A Farewell to Flat Biology. Three-dimensional Cell Culture Models in Cancer Drug Target Identification and Validation

Cells of epithelial origin, e.g. from breast and prostate cancers, effectively differentiate into complex multicellular structures when cultured in three-dimensions (3D) instead of conventional two-dimensional (2D) adherent surfaces. The spectrum of different organotypic morphologies is highly dependent on the culture environment that can be either non-adherent or scaffold-based. When embedded in physiological extracellular matrices (ECMs), such as laminin-rich basement membrane extracts, normal epithelial cells differentiate into acinar spheroids reminiscent of glandular ductal structures. Transformed cancer cells, in contrast, typically fail to undergo acinar morphogenic patterns, forming poorly differentiated or invasive multicellular structures. The 3D cancer spheroids are widely accepted to better recapitulate various tumorigenic processes and drug responses. So far, however, 3D models have been employed predominantly in the Academia, whereas the pharmaceutical industry has yet to adopt a more widely and routine use. This is mainly due to poor characterisation of cell models, lack of standardised workflows and high throughput cell culture platforms, and the availability of proper readout and quantification tools. In this thesis, a complete workflow has been established entailing well-characterised 3D cell culture models for prostate cancer, a standardised 3D cell culture routine based on high-throughput-ready platform, automated image acquisition with concomitant morphometric image analysis, and data visualisation, in order to enable large-scale high-content screens. Our integrated suite of software and statistical analysis tools were optimised and validated using a comprehensive panel of prostate cancer cell lines and 3D models. The tools quantify multiple key cancer-relevant morphological features, ranging from cancer cell invasion through multicellular differentiation to growth, and detect dynamic changes both in morphology and function, such as cell death and apoptosis, in response to experimental perturbations including RNA interference and small molecule inhibitors. Our panel of cell lines included many non-transformed and most currently available classic prostate cancer cell lines, which were characterised for their morphogenetic properties in 3D laminin-rich ECM. The phenotypes and gene expression profiles were evaluated concerning their relevance for pre-clinical drug discovery, disease modelling and basic research. In addition, a spontaneous model for invasive transformation was discovered, displaying a highdegree of epithelial plasticity. This plasticity is mediated by an abundant bioactive serum lipid, lysophosphatidic acid (LPA), and its receptor LPAR1. The invasive transformation was caused by abrupt cytoskeletal rearrangement through impaired G protein alpha 12/13 and RhoA/ROCK, and mediated by upregulated adenylyl cyclase/cyclic AMP (cAMP)/protein kinase A, and Rac/ PAK pathways. The spontaneous invasion model tangibly exemplifies the biological relevance of organotypic cell culture models. Overall, this thesis work underlines the power of novel morphometric screening tools in drug discovery.

Morphology and morphometry of feline bone marrow-derived mesenchymal stem cells in culture

Mesenchymal stem cells (MSC) are increasingly being proposed as a therapeutic option for treatment of a variety of different diseases in human and veterinary medicine. Stem cells have been isolated from feline bone marrow, however, very few data exist about the morphology of these cells and no data were found about the morphometry of feline bone marrow-derived MSCs (BM-MSCs). The objectives of this study were the isolation, growth evaluation, differentiation potential and characterization of feline BM-MSCs by their morphological and morphometric characteristics. in vitro differentiation assays were conducted to confirm the multipotency of feline MSC, as assessed by their ability to differentiate into three cell lineages (osteoblasts, chondrocytes, and adipocytes). To evaluate morphological and morphometric characteristics the cells are maintained in culture. Cells were observed with light microscope, with association of dyes, and they were measured at 24, 48, 72 and 120h of culture (P1 and P3). The non-parametric ANOVA test for independent samples was performed and the means were compared by Tukey's test. On average, the number of mononuclear cells obtained was 12.29 (±6.05x10(6)) cells/mL of bone marrow. Morphologically, BM-MSCs were long and fusiforms, and squamous with abundant cytoplasm. In the morphometric study of the cells, it was observed a significant increase in average length of cells during the first passage. The cell lengths were 106.97±38.16µm and 177.91±71.61µm, respectively, at first and third passages (24 h). The cell widths were 30.79±16.75 µm and 40.18±20.46µm, respectively, at first and third passages (24 h).The nucleus length of the feline BM-MSCs at P1 increased from 16.28µm (24h) to 21.29µm (120h). However, at P3, the nucleus length was 26.35µm (24h) and 25.22µm (120h). This information could be important for future application and use of feline BM-MSCs.

Growth and nutrient concentration in coffee root system under weed species competition

The effects of competition of six weed species on growth, nutrient concentration and nutrient content of coffee plant root system under greenhouse conditions were evaluated. Thirty days after coffee seedling transplantation into 12 L pots with soil level area of 6.5 dm². Weeds were transplanted or sowed in these pots, at densities of 0, 1, 2, 3, 4 and 5 plants per pot. The duration of competition (or weedy periods) from weed transplantation or emergence until plant harvesting, at the weed preflowering stage, were (in days): 77 (Bidens pilosa), 180 (Commelina diffusa), 82 (Leonurus sibiricus), 68 (Nicandra physaloides), 148 (Richardia brasiliensis) and 133 (Sida rhombifolia). Dry matter of coffee plants was linearly reduced with increasing B. pilosa and S. rhombifolia density, with pronounced effect of B. pilosa. C. diffusa was the only weed species whose increasing density in the pots did not diminish crop root dry matter. L. sibiricus, N. physaloides and R. brasiliensis reduced root dry matter of coffee plants by 75, 52 and 47%, respectively, as compared to the weed-free treatment, regardless of weed density. Under competition, even though weed species showed lower macronutrient concentration in the roots (except for P), they accumulated 4.2 (N), 12.3 (P), 4.3 (K), 5.5 (Ca), 7.6 (Mg) and 4.4 (S) times more nutrients in the roots than the coffee plants. Crop and weed nutrient concentration, as well as competition degrees greatly varied depending on both weed species and densities.

Heritage through Fiction: Dracula Tourism in Romania

Heritage and tourism have become inextricably linked. This link can be seen as producing inauthentic and falsified tradition, and it can therefore be seen as a threat to cultural heritage. On the other hand the link can be seen as a positive thing, as something which helps to preserve herit-age, culture and folklore in a changing and globalising world. This dissertation investigates heritage in the context of Dracula Tourism in Romania. Dracula tourism is tourism where tourists visit places connected with either the fictional vampire Dracula or the historical Dracula, a Romanian historical ruler Vlad the Impaler. The main research question of this study is how can Romanian heritage and culture be shown and promoted through a seemingly superficial Dracula tourism which is based on Western popular cul-ture? And is it possible to find Romanian heritage through popular fiction in Dracula tourism? The main sources for this work are based on the fieldwork done by the author in 2010 and 2011 and the web pages of ten Romanian travel agencies that offer Dracula tourism. The stories and images found on the web pages and used by the tour guides form the bulk of the research material. The emphasis and perspective of this research is folkloristic. Critical discourse analysis and multimodal discourse analysis form the main theoretical approach of this dissertation. In addition the research material is approached through intertextuality, folklore process, hybridisation, authenticity and social constructionism. This dissertation aims to offer new perspectives on the research literature concerning tourism and heritage and to offer a folkloristic view of tourism research. It also aims to offer new perspectives to folkloristics in terms of the research on the use of folklore and tradition and offer new perspectives on the use and definition of the concept of authenticity. Although the research subject of this thesis is Dracula tourism in Romania, the findings can be utilised and applied in a larger context and field of research. The key research findings show that heritage can be found within Dracula Tourism in three forms: as defined from above (UNESCO World Heritage Sites), as local heritage and as a form of opposition. The Romanian travel agencies researched in this dissertation use Dracula tourism as a gateway into Romanian history, culture, tradition and heritage. Kulttuuriperintö ja turismi yhdistyvät toisiinsa erottamattomasti. Toisaalta tämän yhteyden on nähty tuottavan epäautenttista ja väärennettyä perinnettä, ja tällöin sen on koettu muodostavan uhan kulttuuriperinnölle. Toisaalta yhteys on mielletty myös positiivisena asiana, sillä sen on nähty toimivan globalisoituvassa maailmassa kulttuuriperintöä, kulttuuria ja kansanperinnettä säilyttävänä tekijänä. Väitöskirjassa tutkitaan sitä, miten kulttuuriperintö ilmenee Dracula-turismissa Romaniassa. Dracula- turismi on turismia, joka liittyy joko fiktiiviseen vampyyrikreivi Draculaan tai historiallisena Draculana tunnettuun romanialaiseen hallitsijaan Vlad Seivästäjään. Väitöskirjan päätutkimuskysymyksenä on, miten romanialaista kulttuuriperintöä ja kulttuuria voidaan tuoda esiin näennäisesti pinnallisen ja länsimaiseen populaarikulttuuriin pohjautuvan Dracula-turismin kautta. Työn päälähteet pohjautuvat tutkijan vuosina 2010 ja 2011 tekemiin kenttätöihin sekä kymmenen Dracula-turismia tarjoavan romanialaisen matkatoimiston WWW-sivustoihin. Internet-sivuilta löytyvät tarinat ja kuvat sekä matkaoppaiden käyttämät tarinat muodostavat tutkimuksen tutkimusaineiston. Tutkimuksen painotus ja näkökulma ovat folkloristisia. Tutkimuksen teoreettinen viitekehys muodostuu kriittisestä diskurssianalyysistä sekä multimodaalisesta diskurssianalyysistä. Näiden lisäksi tutkimusaineistoa analysoidaan intertekstuaalisuuden, folkloreprosessin, hybridisaation, autenttisuuden ja sosiaalisen konstruktion käsitteiden avulla. Väitöskirjatutkimus tarjoaa uusia näkökulmia turismia ja kulttuuriperintöä käsittelevään tutkimukseen sekä tarjoaa matkailun tutkimukseen folkloristisen lisänäkökulman. Folkloristisen tutkimuksen näkökulmasta työn keskiössä ovat perinteen ja folkloren hyödyntäminen sekä autenttisuuden käsitteen määrittely ja käyttö, joihin työssä otetaan kantaa. Vaikka tutkimus käsittelee Dracula-turismia Romaniassa, ovat tutkimustulokset käytettävissä ja sovellettavissa myös laajemmin. Työn keskeiset tutkimustulokset osoittavat, että kulttuuriperintö ilmenee Dracula-turismissa kolmella eri tavalla: ylätasolla kuten esimerkiksi UNESCO:n kohteissa, alatasolla paikallisten ihmisten tai ihmisryhmien määrittelemänä sekä vastustuksen muotona. Väitöskirjassa tutkitut matkatoimistot käyttävät Dracula-turismia ikään kuin porttina romanialaiseen historiaan, kulttuuriin, perinteeseen ja kulttuuriperinteeseen.

Westerners in search of the legendary potion : ayahuasca travel in the borderland between tourism and pilgrimage

The thesis examines the phenomenon most commonly known as “ayahuasca tourism” – i.e. the practice of westerners traveling to South America and partaking in ceremonies in which a powerful entheogenic brew, ayahuasca, is consumed. While this popular phenomenon has been steadily increasing during the last decades, it has, however, been insufficiently studied by scholars. An important question which has not been properly addressed in earlier studies is how ayahuasca tourism relates to the wider occurrence of travel and how it should be perceived with reference to the theoretical frameworks on the subject of travel. Drawing on theories regarding pilgrimage and tourism, the main purpose of this thesis is to examine the relationship between ayahuasca tourism and the broader spectrum of travel. In particular, the study tests the designations “pilgrimage”, “religious tourism” and “spiritual tourism” with reference to ayahuasca tourism. Utilizing earlier literature as well as ayahuasca tourists‟ reports obtained from an Internet forum as a basis for analysis, I search for a suitable terminology to be used for the phenomenon. The study lays special emphasis on the protagonists‟ motivations, experiences and outcomes in order to take note of various aspects of the wide-ranging occurrence of ayahuasca tourism. Key findings indicate that ayahuasca tourism is best understood as a combination of pilgrimage and tourism. On the basis of the analysis I argue that ayahuasca tourism should be labeled as “pilgrimage” and/or “spiritual tourism”, and the tourists respectively as “pilgrims” and/or “spiritual tourists”. The category of “religious tourism/tourist”, on the other hand, turns out to be an inappropriate designation when describing the phenomenon. In general, through my study I show that the results are consistent with the present trend in the study of travel to perceive pilgrimage and tourism as theoretically similar phenomena. The study of ayahuasca tourism serves thus as living proof of contemporary travel, in which the categories of pilgrimage and tourism are often indistinguishable. I suggest that ayahuasca tourism is by no means exceptional on this point, but can rather be used as an illustration of modern travel forms on a general level. Thus, the present study does not only add to the research of ayahuasca tourism, but also provides additional insights into the study of travel.

Carbon/Nitrogen Ratio Optimization and Periphyton Development on the Production and Sustainability of Penaeus Monodon(Fabricius)in Extensive Culture System

Controlling the inorganic nitrogen by manipulating carbon / nitrogen ratio is a method gaining importance in aquaculture systems. Nitrogen control is induced by feeding bacteria with carbohydrates and through the subsequent uptake of nitrogen from the water for the synthesis of microbial proteins. The relationship between addition of carbohydrates, reduction of ammonium and the production of microbial protein depends on the microbial conversion coefficient. The carbon / nitrogen ratio in the microbial biomass is related to the carbon contents of the added material. The addition of carbonaceous substrate was found to reduce inorganic nitrogen in shrimp culture ponds and the resultant microbial proteins are taken up by shrimps. Thus, part of the feed protein is replaced and feeding costs are reduced in culture systems.The use of various locally available substrates for periphyton based aquaculture practices increases production and profitability .However, these techniques for extensive shrimp farming have not so far been evaluated. Moreover, an evaluation of artificial substrates together with carbohydrate source based farming system in reducing inorganic nitrogen production in culture systems has not yet been carried-out. Furthermore, variations in water and soil quality, periphyton production and shrimp production of the whole system have also not been determined so-far.This thesis starts with a general introduction , a brief review of the most relevant literature, results of various experiments and concludes with a summary (Chapter — 9). The chapters are organised conforming to the objectives of the present study. The major objectives of this thesis are, to improve the sustainability of shrimp farming by carbohydrate addition and periphyton substrate based shrimp production and to improve the nutrient utilisation in aquaculture systems.

Studies on The Ecology And Production of Algae in Prawn Culture Systems

Existing method of culture were largely based on empirical knowledge. Lacking a scientific basis as such methods did, they were often wasteful and suffered severe limitation. Modern methods of fish and prawn culture based on scientific research, have revolutioned the industry in recent years and not only extended its scope to cover the whole country but led to increased fish and prawn production. An understanding of the biological capability of the water in the perennial and seasonal culture ponds, and the nature and extent of the influence of the abiotic factors on the production of organisms in the primary level of food chain would contribute to effectively implement management measures in the stocking strategies and in the evaluation of economics of production of prawns. It is against this background that the present topic of investigation "Studies on the ecology and production of algae in prawn culture systems” was selected.

Forecasting the spot prices of various coffee types using linear and non-linear error correction models

Este documento estima modelos lineales y no-lineales de corrección de errores para los precios spot de cuatro tipos de café. En concordancia con las leyes económicas, se encuentra evidencia que cuando los precios están por encima de su nivel de equilibrio, retornan a éste mas lentamente que cuando están por debajo. Esto puede reflejar el hecho que, en el corto plazo, para los países productores de café es mas fácil restringir la oferta para incrementar precios, que incrementarla para reducirlos. Además, se encuentra evidencia que el ajuste es más rápido cuando las desviaciones del equilibrio son mayores. Los pronósticos que se obtienen a partir de los modelos de corrección de errores no lineales y asimétricos considerados en el trabajo, ofrecen una leve mejoría cuando se comparan con los pronósticos que resultan de un modelo de paseo aleatorio.

La diplomacia cultural como herramienta para el mejoramiento de la relación económica de Colombia con Corea del Sur y Australia

Esta investigación se preocupa por dilucidar la función de la diplomacia cultural como herramienta para mejorar la relación económica de Colombia con Corea del Sur y Australia. Así, se analiza a la diplomacia cultural y lo que hace Colombia en esta materia en ambos países; así como el estado de la relación económica de Colombia en un periodo de ocho años con dichos países, y cómo las acciones culturales colombianas podrían llegar a mejorar dicha relación económica. De esta manera además del desarrollo conceptual de diplomacia cultural y los indicadores económicos, a saber; exportaciones; Inversión Extranjera Directa y turismo; se corrió un modelo de regresión lineal para saber si efectivamente hay relación entre ambas variables y una contribución final que consiste en una propuesta de generación de indicadores de gestión a utilizarse al momento de implementar la diplomacia cultural como herramienta en política exterior.

Disabled access and heritage attractions

In a UK context, the importance of heritage tourism, the potential of the disabled market, and government policies concerning tourism, social inclusion, and the historic environment provide the setting within which access improvements at heritage attractions for disabled visitors are studied. At issue is how disabled access and conservation can be reconciled. The stakeholders range from the central actors, the disabled tourists and the heritage tourism service providers, through to the gatekeeper and lobby players in the conservation, disability, and tourism contexts. The critical power structures are identified. Changes to the historic environment are managed through the conservation planning system in which disability interests are not formally represented. Recent disability discrimination legislation has not altered this balance of power, and is a source of uncertainty over the access standards that should apply to heritage attractions. An evaluation of progress in implementing access improvements at heritage attractions reveals the limited extent of improvements undertaken to date. Consideration is given not only to physical access but also to alternative methods (intellectual access) of providing the heritage tourism service. In conclusion, the situation is examined from three perspectives. From the disabled tourists' perspective, choice of heritage attractions to visit remains restricted compared to that of nondisabled tourists. The lack of consultation with disabled stakeholders in the access improvements decision-making process is discussed, including the acceptability of alternative methods of service delivery to disabled tourists. The uncertainties facing heritage tourism service providers arising from the disability discrimination legislation are considered but, to ensure a more balanced recognition of disability interests, both conservation planning and disability discrimination legislation need to be amended, adjusting the roles of the legislative gatekeepers.

The UK Department of National Heritage: a model for the development and integration of leisure policy in the new Europe?

Leisure is in the vanguard of a social and cultural revolution which is replacing the former East/West political bipolarity with a globalised economic system in which the new Europe has a central rôle. Within this revolution, leisure, including recreation, culture and tourism, is constructed as the epitome of successful capitalist development; the very legitimisation of the global transmogrification from a production to a consumption orientation. While acting as a direct encouragement to the political transformation in many eastern European states, it is uncertain how the issue of leisure policy is being handled, given its centrality to the new economic order. This paper therefore examines the experience of western Europe, considering in particular the degree to which the newly-created Department of National Heritage in the UK provides a potential model for leisure development and policy integration in the new Europe. Despite an official rhetoric of support and promotion of leisure activities, reflecting the growing economic significance of tourism and the positive relationship between leisure provision and regional economic development, the paper establishes that in the place of the traditional rôle of the state in promoting leisure interests, the introduction of the Department has signified a shift to the use of leisure to promote the Government's interests, particularly in regenerating citizen rights claims towards the market. While an institution such as the Department of National Heritage may have relevance to emerging states as a element in the maintenance of political hegemony, therefore, it is questionable how far it can be viewed as a promoter or protector of leisure as a signifier of a newly-won political, economic and cultural freedom throughout Europe.

Interaction between attaching and effacing Escherichia coli serotypes O157:H7 and O26:K60 in cell culture

Ruminants harbour both O157:H7 and non-O157 Attaching Effacing Escherichia coli (AEEC) strains but to date only nonO157 AEEC have been shown to induce attaching effacing lesions in naturally infected animals. However, O157 may induce lesions in deliberate oral inoculation studies and persistence is considered dependent upon the bacterially encoded locus for enterocyte effacement. In concurrent infections in ruminants it is unclear whether non-O157 AEEC contribute either positively or negatively to the persistence of E. coli O157:H7. To investigate this, and prior to animal studies, E. coli O157:H7 NCTC 12900, a non-toxigenic strain that persists in conventionally reared sheep, and non-toxigenic AEEC O26:K60 isolates of sheep origin were tested for adherence to Hep-2 tissue culture alone and in competition one with another. Applied together, both strains adhered in similar numbers but lower than when either was applied separately. Pre-incubation of tissue culture with either one strain reduced significantly (P < 0.05) the extent of adherence of the strain that was applied second. It was particularly noticeable that AEEC O26 when applied first reduced adherence and inhibited microcolony formation, as demonstrated by confocal microscopy, of E. coli 01 57:H7. The possibility that prior colonisation of a ruminant by non-O157 AEEC such as O26 may antagonise O157 colonisation and persistence in ruminants is discussed. (C) 2004 Elsevier B.V. All rights reserved.

Ecological and social drivers of coffee pollination in Santander, Colombia

Bees and other insects provide pollination services that are key to determining the fruit set on coffee plantations. These pollination services are influenced by local ecology as well as human factors, both social and economic. To better understand these different factors, we assessed their effect on pollinators and coffee pollination services in Santander, Colombia. We quantified the effect of key ecological drivers on pollinator community composition, such as the method of farm management (either conventional or organic) and the surrounding landscape composition, specifically the proximity to forest. We found that ambient levels of pollination services provided by the local pollinator fauna (open pollination) accounted for a 10.5 ± 2.0% increase in final coffee fruit set, and that the various pollinators are affected differently by the differing factors. For example, our findings indicate that conventional farm management, using synthetic inputs, can promote pollinators, especially if they are in close proximity to natural forest fragments. This is particularly true for stingless bees. Honeybee visitation to coffee is also positively influenced by the conventional management of farms. Factors associated with greater numbers of stingless bees on farms include greater shade cover, lower tree densities, smaller numbers and types of trees in bloom, and younger coffee plantations. A forested landscape close to farms appears to enhance these factors, giving increased stability and resilience to the pollinating bees and insects. However we found that organic farms also support diverse pollinator communities, even if distant from forest fragments. The contribution of honeybees to pollination value (US$129.6/ha of coffee) is greater than that of stingless bees (US$16.5/ha of coffee). Since the method of farm management has a major impact on the numbers and types of pollinators attracted to farms, we have analysed the statistically significant social factors that influence farmers’ decisions on whether to adopt organic or conventional practices. These include the availability of technology, the type of landowner (whether married couples or individual owners), the number of years of farmers’ formal education, the role of institutions, membership of community organizations, farm size, coffee productivity and the number of coffee plots per farm. It is hoped that the use of our holistic approach, which combines investigation of the social as well as the ecological drivers of pollination, will help provide evidence to underpin the development of best practices for integrating the management of pollination into sustainable agricultural practices.

Comparison of agar gel immunodiffusion test, rapid slide agglutination test, microbiological culture and PCR for the diagnosis of canine brucellosis

The performance of the rapid slide agglutination test, with and without 2-mercaptoethanol (RSAT and 2ME-RSAT) and agar gel immunodiffusion test (AGID) was evaluated for the diagnosis of brucellosis in naturally infected dogs. The microbiological culture, PCR and clinical parameters were used as reference. A total of 167 dogs were clinically examined and tested by blood culture, culture of semen/vaginal swab and PCR in blood and semen/vaginal swab. According to the results observed the 167 dogs were divided into three groups: Brucella canis infected dogs (Group 1). B. canis non-infected dogs (Group 2) and dogs with suspected brucellosis (Group 3). The dogs were then tested by RSAT, 2ME-RSAT and AGID. Groups 1 and 2 were used to calculate the diagnostic sensitivity and specificity of the serological tests and the results observed in Group 3 were also discussed. The diagnostic sensitivity of RSAT, 2ME-RSAT and AGID was respectively 70.58%, 31.76%, and 52.94%. The diagnostic specificity of RSAT, 2ME-RSAT and AGID was respectively 83.34%, 100%, and 100%. In dogs with suspected brucellosis 15% were RSAT positive, none was 2ME-RSAT positive and 5% were AGID positive. Although the serological tests are the most commonly used methods for brucellosis diagnosis, a significant proportion of false-negative results were observed highlighting the importance of the direct methods of diagnosis, like blood culture and PCR to improve the diagnosis of canine brucellosis. (c) 2008 Elsevier Ltd. All rights reserved.

The stakeholders‟ involvement in the process of building and maintaining a destination brand

A destination is a place that attracts visitors for a temporary stay to participate in tourism related activities or non- activities. Globalization, the increased number of travelers and the increased buying power have increased the competition between the destinations and the destinations have become more substitutable. It has been agreed that destinations can be branded as well as products and to be competitive it is getting common to brand destinations. Destination Marketing Organizations (DMOs) are responsible for the marketing of an identifiable destination. The purpose of this study is to present an exploratory study of how a destination marketing organization creates and builds a strong destination brand and how the stakeholders have been involved in the process. The study is done with a qualitative case study approach. The case study was chosen as the research method to make a detailed and intensive analysis of the research objective, in this case the destination brand of Brazil and its stakeholders.