To Divide or Not to Divide; MicroRNAs and Small Compounds as Modulators of Mitosis

Mitosis is under the stringent quality control of the spindle assembly checkpoint (SAC). However, in cancer cells this control can fail, leading to excessive cellular proliferation and ultimately to the formation of a tumor. Novel cancer cell selective therapies are needed to stop the uncontrolled cell proliferation and tumor growth. The aim of the research presented in this thesis was to identify microRNAs (miRNAs) that could play a role in cancer cell proliferation as well as low molecular weight (LMW) compounds that could interfere with cell division. The findings could be used to develop better cancer diagnostics and therapies in the future. First, a high-throughput screen (HTS) was performed to identify LMW compounds that possess a similar chemical interaction field as rigosertib, an anti-cancer compound undergoing clinical trials. A compound termed Centmitor-1 was discovered that phenocopied the cellular impact of rigosertib by affecting the microtubule dynamics. Next, another HTS aimed at identifying compounds that would target the Hec1 protein, which mediates the interaction between spindle microtubules and chromosomes. Perturbation of this connection should prevent cell division and induce cell death. A compound termed VTT-006 was discovered that abrogated mitosis in several cell line models and exhibited binding to Hec1 in vitro. Lastly, using a cell-based HTS two miRNAs were identified that affected cancer cell proliferation via Aurora B kinase, which is an important mitotic regulator. MiR-378a-5p was found to indirectly suppress the production of the kinase whereas let-7b showed direct binding to the 3’UTR of Aurora B mRNA and repressed its translation. The miRNA-mediated perturbation of Aurora B induced defects in mitosis leading to abnormal chromosome segregation and induction of aneuploidy. The results of this thesis provide new information on miRNA signaling in cancer, which could be utilized for diagnostic purposes. Moreover, the thesis introduces two small compounds that may benefit future drug research.

To divide or differentiate? : nestin-mediated regulation of Cdk5 in cell fate decisions

The molecular functions of the non-cell cycle-related Cyclin-dependent kinase 5 (Cdk5) have been of primary interest within the neuroscience field, but novel undertakings are constantly emerging for the kinase in tissue homeostasis, as well as in diseases such as diabetes and cancer. Although Cdk5 activation is predominantly regulated by specific non-cyclin activator protein binding, additional mechanisms have proved to orchestrate Cdk5 signaling in cells. For example, the interaction between the intermediate filament protein nestin and Cdk5 has been proposed to determine cellular fate during neuronal apoptosis through nestin-dependent adjustment of the sensitive balance and turnover of Cdk5 activators. While nestin constitutes a crucial regulatory scaffold for appropriate Cdk5 activation in apoptosis, Cdk5 itself phosphorylates nestin with the consequence of filament reorganization in both neuronal progenitors and differentiating muscle cells. Interestingly, the two proteins are often found coexpressed in various tissues and cell types, proposing that nestin-mediated scaffolding of Cdk5 and its activators may be applicable to other tissue systems as well. In the literature, the molecular functions of nestin have remained in the shade, as it is mostly exploited as a marker protein for progenitor cells. In light of these studies, the aim of this thesis was to assess the importance of the nestin scaffold in regulation of Cdk5 actions in cell fate decisions. This thesis can be subdivided into two major projects: one that studied the nature of the Cdk5-nestin interplay in muscle, and one that assessed their role in prostate cancer. During differentiation of a myoblast cell line, the filament formation properties of nestin was found to be crucial in directing Cdk5 activity, with direct consequences on the process of differentiation. Also the genetic knockout of nestin was found to influence Cdk5 activity, although differentiation per se was not affected. Instead, the genetic ablation of nestin had broad consequences on muscle homeostasis and regeneration. While the nestin-mediated regulation of Cdk5 in muscle was found to act in multiple ways, the connection remained more elusive in cancer models. Cdk5 was, however, established as a significant determinant of prostate cancer proliferation; a behavior uncharacteristic for this differentiation-associated kinase. Through complex and simultaneous regulation of two major prostate cancer pathways, Cdk5 was placed upstream of both Akt kinase and the androgen receptor. Its action on proliferation was nonetheless mainly exerted through the Akt signaling pathway in various cancer models. In summary, this thesis contributed to the knowledge of Cdk5 regulation and functions in two atypical settings; proliferation (in a cancer framework) and muscle differentiation, which is a poorly understood model system in the Cdk5 field. This balance between proliferation and differentiation implemented by Cdk5 is ultimately regulated (where present) by the dynamics of the cytoskeletal nestin scaffold.

Sphingosine-1-phosphate-evoked invasion of follicular thyroid cancer cells : evidence for the involvement of HIF-Iα, MMP2 and MMP9

Sphingolipids are widely expressed molecules, which traditionally were considered to have majorly structural properties. Nowadays, however, they are implicated in a wide range of different biological processes. The bioactive lipid sphingosine 1-phosphate (S1P) has emerged during the past decade as one of the most studied molecules due to its proliferative and pro-migratory abilities both during normal physiology and in the pathology of a subset of different diseases. Migration and invasion of cancer cells require changes in cell behavior and modulation of the tissue microenvironment. Tumor aggressiveness is markedly enhanced by hypoxia, in which hypoxia inducible transcription factors 1-2α (HIF-1-2α) are activated to promote metabolism, proliferation and migration. Invasion requires degradation of the extracellular matrix (ECM) achieved by several degrading and remodeling enzymes. Matrix metalloproteinases (MMPs) are broadly expressed and well accepted as proteolytic enzymes with essential roles both in normal physiology and in pathology. Previously, S1P was shown to strongly evoke migration of follicular ML-1 thyroid cancer cells. The objective of this study was to further investigate and understand the mechanisms behind this regulation. In the first project it was demonstrated that S1P enhances the expression and activity of HIF-1α. S1P enhanced the expression of HIF-1α by increasing its synthesis and stability. The S1P-increased HIF-1α was mediated via S1P3, Gi/0, PI3K, PKCβI, ERK1/2, mTOR and translation factors p70S6K and eIF4E. Finally, it was shown that HIF-1α mediated S1P-induced migration. The ECM is constituted of a complex and coordinated assembly of many types of proteins. In order to be able to invade, cells need to break down the ECM, therefore several key players in this event were investigated in the second project. S1P increased the secretion and activity of MMP2 and MMP9 via S1P-receptor 1 and 3 and that these MMPs participated in the S1P-facilitated invasion of ML-1 cells. In this interplay, calpains and Rac1 were involved, both of which are crucial players in migration and invasion. The prognosis for some types of thyroid cancer is relatively good. However, there are forms of thyroid cancers, for which there are no treatments or the current available treatments are inefficient. Thus, new medical interventions are urgently needed. In the third project the significance of the S1P-receptor modulating drug FTY720, which is currently used for the treatment of multiple sclerosis (MS), was studied. The effect of FTY720 was tested on several thyroid cancer cell lines, and it inhibited the proliferation and invasion of all cancer cell lines tested. In ML-1 cells, FTY720 attenuated invasion by blocking signaling intermediates important for migration and invasion of the cells. Moreover, FTY720 inhibited the proliferation of ML-1 cells by increasing the expression of p21 and p27, hence, inducing cell arrest in G1 phase of the cell cycle. Thus, it can be suggested that FTY720 could be used in the treatment of thyroid cancer.

Translating with the Web. Professional translators' information-seeking behaviour in translation with online resources

Tämän tutkielman aiheena on ammattikääntäjien tiedonhaku, kun käytettävissä on ainoastaan verkkolähteitä. Tutkimuksessa on tarkasteltu, mistä ja miten ammattikääntäjät etsivät tietoa internetistä kääntäessään lähtötekstiä englannista suomeen. Lisäksi tutkimuksen tarkoituksena on osoittaa, että tiedonhakutaidot ja lähdekriittisyys ovat käännöskompetensseja, joita tulisi sekä ylläpitää että opettaa osana kääntäjäkoulutusta. Tutkimuksen aineisto kerättiin empiirisesti käyttämällä kolmea metodia. Käännösprosessi ja sen aikana tapahtunut tiedonhaku tallennettiin käyttäen Camtasia-näyttövideointiohjelmaa ja Translog-II -näppäilyntallennusohjelmaa. Lisäksi tutkimukseen osallistuneet kääntäjät täyttivät kaksi kyselyä, joista ensimmäinen sisälsi taustatietokysymyksiä ja toinen itse prosessiin liittyviä retrospektiivisiä kysymyksiä. Kyselyt toteutettiin Webropol-kyselytyökalulla. Aineistoa kerättiin yhteensä viidestä koetilanteesta. Tutkimuksessa tarkasteltiin lähemmin kolmen ammattikääntäjän tiedon-hakutoimintoja erottelemalla käännösprosesseista ne tauot, joiden aikana kääntäjät etsivät tietoa internetistä. Käytettyjen verkkolähteiden osalta tutkimuksessa saatiin vastaavia tuloksia kuin aiemmissakin tutkimuksissa: eniten käytettyjä olivat Google, Wikipedia sekä erilaiset verkkosanakirjat. Tässä tutkimuksessa kuitenkin paljastui, että ammattikääntäjien tiedonhaun toimintamallit vaihtelevat riippuen niin kääntäjän erikoisalasta kuin hänen tiedonhakutaitojensa tasosta. Joutuessaan työskentelemään tutun työympäristönsä ja oman erikoisalansa ulkopuolella turvautuu myös osa ammattikääntäjistä alkeellisimpiin tiedonhakutekniikoihin, joita käännöstieteen opiskelijoiden on havaittu yleisesti käyttävän. Tulokset paljastivat myös, että tiedonhaku voi viedä jopa 70 prosenttia koko käännösprosessiin kuluvasta ajasta riippuen kääntäjän aiemmasta lähtötekstin aihepiiriin liittyvästä tietopohjasta ja tiedonhaun tehokkuudesta. Tutkimuksessa saatujen tulosten pohjalta voidaan sanoa, että myös ammattikääntäjien tulisi kehittää tiedonhakutaitojaan pitääkseen käännösprosessinsa tehokkaana. Lisäksi kääntäjien pitäisi muistaa arvioida kriittisesti käyttämiään tietolähteitä: lähdekritiikki on tarpeen erityisesti verkkolähteitä käytettäessä. Tästä syystä tiedonhakutaitoja ja lähdekriittisyyttä tulisikin opettaa ja harjoitella jo osana kääntäjäkoulutusta. Kääntäjien ei myöskään pidä jättää tiedonhakua pelkkien verkkolähteiden varaan, vaan jatkossakin käyttää hyväkseen niin painettuja tietolähteitä kuin myös henkilölähteitä.

Metal-Ion-Binding Oligonucleotides: High-Affinity Probes for Nucleic Acid Sequences

Small non-coding RNAs have numerous biological functions in cell and are divided into different classes such as: microRNA, snoRNA, snRNA and siRNA. MicroRNA (miRNA) is the most studied non-coding RNA to date and is found in plants, animals and some viruses. miRNA with short sequences is involved in suppressing translation of target genes by binding to their mRNA post-transcriptionally and silencing it. Their function besides silencing of the viral gene, can be oncogenic and therefore the cause of cancer. Hence, their roles are highlighted in human diseases, which increases the interest in using them as biomarkers and drug targets. One of the major problems to overcome is recognition of miRNA. Owing to a stable hairpin structure, chain invasion by conventional Watson-Crick base-pairing is difficult. One way to enhance the hybridization is exploitation of metal-ion mediated base-pairing, i. e. oligonucleotide probes that tightly bind a metal ions and are able to form a coordinative bonds between modified and natural nucleobases. This kind of metallo basepairs containing short modified oligonucleotides can also be useful for recognition of other RNA sequences containing hairpin-like structural motives, such as the TAR sequence of HIV. In addition, metal-ion-binding oligonucleotides will undoubtedly find applications in DNA-based nanotechnology. In this study, the 3,5-dimethylpyrazol-1-yl substituted purine derivatives were successfully incorporated within oligonucleotides, into either a terminal or non-terminal position. Among all of the modified oligonucleotides studied, a 2-(3,5-dimethylpyrazol-1-yl)-6-oxopurine base containing oligonucleotide was observed to bind most efficiently to their unmodified complementary sequences in the presence of both Cu2+ or Zn2+. The oligonucleotide incorporating 2,6-bis(3,5-dimethylpyrazol-1-yl)purine base also markedly increased the stability of duplexes in the presence of Cu2+ without losing the selectivity.

A Diachronic Study of the Practises of Translation of Gerunds and Gerundives from Latin into Old English and Middle English in History Narratives

This is a philologically oriented thesis which studies the possible adoption of a grammatical feature from one language into another from historical linguistic perspective. The foci of the study are, on the one hand, the Latin gerund and gerundive and, on the other hand, the English gerund. The material of this study consists of excerpts from two British history narratives in Latin and from the Old English and Middle English translations of these history narratives. The British history narratives selected for the material of this thesis are the 8th century Historia ecclesiastica gentis Anglorum by Bede and the 14th century Polychronicon by Ranulf Higden. Historia ecclesiastica gentis Anglorum has been compared with its Old English translation from the 11th century, the author of which is unknown. The Polychronicon, on the other hand, has been compared with two different Middle English translations: one from the 14th century, by John Trevisa; the other from the 15th century, the author of which is also unknown. The purpose of this thesis is to investigate whether the gerund, which was adopted into English by the Middle English period, has been used to translate the Latin gerunds and gerundives. At the basis of the study is the hypothesis that the English gerund has been used to translate the Latin gerunds and gerundives at least occasionally. The methodology of this thesis consists of detailed and qualitative study of the primary material. The primary material has been studied from synchronic, diachronic and paratextual perspective. The results of this thesis confirm that the English gerund has occasionally been used to translate the Latin gerunds and gerundives. The instances that confirm with the hypothesis are so rare, however, that the relationship between the English gerund and the Latin gerund and gerundive seems to be indirect or at least enshadowed by wide-ranging grammatical differences.