Structural and functional characterisation of cyclin dependent kinase 1 containing complexes

PhD ThesisCDK1 belongs to the family of serine/threonine kinases that require cyclin binding and phosphorylation for activation. CDK1 is partnered by cyclin A or cyclin B and is the only essential CDK that is required to drive the eukaryotic cell cycle. Because of their roles in driving cell growth and division, the activities of some members of the CDK family are upregulated in different cancer types making them attractive targets for therapy. The structure of monomeric CDK1 bound to CKS1 and in a complex with cyclin B are presented. These structures confirm the conserved inactive monomeric CDK fold and show how it can be remodelled by cyclin binding. These structures have revealed important insights for our understanding of CDK function and regulation as well as for the development of new more potent and specific drugs. A range of biophysical and biochemical techniques have been used to confirm and develop hypotheses as to the regulation and activity of CDK1 proposed by the structures. The melting temperature of CDK1 was estimated and compared to that of the cyclin B bound complex, CDK2 and CDK2-cyclin A to determine if the smaller interface between CDK1 and cyclin B may have implications for protein stability. The CDK1–cyclin B structures also reveal potential novel protein interaction sites that might regulate CDK1 activity. These findings inspired further investigations to explore binding sites by structural biology methods. CDK1 can phosphorylate the largest number of substrates in comparison to other CDKs. This relaxed substrate specificity was explored with reference to the insights provided by the CDK1-cyclin B-CKS2 structure. The similarities in sequence and secondary structure exhibited by the CDK family pose challenges for inhibitor design. The binding modes of a diverse set of inhibitors were characterized by a range of biophysical methods and their structures were determined by X-ray crystallography. The results of these experiments highlight the importance of conformational plasticity of the whole molecule in shaping the ATP binding site

Human ex vivo prostate tissue model system identifies ING3 as an oncoprotein

Background: Although the founding members of the INhibitor of Growth (ING) family of histone mark readers, ING1 and ING2, were defined as tumour suppressors in animal models, the role of other ING proteins in cellular proliferation and cancer progression is unclear. Methods: We transduced ex vivo benign prostate hyperplasia tissues with inducible lentiviral particles to express ING proteins. Proliferation was assessed by H3S10phos immunohistochemistry (IHC). The expression of ING3 was assessed by IHC on a human prostate cancer tissue microarray (TMA). Gene expression was measured by DNA microarray and validated by real-time qPCR. Results: We found that ING3 stimulates cellular proliferation in ex vivo tissues, suggesting that ING3 could be oncogenic. Indeed, ING3 overexpression transformed normal human dermal fibroblasts. We observed elevated levels of ING3 in prostate cancer samples, which correlated with poorer patient survival. Consistent with an oncogenic role, gene-silencing experiments revealed that ING3 is required for the proliferation of breast, ovarian, and prostate cancer cells. Finally, ING3 controls the expression of an intricate network of cell cycle genes by associating with chromatin modifiers and the H3K4me3 mark at transcriptional start sites. Conclusions: Our investigations create a shift in the prevailing view that ING proteins are tumour suppressors and redefine ING3 as an oncoprotein

A new method of quantifying glutathione levels in freshly isolated single superfused rat cardiomyocytes

Introduction: Glutathione (GSH) is an important antioxidant in the heart whose content changes during cardiac insults. However, there are currently no methods for continuously monitoring free cytoplasmic GSH levels in single isolated and superfused cardiomyocytes exposed to normal and pathological conditions. Methods: GSH was measured using CellTracker™ Blue CMAC (Molecular Probes), a member of a new family of thiol-sensitive dyes. The fluorescence of 5 μM CellTracker™ Blue CMAC was measured in various solutions containing glutathione- S-transferase and in freshly isolated single superfused cardiomyocytes using an inverted fluorescence microscope. The cardiomyocytes were isolated by standard procedures and loaded with either CellTracker™ BlueCMAC or monochlorobimane by 15 min of shaking incubation in the dark at room temperature followed by centrifugation with resuspension of the cells in dye-free media. Cell volume was calculated from the ³H₂O and [¹⁴C]sucrose space. Results: CellTrackerk™ Blue CMAC fluorescence was linearly proportional to 0–100 μM GSH, as described by the equation: Y= 182.2 (X) + 681.6 (r²=.99,

Kuntouttavan työtoiminnan prosessien kehittäminen

Tämän opinnäytetyön tarkoituksena oli kehittää kuntouttavan työtoiminnan prosesseja Pohjois-Satakunnan peruspalveluliikelaitoskuntayhtymässä. Kehittämistehtävässä kehitettiin toimivat mallit kuntouttavan työtoiminnan palveluun kunta -ja palvelurakenneuudistuksen mukanaan tuomassa uudessa organisaatiossa. Kuntouttavan työtoiminnan palvelua kehitettiin toimintatutkimuksen menetelmillä, jossa toimintatutkimuksen syklit, suunnittelu, toiminta, havainnointi ja reflektointi seurasivat toisiaan. Kehittämistehtävässä tarkasteltiin kuntouttavan työtoiminnan palvelua organisaation ja työntekijöiden näkökulmasta, mutta myös asiakasnäkökulma otettiin huomioon. Tutkimuksen aineistona käytettiin reflektoivaa päiväkirjaa sekä työntekijöiden ja asiakkaiden haastatteluja. Haastattelut tehtiin teemahaastattelun menetelmällä. Kehittämistehtävässä muodostettiin prosessikaaviot aktivointisuunnitelman laatimisesta, kuntouttavan työtoiminnan prosessista sekä työnjaosta eri toimipisteiden välillä. Lisäksi kehitettiin työntekijöiden työn yhtenäistämiseksi erilaisia malleja kuntouttavan työtoiminnan ohjaukseen. Kehittämistehtävän perusteella voidaan kuntayhtymän eri toimipisteissä tarjota tasavertaista ja yhdenmukaista palvelua ja asiakkaiden saatavilla ovat laadukkaat ja kattavat kuntouttavan työtoiminnan palvelut. Kehittämistehtävän mukaan tulevaisuuden haasteena on kuntouttavan työtoiminnan asiakasmäärien kasvu, osaamista vastaavien työtoimintapaikkojen löytyminen sekä riittävä ohjausresurssi, myös aktiivisen sosiaalipolitiikan mukanaan tuomat lakimuutokset tuovat haastetta kuntouttavan työtoiminnan järjestämiseen. Kehittämisen keskeisinä käsitteinä olivat kunta- ja palvelurakenneuudistus, aktiivinen sosiaalipolitiikka, kuntouttava työtoiminta, osallisuus ja kuntouttavan työtoiminnan vaikuttavuus Avainsanat: sosiaalipolitiikka, työllisyyspolitiikka, työttömät, kuntouttava työtoiminta, palvelurakenne.The aim of this development project was to improve rehabilitative employment activities and working processes. This development project was executed in the new organization of Social and Healthcare federation of municipalities in Pohjois-Satakunta. The rehabilitative employment activity and its processes were improved with methods of activity analysis. There planning, action, observation and reflection had been followed by each other. The aim of the rehabilitative employment activity is working life or maintaining the unemployed person’s ability to work. The materials of this developments project were collected with interviews and reflective diary. During this development project had been created new patterns how to guide new clients to the rehabilitative employment activities. This development project is also description of that process. Keywords: rehabilitative employment activity, activation policy, unemployment, social politics

Oxidative Stress, Gene Expression, and Protein Changes Induced in the Human Placenta during Labor

Malperfusion of the placenta has been implicated as a cause of oxidative stress in complications of human pregnancy, leading to release of proinflammatory cytokines and anti-angiogenic factors into the maternal circulation. Uterine contractions during labor are known to be associated with intermittent utero-placental perfusion. We therefore tested whether oxidative stress, proinflammatory cytokines, and angiogenic regulators were increased in placentas subjected to short (<5 hours) and long (>15 hours) labor compared with nonlabored controls delivered by cesarean section. In addition, broader changes in gene transcripts were assessed by microarray analysis. Oxidative stress, activation of the nuclear factor-κB pathway, tumor necrosis factor-α and interleukin 1β all increased in placental tissues after labor. Stabilization of hypoxia-inducible factor-1α and increased vascular endothelial growth factor soluble receptor-1 were also observed. By contrast, tissue levels of placenta growth factor decreased. Apoptosis was also activated in labored placentas. The magnitude of these changes related to the duration of labor. After labor, 55 gene transcripts were up-regulated and 35 down-regulated, and many of these changes were reflected at the protein level. In conclusion, labor is a powerful inducer of placental oxidative stress, inflammatory cytokines, and angiogenic regulators. Our findings are consistent with intermittent perfusion being the initiating cause. Placentas subjected to labor do not reflect the normal in vivo state at the molecular level