be achieved and can this co-operation be justified by sustainability gains. At the core of this thesis is finding out what value can sustainability in competition law have and can it be considered under the Article 101(3) TFEU requirement for a “fair share for consumers". This question is up-to-date as the Commission is currently in the process of renewing the horizontal guidelines. This thesis presents current views from stakeholders, which previously have not been analysed as a part of a master’s thesis. The thesis consists of seven chapters. The first chapter contains introduction. The scope, limitations and research questions of the thesis are presented in the second chapter. This is followed by a chapter containing a brief history and introduction to the concept of sustainability. The fourth chapter entails the limitations and demands that competition law sets for taking sustainability into account. The fifth chapter contains a summary and analysis of the Commission’s recent call for contribution on the matter and a brief analysis on recently published draft guidelines. This chapter also contains current and future trends on the issue. The sixth chapter contains de lege ferenda -section with writer’s view on how the situation should be handled and current tools amended. This thesis concludes with an analysis answering the research questions and summarising the findings, as well as containing suggestions for future actions. This thesis shows that there is legal support for taking environmental issues into account in every policy, including competition policy. Also, competition law provisions leave room for interpretation and there is no rule against taking sustainability into account. In fact, quite the opposite: primary law presupposes for it to be considered and emphasized when possible as TFEU 191 (2) and supporting case law points out. The CJEU follows its own rules but reflects the society at large. Taking current political climate into account, the CJEU would most likely rule in favour of sustainability where possible. Legal uncertainty and the chilling effect of certain rules have not made it compelling for undertakings to take action in the field of sustainability. The Commission has pointed out in the Communication for draft guidelines, that sustainability makes an excellent argument in the balancing of benefits under Article 101(3). The next step after the guidelines have been finalised, would be that someone would dare to lead and be brave enough to test the boundaries and current legal environment. Article 101(3) combined with sustainability should complement other means for striving towards a more sustainable future, not substitute them. The value of sustainability is priceless, and sustainability should be pursued with this in mind

Human papillomavirus 16 E5 modulates the expression of host microRNAs

Peer reviewe

Detection of Human Papillomaviruses by Polymerase Chain Reaction and Ligation Reaction on Universal Microarray

Peer reviewe

Monien mahdollisuuksien soveltava pystylaskettelu

Toimeksiantajana opinnäytetyölle toimi Suomen Hiihdonopettajat ry (SHOry). Opinnäytetyön tavoitteena oli kerätä tutkittuun tietoon pohjautuvaa aineistoa. Kerättyä aineistoa hyödyntäen tuotettiin opas soveltavan pystylaskettelun ohjaamiseen. Opinnäytetyön tarkoituksena oli tuottaa tietoa soveltavasta pystylaskettelusta eri alojen käyttöön oppaan muodossa. Toimeksiantaja voi hyödyntää opasta kehittäessään soveltavaa pystylaskettelua ja kouluttaessa uusia soveltavan alppihiihdon ohjaajia. Tämä opinnäytetyö on toiminnallinen työ, joka koostuu toiminnallisesta osiosta ja raporttiosiosta. Työn lopputuotteena on ”Riemua Rinteessä” – monien mahdollisuuksien soveltava pystylaskettelu- opas. Opas on toteutettu Jämsä & Manninen (2000) sosiaali- ja terveysalojen tuotteistamisprosessin mallia hyödyntäen. Raporttiosio pitää sisällään teoreettisen viitekehyksen ja oppaan tuotteistamisprosessin. Moniammatillisesti tuotetulla työllä pyrimme kehittämään fysioterapia- ja liikunta-alojen yhteistyötä. Oppaassa tuodaan esille tietoa soveltavasta pystylaskettelusta, ohjaamisen soveltamisesta, laskettelusta osana kuntoutusta ja soveltavasta pystylaskettelusta kilpaurheiluna pohjautuen ICF-luokitukseen ja nykyaikaiseen taidon oppimisen malliin. Tämän lisäksi oppaassa käsitellään apuvälineitä ja rinteessä tapahtuvaa viestintää. Oppaan sisältö on sovellettavissa niin aisti-, kehitys- kuin liikuntavammaisille henkilöille.This thesis was commissioned by Finnish National Association of Ski Instructors. The aim of this thesis was to collect data based on existing research and to write a guidebook based on this data. The purpose of this thesis was to produce new material for different professional sectors. The commissioner can use this guidebook in the development of adaptive alpine skiing. This is a collaborative study. The thesis includes a report and a guidebook. The title of the guidebook is “Joy of the Slope” – many possibilities of adaptive alpine skiing upright. The productization process was based on the model developed by Jämsä and Manninen. This report includes a theoretical part and the productization process. By producing this work multi-professionally we aim to develop the cooperation between physiotherapy and sport. The guidebook gives information and instructions about adaptive alpine skiing upright, adaptive downhill skiing therapy and disability sport based on ICF classification and skills learning. Furthermore, the guidebook gives information about the adaptive equipment and communication used in adaptive alpine skiing upright. The contents of the guidebook can be applied to the sensory disabled, the mentally disabled and the physically disabled

Cell culture model predicts human disease: Altered expression of junction proteins and matrix metalloproteinases in cervical dysplasia

<p>Abstract</p> <p>Background</p> <p>Cervical cancer is necessarily caused by human papillomaviruses, which encode three oncogenes manifesting their functions by interfering with a number of cellular proteins and pathways: the E5, E6, and E7 proteins. We have earlier found in our microarray studies that the E5 oncogene crucially affects the expression of cellular genes involved in adhesion and motility of epithelial cells.</p> <p>Methods</p> <p>In order to biologically validate our previous experimental findings we performed immunohistochemical staining of a representative set of tissue samples from different grades of high-risk human papillomavirus associated cervical disease as well as normal squamous and columnar cervical epithelium. Three-dimensional collagen raft cultures established from E5-expressing and control epithelial cells were also examined. The expression of p16, matrix metalloproteinase (MMP) -7, MMP-16, cytokeratin (CK) 8/18, laminin, E-cadherin and beta-catenin was studied.</p> <p>Results</p> <p>In agreement with our previous microarray studies, we found intense staining for E-cadherin and beta-catenin in adherens junctions even in high-grade cervical lesions. Staining for MMP-16 was increased in severe disease as well. No significant change in staining for MMP-7 and cytokeratin 8/18 along with the grade of cervical squamous epithelial disease was observed.</p> <p>Conclusions</p> <p>Here we have confirmed, using tissue material from human papillomavirus associated lesions, some of the cellular gene expression modifications that we earlier reported in an experimental system studying specifically the E5 oncogene of papillomaviruses. These findings were partially surprising in the context of cervical carcinogenesis and emphasize that the complexity of carcinogenesis is not yet fully understood. Microarray approaches provide a wide overwiev of gene expression in experimental settings, which may yield biologically valid biomarkers for disease diagnostics, prognosis, and follow-up.</p

Detection of Human Papillomaviruses by Polymerase Chain Reaction and Ligation Reaction on Universal

Sensitive and specific detection of human papillomaviruses (HPV) in cervical samples is a useful tool for the early diagnosis of epithelial neoplasia and anogenital lesions. Recent studies support the feasibility of HPV DNA testing instead of cytology (Pap smear) as a primary test in population screening for cervical cancer. This is likely to be an option in the near future in many countries, and it would increase the efficiency of screening for cervical abnormalities. We present here a microarray test for the detection and typing of 15 most important high-risk HPV types and two low risk types. The method is based on type specific multiplex PCR amplification of the L1 viral genomic region followed by ligation detection reaction where two specific ssDNA probes, one containing a fluorescent label and the other a flanking ZipCode sequence, are joined by enzymatic ligation in the presence of the correct HPV PCR product. Human beta-globin is amplified in the same reaction to control for sample quality and adequacy. The genotyping capacity of our approach was evaluated against Linear Array test using cervical samples collected in transport medium. Altogether 14 out of 15 valid samples (93%) gave concordant results between our test and Linear Array. One sample was HPV56 positive in our test and high-risk positive in Hybrid Capture 2 but remained negative in Linear Array. The preliminary results suggest that our test has accurate multiple HPV genotypin

Comparison of the PGMY-t and the original PGMY multiplex PCR primer mixes as measured by HPV LDR signals on microarray.

<p>Both primer mixes at 0.2 µM primer concentration amplify all five HPV types from 1 ng of template (A) & (B) and from 1 pg of template (C) & (D). With the original PGMY mix at 1 ng template concentration, HPV 59 gives a false positive signal (B). HPV 33 signal is also relatively weak with the original PGMY at 1 pg template concentrations (D). Data are presented as means±SD from two independent microarrays. Y-axis shows signal intensity in arbitrary units. Asterisk (*) indicates the target HPV types present in the experiment.</p

The principle of ligation detection reaction.

<p>The discriminating probe (DP) and the common probe (CP) are designed to hybridize adjacently on the template DNA and are joined by ligase in the presence of a matching template (HPV PCR product). The discriminating 3′ base can be A, C, T or G. The reaction is thermally cycled and ligation products addressed on microarray spots by the unique ZipCode sequences flanking each CP. Hybridization control probe carries a different label (6-Fam) than the DP (Cy3).</p