Dracocephalum moldavica L. and Melissa officinalis L. : Chemistry and Bioactivities Relevant in Alzheimer’s Disease Therapy

Oksidatiivisen stressin eli liiallisen reaktiivisten happiyhdisteiden määrän soluissa on jo pitkään arveltu olevan tärkeä Alzheimerin taudin kehittymiseen ja etenemiseen vaikuttava tekijä. Tämän vuoksi kiinnostus erilaisten antioksidanttien (yhdisteitä, jotka neutraloivat näitä happiradikaaleja soluissa) mahdollisia terapeuttisia ominaisuuksia Alzheimerin taudin hoidossa on tutkittu laajalti. Tähän mennessä ei kuitenkaan ole vielä onnistuttu löytämään antioksidanttia, joka olisi hyödyksi Alzheimerin taudin hoidossa. Tämän vuoksi on tärkeää pyrkiä löytämään uusia antioksidanttien lähteitä sekä tutkia niistä löytyviä aktiivisia yhdisteitä. Kiinnostus luonnon antioksidantteja kohtaan on kasvanut voimakkaasti viime aikoina. Huomio on kiinnittynyt erityisesti aromaattisista sekä lääkekasveista löytyviin antioksidantteihin. Lamiaceae- perheeseen kuuluvia tuoksuampiaisyrttiä (Dracocephalum moldavica L.) ja sitruunamelissaa (Melissa officinalis L.) on käytetty Iranissa pitkään sekä ruoanlaitossa että lääkinnässä, minkä vuoksi näiden kasvien uutteiden antioksidanttisisältöä päätettiin analysoida käyttäen useaa erilaista in vitro- menetelmää. Näissä kokeissa ilmeni, että uutteilla oli useita antioksidanttisia vaikutuksia. Näistä antioksidanttisista vaikutuksista vastaavia yhdisteitä pyrittiin tunnistamaan käyttäen HPLC-PDA- tekniikkaa, minkä seurauksena niiden havaittiin sisältävän erilaisia polyfenoleita, kuten hydroksyloituneita bentsoeeni- ja cinnamamidihapon johdannaisia sekä flavonoideja. Kummankin kasvin uutteissa runsaimmin esiintynyt yhdiste oli rosmariinihappo. Sitruunamelissaa (M. officinalis) on käytetty antiikin ajoista alkaen kognitiivisten toimintojen häiriöiden hoidossa. Perustuen tietoon kasvin käytöstä perinteisessä lääkinnässä, sen tehoa Alzheimerin taudin hoidossa on tutkittu viime aikoina kliinisin kokein. Sitruunamelissan todettiinkin olevan hyödyksi lievää ja keskivaikeaa Alzheimeimerin tautia sairastavien potilaiden hoidossa. Väitöskirjan osanan olevasta kooste-artikkelista käy ilmi, että tutkimalla lääkekasvien ominaisuuksia voidaan saada arvokkaita suuntaa-antavia vihjeitä Alzheimerin taudin lääkehoidon kehittämiseen. Tämän perusteella päätettiinkin testatata myös sitruunamelissauutteen kykyä estää asetyylikoliiniesteraasin (AChE) toimintaa, koska tämän entsyymin toiminna estämisen tiedetään olevan hyödyksi Alzheimerin taudin hoidossa. Uute kykeni estämään AChE:n toimintaa, minkä vuoksi uutteen sisältämiä komponentteja päätettiin tutkia terkemmin. Uute jaettiin erilaisiin fraktioihin käyttäen HPLC-menetelmää, minkä jälkeen testattiin jokaisen fraktion kykyä inhiboida AchE. Suurin osa fraktioista kykeni inhiboimaan AChE:n toimintaa selkeästi tehokkaammin, kuin raakauute. Kaikista tehokkainta fraktiota analysoitiin tarkemmin sen aktiivisten yhdisteiden tunnistamiseksi, minkä seurauksena sen sisältämät yhdisteet tunnistettiin cis ja trans-rosmariinihapoiksi. Tässä tutkimuksessa tunnistettujen yhdisteiden hyödyllisyyttä Alzheimerin taudin hoidossa tulisi seuraavaksi tutkia erilaisissa in vivo-malleissa. Lisäksi jäljellä olevien fraktioiden kemiallinen koostumus tulisi selvittää sekä antioksidanttiaktiivisuuden ja AChE:n toiminnan inhiboinnin välistä mahdollista yhteyttä tulisi tutkia tarkemmin. Tämä tutkimus osoittaa tuoksuampiasyrtin (D. moldavica) sekä sitruunamelissan (M. officinalis) sisältävän monenlaisia aktiivisia antioksidantteja. Lisäksi sitruunamelissan sisältämät yhdisteet kykenivät estämään asetyylikoliiniesteraasin (AchE) toimintaa. Nämä tulokset tukevat osaltaan väitöskirjan osana olevan kooste-artikkelin johtopäätöksiä, joiden mukaan etnofarmakologinen kasvitutkimus voi osoittautua erittäin hyödylliseksi kehitettäessä uutta lääkehoitoa Alzheimerin tautiin. Lisäksi tässä väitöskirjassa kuvattu tutkimus osoittaakin, että perinteisesti lääkekasvina käytettyä sitruunamelissaa voidaan mahdollisesti hyödyntää uusien Alzheimerin taudin hoitoon käytettävien lääkkeiden kehityksessä.

Genomic Profiling of Gastric Cancer

Gastric cancer is the fourth most common cancer and the second most common cause of cancer-related death worldwide. Due to lack of early symptoms, gastric cancer is characterized by late stage diagnosis and unsatisfactory options for curative treatment. Several genomic alterations have been identified in gastric cancer, but the major factors contributing to initiation and progression of gastric cancer remain poorly known. Gene copy number alterations play a key role in the development of gastric cancer, and a change in gene copy number is one of the fundamental mechanisms for a cancer cell to control the expression of potential oncogenes and tumor suppressor genes. This thesis aims at clarifying the complex genomic alterations of gastric cancer to identify novel molecular biomarkers for diagnostic purposes as well as for targeted treatment. To highlight genes of potential biological and clinical relevance, we carried out a systematic microarray-based survey of gene expression and copy number levels in primary gastric tumors and gastric cancer cell lines. Results were validated using immunohistochemistry, real-time qRT-PCR, and affinity capture-based transcript (TRAC) assay. Altogether 192 clinical gastric tissue samples and 7 gastric cancer cell lines were included in this study. Multiple chromosomal regions with recurrent copy number alterations were detected. The most frequent chromosomal alterations included gains at 7q, 8q, 17q, 19q, and 20q and losses at 9p, 18q, and 21q. Distinctive patterns of copy number alterations were detected for different histological subtypes (intestinal and diffuse) and for cancers located in different parts of the stomach. The impact of copy number alterations on gene expression was significant, as 6-10% of genes located in the regions of gains and losses also showed concomitant alterations in their expression. By combining the information from the DNA- and RNA-level analyses many novel gastric cancer-related genes, such as ALPK2, ENAH, HHIPL2, and OSMR, were identified. Independent genome-wide gene expression analysis of Finnish and Japanese gastric tumors revealed an additional set of genes that was differentially expressed in cancerous gastric tissues compared with normal tissue. Overexpression of one of these genes, CXCL1, was associated with an improved survival of gastric cancer. Thus, using an integrative microarray analysis, several novel genes were identified that may be critically important for gastric carcinogenesis. Further studies of these genes may lead to novel biomarkers for gastric cancer diagnosis and targeted therapy.

Lionhearts of the Playworld : An ethnographic case study of the development of agency in play pedagogy

This is an ethnographic case study of the creation and emergence of a playworld – a pedagogical approach aimed at promoting children’s development and learning in early education settings through the use of play and drama. The data was collected in a Finnish experimental mixed-age elementary school classroom in the school year 2003-2004. In the playworld students and teachers explore different social and cultural phenomena through taking on the roles of characters from a story or a piece of literature and acting inside the frames of an improvised plot. The thesis takes under scrutiny the notion of agency in education. It produces theoretically grounded empirical knowledge of the ways in which children struggle to become recognized and agentive actors in early education settings and how their agency develops in their interaction with adults. The study builds on the activity theoretical and sociocultural tradition and develops a methodological framework called video-based narrative interaction analysis for studying student agency as developing over time but manifesting through the situational material and discursive local interactions. The research questions are: 1. What are the children’s ways of enacting their agency in the playworld? 2. How do the children’s agentive actions change and develop over the spring? 3. What are the potentials and challenges of the playworld for promoting student agency? 4. How do the teachers and the children deal with the contradiction between control and agency in the playworld? The study consists of a summary part and four empirical articles which each have a particular viewpoint. Articles I and II deal with individual students’ paths to agency. In Article I the focus is on the role of resistance and questioning in enabling important spaces for agency. Article II takes a critical gender perspective and analyzes how two girls struggled towards recognition in the playworld. It also illuminates the role of imagination in developing a sense of agency. Article III examines how the open-ended and improvisational nature of the playworld interaction provided experiences and a sense of ‘shared agency’ for the students and teachers in the class. Article IV turns the focus on the teachers and analyzes how their role actions in the playworld helped the children to enact agency. It also discusses the challenges that the teachers faced in this work and asks what makes the playworld activity sustainable in the class. The summary part provides a critical literature review on the concept of agency and argues that the inherently contradictory nature of the phenomenon of agency has not been sufficiently theorized. The summary part also locates the playworld intervention in a historical frame by discussing the changing conceptions of adulthood and childhood in the West. By focusing on the changing role of play and art in both adults’ and children’s contemporary lives, the thesis opens up an important but often neglected perspective on the problem of promoting student agency in education. The results illustrate how engaging in a collectively imagined and dramatized pretend play space together with the children enabled the teachers to momentarily put aside their “knower” positions in the classroom. The fictive roles and the narrative plot helped them to create a necessary incompleteness and open-endedness in the activity that stimulated the children’s initiatives. This meant that the children too could momentarily step out of their traditional classroom positions as pupils and initiate action to further the collective play. Engaging in this kind of unconventional activity and taking up and enacting agency was, however, very challenging for the participating children and teachers. It often contradicted the need to sustain control and order in the classroom. The study concludes that play- and drama-based pedagogies offer a unique but undeveloped potential for developing educational spaces that help teachers and children deal with the often contradictory requirements of schooling.

Adenoviral Gene Therapy for Advanced Head and Neck Cancer

Advanced stage head and neck cancers (HNC) with distant metastasis, as well as prostate cancers (PC), are devastating diseases currently lacking efficient treatment options. One promising developmental approach in cancer treatment is the use of oncolytic adenoviruses, especially in combination therapy with conventional cancer therapies. The safety of the approach has been tested in many clinical trials. However, antitumor efficacy needs to be improved in order to establish oncolytic viruses as a viable treatment alternative. To be able to test in vivo the effects on anti-tumor efficiency of a multimodal combination therapy of oncolytic adenoviruses with the standard therapeutic combination of radiotherapy, chemotherapy and Cetuximab monoclonal antibody (mAb), a xenograft HNC tumor model was developed. This model mimics the typical clinical situation as it is initially sensitive to cetuximab, but resistance develops eventually. Surprisingly, but in agreement with recent findings for chemotherapy and radiotherapy, a higher proportion of cells positive for HNC cancer stem cell markers were found in the tumors refractory to cetuximab. In vitro as well as in vivo results found in this study support the multimodal combination therapy of oncolytic adenoviruses with chemotherapy, radiotherapy and monoclonal antibody therapy to achieve increased anti-tumor efficiency and even complete tumor eradication with lower treatment doses required. In this study, it was found that capsid modified oncolytic viruses have increased gene transfer to cancer cells as well as an increased antitumor effect. In order to elucidate the mechanism of how oncolytic viruses promote radiosensitization of tumor cells in vivo, replicative deficient viruses expressing several promising radiosensitizing viral proteins were tested. The results of this study indicated that oncolytic adenoviruses promote radiosensitization by delaying the repair of DNA double strand breaks in tumor cells. Based on the promising data of the first study, two tumor double-targeted oncolytic adenoviruses armed with the fusion suicide gene FCU1 or with a fully human mAb specific for human Cytotoxic T Lymphocyte-Associated Antigen 4 (CTLA-4) were produced. FCU1 encodes a bifunctional fusion protein that efficiently catalyzes the direct conversion of 5-FC, a relatively nontoxic antifungal agent, into the toxic metabolites 5-fluorouracil and 5-fluorouridine monophosphate, bypassing the natural resistance of certain human tumor cells to 5-fluorouracil. Anti-CTLA4 mAb promotes direct killing of tumor cells via apoptosis and most importantly immune system activation against the tumors. These armed oncolytic viruses present increased anti-tumor efficacy both in vitro and in vivo. Furthermore, by taking advantage of the unique tumor targeted gene transfer of oncolytic adenoviruses, functional high tumor titers but low systemic concentrations of the armed proteins were generated. In addition, supernatants of tumor cells infected with Ad5/3-24aCTLA4, which contain anti-CTLA4 mAb, were able to effectively immunomodulate peripheral blood mononuclear cells (PBMC) of cancer patients with advanced tumors. -- In conclusion, the results presented in this thesis suggest that genetically engineered oncolytic adenoviruses have great potential in the treatment of advanced and metastatic HNC and PC.

The role of catechol-O-methyltransferase (COMT) and the effects of COMT inhibition in brain and in cardiovascular and renal pathophysiology

Catechol-O-methyltransferase (COMT) metabolizes catecholamines such as dopamine (DA), noradrenaline (NA) and adrenaline, which are vital neurotransmitters and hormones that play important roles in the regulation of physiological processes. COMT enzyme has a functional Val158Met polymorphism in humans, which affects the subjects COMT activity. Increasing evidence suggests that this functional polymorphism may play a role in the etiology of various diseases from schizophrenia to cancers. The aim of this project was to provide novel biochemical information on the physiological and especially pathophysiological roles of COMT enzyme as well as the effects of COMT inhibition in the brain and in the cardiovascular and renal system. To assess the roles of COMT and COMT inhibition in pathophysiology, we used four different study designs. The possible beneficial effects of COMT inhibition were studied in double-transgenic rats (dTGRs) harbouring human angiotensinogen and renin genes. Due to angiotensin II (Ang II) overexpression, these animals exhibit severe hypetension, cardiovascular and renal end-organ damage and mortality of approximately 25-40% at the age of 7-weeks. The dTGRs and their Sprague-Dawley controls tissue samples were assessed with light microscopy, immunohistochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and high-pressure liquid chromatography (HPLC) to evaluate the tissue damages and the possible protective effects pharmacological intervention with COMT inhibitors. In a second study, the consequence of genetic and pharmacological COMT blockade in blood pressure regulation during normal and high-sodium was elucidated using COMT-deficient mice. The blood pressure and the heart rate were measured using direct radiotelemetric blood pressure surveillance. In a third study, the effects of acute and subchronic COMT inhibition during combined levodopa (L-DOPA) + dopa decarboxylase inhibitor treatment in homocysteine formation was evaluated. Finally, we assessed the COMT enzyme expression, activity and cellular localization in the CNS during inflammation-induced neurodegeneration using Western blotting, HPLC and various enzymatic assays. The effects of pharmacological COMT inhibition on neurodegeneration were also studied. The COMT inhibitor entacapone protected against the Ang II-induced perivascular inflammation, renal damage and cardiovascular mortality in dTGRs. COMT inhibitors reduced the albuminuria by 85% and prevented the cardiovascular mortality completely. Entacapone treatment was shown to ameliorate oxidative stress and inflammation. Furthermore, we established that the genetic and pharmacological COMT enzyme blockade protects against the blood pressure-elevating effects of high sodium intake in mice. These effects were mediated via enhanced renal dopaminergic tone and suggest an important role of COMT enzyme, especially in salt-sensitive hypertension. Entacapone also ameliorated the L-DOPA-induced hyperhomocysteinemia in rats. This is important, since decreased homocysteine levels may decrease the risk of cardiovascular diseases in Parkinson´s disease (PD) patients using L-DOPA. The Lipopolysaccharide (LPS)-induced inflammation and subsequent delayed dopaminergic neurodegeneration were accompanied by up-regulation of COMT expression and activity in microglial cells as well as in perivascular cells. Interestingly, similar perivascular up-regulation of COMT expression in inflamed renal tissue was previously noted in dTGRs. These results suggest that inflammation reactions may up-regulate COMT expression. Furthermore, this increased glial and perivascular COMT activity in the central nervous system (CNS) may decrease the bioavailability of L-DOPA and be related to the motor fluctuation noted during L-DOPA therapy in PD patients.

Molecular Mechanisms of Androgen Receptor Interactions

The androgen receptor (AR) mediates the effects of the male sex-steroid hormones (androgens), testosterone and 5?-dihydrotestosterone. Androgens are critical in the development and maintenance of male sexual characteristics. AR is a member of the steroid receptor ligand-inducible transcription factor family. The steroid receptor family is a subgroup of the nuclear receptor superfamily that also includes receptors for the active forms of vitamin A, vitamin D3, and thyroid hormones. Like all nuclear receptors, AR has a conserved modular structure consisting of a non-conserved amino-terminal domain (NTD), containing the intrinsic activation function 1, a highly conserved DNA-binding domain, and a conserved ligand-binding domain (LBD) that harbors the activation function 2. Each of these domains plays an important role in receptor function and signaling, either via intra- and inter-receptor interactions, interactions with specific DNA sequences, termed hormone response elements, or via functional interactions with domain-specific proteins, termed coregulators (coactivators and corepressors). Upon binding androgens, AR acquires a new conformational state, translocates to the nucleus, binds to androgen response elements, homodimerizes and recruits sequence-specific coregulatory factors and the basal transcription machinery. This set of events is required to activate gene transcription (expression). Gene transcription is a strictly modulated process that governs cell growth, cell homeostasis, cell function and cell death. Disruptions of AR transcriptional activity caused by receptor mutations and/or altered coregulator interactions are linked to a wide spectrum of androgen insensitivity syndromes, and to the pathogenesis of prostate cancer (CaP). The treatment of CaP usually involves androgen depletion therapy (ADT). ADT achieves significant clinical responses during the early stages of the disease. However, under the selective pressure of androgen withdrawal, androgen-dependent CaP can progress to an androgen-independent CaP. Androgen-independent CaP is invariably a more aggressive and untreatable form of the disease. Advancing our understanding of the molecular mechanisms behind the switch in androgen-dependency would improve our success of treating CaP and other AR related illnesses. This study evaluates how clinically identified AR mutations affect the receptor s transcriptional activity. We reveal that a potential molecular abnormality in androgen insensitivity syndrome and CaP patients is caused by disruptions of the important intra-receptor NTD/LBD interaction. We demonstrate that the same AR LBD mutations can also disrupt the recruitment of the p160 coactivator protein GRIP1. Our investigations reveal that 30% of patients with advanced, untreated local CaP have somatic mutations that may lead to increases in AR activity. We report that somatic mutations that activate AR may lead to early relapse in ADT. Our results demonstrate that the types of ADT a CaP patient receives may cause a clustering of mutations to a particular region of the receptor. Furthermore, the mutations that arise before and during ADT do not always result in a receptor that is more active, indicating that coregulator interactions play a pivotal role in the progression of androgen-independent CaP. To improve CaP therapy, it is necessary to identify critical coregulators of AR. We screened a HeLa cell cDNA library and identified small carboxyl-terminal domain phosphatase 2 (SCP2). SCP2 is a protein phosphatase that directly interacts with the AR NTD and represses AR activity. We demonstrated that reducing the endogenous cellular levels of SCP2 causes more AR to load on to the prostate specific antigen (PSA) gene promoter and enhancer regions. Additionally, under the same conditions, more RNA polymerase II was recruited to the PSA promoter region and overall there was an increase in androgen-dependent transcription of the PSA gene, revealing that SCP2 could play a role in the pathogenesis of CaP.

Improving oncolytic adenoviral therapies for gastrointestinal cancers and tumor initiating cells

Although the treatment of most cancers has improved steadily, only few metastatic solid tumors can be cured. Despite responses, refractory clones often emerge and the disease becomes refractory to available treatment modalities. Furthermore, resistance factors are shared between different treatment regimens and therefore loss of response typically occurs rapidly, and there is a tendency for cross-resistance between agents. Therefore, new agents with novel mechanisms of action and lacking cross-resistance to currently available approaches are needed. Modified oncolytic adenoviruses, featuring cancer-celective cell lysis and spread, constitute an interesting drug platform towards the goals of tumor specificity and the implementation of potent multimodal treatment regimens. In this work, we demonstrate the applicability of capsid-modified, transcriptionally targeted oncolytic adenoviruses in targeting gastric, pancreatic and breast cancer. A variety of capsid modified adenoviruses were tested for transductional specificity first in gastric and pancreatic cancer cells and patient tissues and then in mice. Then, oncolytic viruses featuring the same capsid modifications were tested to confirm that successful transductional targeting translates into enhanced oncolytic potential. Capsid modified oncolytic viruses also prolonged the survival of tumor bearing orthotopic models of gastric and pancreatic cancer. Taken together, oncolytic adenoviral gene therapy could be a potent drug for gastric and pancreatic cancer, and its specificity, potency and safety can be modulated by means of capsid modification. We also characterized a new intraperitoneal virus delivery method in benefit for the persistence of gene delivery to intraperitoneal gastric and pancreatic cancer tumors. With a silica implant a steady and sustained virus release to the vicinity of the tumor improved the survival of the orthotopic tumor bearing mice. Furthermore, silica gel-based virus delivery lowered the toxicity mediating proimflammatory cytokine response and production of total and anti-adenovirus neutralizing antibodies (NAbs). On the other hand, silica shielded the virus against pre-excisting NAbs, resulting in a more favourable biodistribution in the preimmunized mice. The silica implant might therefore be of interest in treating intraperitoneally disseminated disease. Cancer stem cells are thought to be resistant to conventional cancer drugs and might play an important role in cancer relapse and the formation of metastasis. Therefore, we examined if transcriptionally modified oncolytic adenoviruses are able to kill these cells. Complete eradication of CD44+CD24-/low putative breast cancer stem cells was seen in vitro, and significant antitumor activity was detected in CD44+CD24-/low –derived tumor bearing mice. Thus, genetically engineered oncolytic adenoviruses have potential in destroying cancer initiating cells, which may have relevance for the elimination of cancer stem cells in humans.

Adenoviral gene therapy for non-small cell lung cancer

Adenoviral gene therapy is an experimental approach to cancer refractory to standard cancer therapies. Adenoviruses can be utilized as vectors to deliver therapeutic transgenes into cancer cells, while gene therapy with oncolytic adenoviruses exploits the lytic potential of viruses to kill tumor cells. Although adenoviruses demonstrate several advantages over other vectors - such as the unparalleled transduction efficacy and natural tropism to a wide range of tissues - the gene transfer efficacy to cancer cells has been limited, consequently restricting the therapeutic effect. There are, however, several approaches to circumvent this problem. We utilized different modified adenoviruses to obtain information on adenovirus tropism towards non-small cell lung cancer (NSCLC) cells. To enhance therapeutic outcome, oncolytic adenoviruses were evaluated. Further, to enhance gene delivery to tumors, we used mesenchymal stem cells (MSCs) as carriers. To improve adenovirus specificity, we investigated whether widely used cyclooxygenase 2 (Cox-2) promoter is induced by adenovirus infection in nontarget cells and whether selectivity can be retained by the 3 untranslated region (UTR) AU-rich elements. In addition, we investigated whether switching adenovirus fiber can retain gene delivery in the presence of neutralizing antibodies. Our results show that adenoviruses, whose capsids were modified with arginine-glycine-aspartatic acid (RGD-4C), the serotype 3 knob, or polylysins displayed enhanced gene transfer into NSCLC cell lines and fresh clinical specimens from patients. The therapeutic efficacy was further improved by using respective oncolytic adenoviruses with isogenic 24bp deletion in the E1A gene. Cox-2 promoter was also shown to be induced in normal and tumor cells following adenovirus infection, but utilization of 3 UTR elements can increase the tumor specificity of the promoter. Further, the results suggested that use of MSCs could enhance the bioavailability and delivery of adenoviruses into human tumors, although cells had no tumor tropism per se. Finally, we demonstrated that changing adenovirus fiber can allow virus to escape from existing neutralizing antibodies when delivered systemically. In conclusion, these results reveal that adenovirus gene transfer and specificity can be increased by using modified adenoviruses and MSCs as carriers, and fiber modifications simultaneously decrease the effect of neutralizing antibodies. This promising data suggest that these approaches could translate into clinical testing in patients with NSCLC refractory to current modalities.

Genetically Engineered Oncolytic Adenoviruses for the Treatment of Kidney and Breast Cancer

Metastatic kidney and breast cancer are devastating diseases currently lacking efficient treatment options. One promising developmental approach in cancer treatment are oncolytic adenoviruses, which have demonstrated excellent safety in many clinical trials. However, antitumor efficacy needs to be improved in order to make oncolytic viruses a viable treatment alternative. To be able to follow oncolytic virus replication in vivo, we set up a non-invasive imaging system based on coinjection of a replication deficient luciferase expressing virus and a replication competent virus. The system was validated in vitro and in vivo and used in other projects of the thesis. In another study we showed that capsid modifications on adenoviruses result in enhanced gene transfer and increased oncolytic effect on renal cancer cells in vitro. Moreover, capsid modified oncolytic adenoviruses demonstrated significantly improved antitumor efficacy in murine kidney cancer models. To transcriptionally target kidney cancer tissue we evaluated two hypoxia response elements for their usability as tissue specific promoters using a novel dual luciferase imaging system. Based on the results of the promoter evaluation and the studies on capsid modifications, we constructed a transcriptionally and transductionally targeted oncolytic adenovirus armed with an antiangiogenic transgene for enhanced renal cell cancer specificity and improved antitumor efficacy. This virus exhibited kidney cancer specific replication and significantly improved antitumor effect in a murine model of intraperitoneal disseminated renal cell cancer. Cancer stem cells are thought to be resistant to conventional cancer drugs and might play an important role in breast cancer relapse and the formation of metastasis. Therefore, we examined if capsid modified oncolytic adenoviruses are able to kill these cells proposed to be breast cancer initiating. Efficient oncolytic effect and significant antitumor efficacy on tumors established with breast cancer initiating cells was observed, suggesting that oncolytic adenoviruses might be able to prevent breast cancer relapse and could be used in the treatment of metastatic disease. In conclusion, the results presented in this thesis suggest that genetically engineered oncolytic adenoviruses have great potential in the treatment of metastatic kidney and breast cancer.

Actin Dynamics in Muscle Cells

In every cell, actin is a key component involved in migration, cytokinesis, endocytosis and generation of contraction. In non-muscle cells, actin filaments are very dynamic and regulated by an array of proteins that interact with actin filaments and/or monomeric actin. Interestingly, in non-muscle cells the barbed ends of the filaments are the predominant assembly place, whereas in muscle cells actin dynamics was reported to predominate at the pointed ends of thin filaments. The actin-based thin filament pointed (slow growing) ends extend towards the middle of the sarcomere's M-line where they interact with the thick filaments to generate contraction. The actin filaments in muscle cells are organized into a nearly crystalline array and are believed to be significantly less dynamic than the ones in other cell types. However, the exact mechanisms of the sarcomere assembly and turnover are largely unknown. Interestingly, although sarcomeric actin structures are believed to be relatively non-dynamic, many proteins promoting actin dynamics are expressed also in muscle cells (e.g ADF/cofilin, cyclase-associated protein and twinfilin). Thus, it is possible that the muscle-specific isoforms of these proteins promote actin dynamics differently from their non-muscle counterparts, or that actin filaments in muscle cells are more dynamic than previously thought. To study protein dynamics in live muscle cells, I used primary cell cultures of rat cardiomyocytes. My studies revealed that a subset of actin filaments in cardiomyocyte sarcomeres displays rapid turnover. Importantly, I discovered that the turnover of actin filaments depends on contractility of the cardiomyocytes and that the contractility-induced actin dynamics plays an important role in sarcomere maturation. Together with previous studies those findings suggest that sarcomeres undergo two types of actin dynamics: (1) contractility-dependent turnover of whole filaments and (2) regulatory pointed end monomer exchange to maintain correct thin filament length. Studies involving an actin polymerization inhibitor suggest that the dynamic actin filament pool identified here is composed of filaments that do not contribute to contractility. Additionally, I provided evidence that ADF/cofilins, together with myosin-induced contractility, are required to disassemble non-productive filaments in developing cardiomyocytes. In addition, during these studies we learned that isoforms of actin monomer binding protein twinfilin, Twf-1 and Twf-2a localise to myofibrils in cardiomyocytes and may thus contribute to actin dynamics in myofibrils. Finally, in collaboration with Roberto Dominguez s laboratory we characterized a new actin nucleator in muscle cells - leiomodin (Lmod). Lmod localises towards actin filament pointed ends and its depletion by siRNA leads to severe sarcomere abnormalities in cardiomyocytes. The actin filament nucleation activity of Lmod is enhanced by interactions with tropomyosin. We also revealed that Lmod expression correlates with the maturation of myofibrils, and that it associates with sarcomeres only at relatively late stages of myofibrillogenesis. Thus, Lmod is unlikely to play an important role in myofibril formation, but rather might be involved in the second step of the filament arrangement and/or maintenance through its ability to promote tropomyosin-induced actin filament nucleation occurring at the filament pointed ends. The results of these studies provide valuable new information about the molecular mechanisms underlying muscle sarcomere assembly and turnover. These data offer important clues to understanding certain physiological and pathological behaviours of muscle cells. Better understanding of the processes occurring in muscles might help to find strategies for determining, diagnosis, prognosis and therapy in heart and skeletal muscles diseases.

CMV-, EBV-, and HHV-6-DNAemia after liver transplantation

Viral infections caused by herpesviruses are common complications after organ transplantation and they are associated with substantial morbidity and even mortality. Herpesviruses remain in a latent state in a host after primary infection and may reactivate later. CMV infection is the most important viral infection after liver transplantation. Less is known about the significance of human herpesvirus-6 (HHV-6). EBV is believed to play a major role in the development of post-transplant lymphoproliferative disorders (PTLD). The aim of this study was to investigate the CMV-, EBV- and HHV-6 DNAemia after liver transplantation by frequent monitoring of adult liver transplant patients. The presence of CMV, EBV and HHV-6 DNA were demonstrated by in situ hybridization assays and by real-time PCR methods from peripheral blood specimens. CMV and HHV-6 antigens were demonstrated by antigenemia assays and compared to the viral DNAemia. The response to antiviral therapy was also investigated. CMV-DNAemia appeared earlier than CMV pp65-antigenemia after liver transplantation. CMV infections were treated with ganciclovir. However, most of the treated patients demonstrated persistence of CMV-DNA for up to several months. Continuous CMV-DNA expression of peripheral blood leukocytes showed that the virus is not eliminated by ganciclovir and recurrences can be expected during several months after liver transplantation. HHV-6 DNAemia / antigenemia was common and occurred usually within the first three months after liver transplantation together with CMV. The HHV-6 DNA expression in peripheral blood mononuclear cells correlated well with HHV-6 antigenemia. Antiviral treatment significantly decreased the number of HHV-6 DNA positive cells, demonstrating the response to ganciclovir treatment. Clinically silent EBV reactivations with low viral loads were relatively common after liver transplantation. These EBV-DNAemias usually appeared within the first three months after liver transplantation together with betaherpesviruses (CMV, HHV-6, HHV-7). One patient developed high EBV viral loads and developed PTLD. These results indicate that frequent monitoring of EBV-DNA levels can be useful to detect liver transplant patients at risk of developing PTLD.

Postoperative Volume Therapy in Cardiac Surgery : Effects on Hemostatic and Circulatory Variables

Background: Patients may need massive volume-replacement therapy after cardiac surgery because of large fluid transfer perioperatively, and the use of cardiopulmonary bypass. Hemodynamic stability is better maintained with colloids than crystalloids but colloids have more adverse effects such as coagulation disturbances and impairment of renal function than do crystalloids. The present study examined the effects of modern hydroxyethyl starch (HES) and gelatin solutions on blood coagulation and hemodynamics. The mechanism by which colloids disturb blood coagulation was investigated by thromboelastometry (TEM) after cardiac surgery and in vitro by use of experimental hemodilution. Materials and methods: Ninety patients scheduled for elective primary cardiac surgery (Studies I, II, IV, V), and twelve healthy volunteers (Study III) were included in this study. After admission to the cardiac surgical intensive care unit (ICU), patients were randomized to receive different doses of HES 130/0.4, HES 200/0.5, or 4% albumin solutions. Ringer’s acetate or albumin solutions served as controls. Coagulation was assessed by TEM, and hemodynamic measurements were based on thermodilutionally measured cardiac index (CI). Results: HES and gelatin solutions impaired whole blood coagulation similarly as measured by TEM even at a small dose of 7 mL/kg. These solutions reduced clot strength and prolonged clot formation time. These effects were more pronounced with increasing doses of colloids. Neither albumin nor Ringer’s acetate solution disturbed blood coagulation significantly. Coagulation disturbances after infusion of HES or gelatin solutions were clinically slight, and postoperative blood loss was comparable with that of Ringer’s acetate or albumin solutions. Both single and multiple doses of all the colloids increased CI postoperatively, and this effect was dose-dependent. Ringer’s acetate had no effect on CI. At a small dose (7 mL/kg), the effect of gelatin on CI was comparable with that of Ringer’s acetate and significantly less than that of HES 130/0.4 (Study V). However, when the dose was increased to 14 and 21 mL/kg, the hemodynamic effect of gelatin rose and became comparable with that of HES 130/0.4. Conclusions: After cardiac surgery, HES and gelatin solutions impaired clot strength in a dose-dependent manner. The potential mechanisms were interaction with fibrinogen and fibrin formation, resulting in decreased clot strength, and hemodilution. Although the use of HES and gelatin inhibited coagulation, postoperative bleeding on the first postoperative morning in all the study groups was similar. A single dose of HES solutions improved CI postoperatively more than did gelatin, albumin, or Ringer’s acetate. However, when administered in a repeated fashion, (cumulative dose of 14 mL/kg or more), no differences were evident between HES 130/0.4 and gelatin.