Gene expression profile of epithelial cells and mesenchymal cells derived from limbal explant culture

Purpose: Limbal stem cell deficiency is a challenging clinical problem and the current treatment involves replenishing the depleted limbal stem cell (LSC) pool by either limbal tissue transplantation or use of cultivated limbal epithelial cells (LEC). Our experience of cultivating the LEC on denuded human amniotic membrane using a feeder cell free method, led to identification of mesenchymal cells of limbus (MC-L), which showed phenotypic resemblance to bone marrow derived mesenchymal stem cells (MSC-BM). To understand the transcriptional profile of these cells, microarray experiments were carried out.Methods: RNA was isolated from cultured LEC, MC-L and MSC-BM and microarray experiments were carried out by using Agilent chip (4x44 k). The microarray data was validated by using Realtime and semiquntitative reverse transcription polymerase chain reaction. Results: The microarray analysis revealed specific gene signature of LEC and MC-L, and also their complementary role related to cytokine and growth factor profile, thus supporting the nurturing roles of the MC-L. We have also observed similar and differential gene expression between MC-L and MSC-BM.Conclusions: This study represents the first extensive gene expression analysis of limbal explant culture derived epithelial and mesenchymal cells and as such reveals new insight into the biology, ontogeny, and in vivo function of these cells.

Elämänmuutoksen, jännityksen ja asiantuntijuuden representointi Veikkaus Oy:n mainonnassa vuosina 1970— 2006

Aim of this master's thesis paper for consumer economics, is to research gambling advertisements in Finland over a period of 35 years, from 1970 to 2006. Veikkaus Oy (later Veikkaus), was founded in 1940, as one of the three licensed gambling organizations in Finland. Material for the current research comprised 1494 advertisements published by Veikkaus in newspapers and magazines at that time. Veikkaus has the exclusive licence to organize lotto games, sport games, instant games and other draw games in Finland. The other two operators, The Finnish Slot Machine Association RAY and Fintoto (on-track horse betting), were not included in the current analysis. This study has been completed according to research contract and grand by the Finnish Foundation for Gaming Research (Pelitoiminnan tutkimussäätiö). In general, advertisements reflect surrounding culture and time, and their message is built on stratified meanings, symbols and codes. Advertising draws the viewer's attention, introduces the advertised subject, and finally, affects the individual's consumption habits. However, advertisements not only work on individual level, but also influence public perception of the advertised product. Firstly, in order to assess gambling as a phenomenon, this paper discusses gambling as consumer behaviour, and also reviews history of gambling in Finland. Winning is a major feature of gambling, and dreaming about positive change of life is a centre of most gambling ads. However, perceived excitement through risk of losing can also be featured in gambling ads. Secondly, this study utilizes Veikkaus’ large advertising archives, were advertising data is analyzed by content analysis and the semiotic analysis. Two methods have been employed to support analyzing outcome in a synergistic way. Content analysis helps to achieve accuracy and comprehensiveness. Semiotic analysis allows deeper and more sensitive analysis to emerged findings and occurrences. It is important to understand the advertised product, as advertising is bound to the culture and time. Hence, to analyze advertising, it is important to understand the environment where the ads appear. Content analysis of Veikkaus data discovered the main gambling and principal advertisement style for each.period. Interestingly, nearly half of Veikkaus’ advertisements promoted topic other than “just winning the bet”. Games of change, like Lotto, typically advertised indirectly represented dreams about winning. In the category of skill gambling, features were represented as investment, and the excitement of sporting expertise was emphasized. In addition, there were a number of gambling ads that emphasize social responsibility of Veikkaus as a government guided organization. Semiotic methods were employed to further elaborate on findings of content analysis. Dreaming in the advertisements was represented by the product of symbols, (e.g. cars and homes) that were found to have significance connection with each other. Thus, advertising represents change of life obtained by the winning. Interestingly, gambling ads promoting jackpots were often representing religious symbolisms. Ads promoting social responsibility were found to be the most common during economical depression of the 90’s. Deeper analysis showed that at that time, advertisements frequently represented depression-related meanings, such as unemployment and bank loans. Skill gaming ads were often represented by sports expertise – late 90’s, their number started sky rocketing, and continued increasing until 2006 (when this study ended). One may conclude that sport betting draws its meanings from the relevant consumer culture, and from the rules and features of the betted sport.

Accurate mass-based analysis in human sport doping control : Application of liquid chromatography - time-of-flight mass spectrometry

Human sport doping control analysis is a complex and challenging task for anti-doping laboratories. The List of Prohibited Substances and Methods, updated annually by World Anti-Doping Agency (WADA), consists of hundreds of chemically and pharmacologically different low and high molecular weight compounds. This poses a considerable challenge for laboratories to analyze for them all in a limited amount of time from a limited sample aliquot. The continuous expansion of the Prohibited List obliges laboratories to keep their analytical methods updated and to research new available methodologies. In this thesis, an accurate mass-based analysis employing liquid chromatography - time-of-flight mass spectrometry (LC-TOFMS) was developed and validated to improve the power of doping control analysis. New analytical methods were developed utilizing the high mass accuracy and high information content obtained by TOFMS to generate comprehensive and generic screening procedures. The suitability of LC-TOFMS for comprehensive screening was demonstrated for the first time in the field with mass accuracies better than 1 mDa. Further attention was given to generic sample preparation, an essential part of screening analysis, to rationalize the whole work flow and minimize the need for several separate sample preparation methods. Utilizing both positive and negative ionization allowed the detection of almost 200 prohibited substances. Automatic data processing produced a Microsoft Excel based report highlighting the entries fulfilling the criteria of the reverse data base search (retention time (RT), mass accuracy, isotope match). The quantitative performance of LC-TOFMS was demonstrated with morphine, codeine and their intact glucuronide conjugates. After a straightforward sample preparation the compounds were analyzed directly without the need for hydrolysis, solvent transfer, evaporation or reconstitution. The hydrophilic interaction technique (HILIC) provided good chromatographic separation, which was critical for the morphine glucuronide isomers. A wide linear range (50-5000 ng/ml) with good precision (RSD<10%) and accuracy (±10%) was obtained, showing comparable or better performance to other methods used. In-source collision-induced dissociation (ISCID) allowed confirmation analysis with three diagnostic ions with a median mass accuracy of 1.08 mDa and repeatable ion ratios fulfilling WADA s identification criteria. The suitability of LC-TOFMS for screening of high molecular weight doping agents was demonstrated with plasma volume expanders (PVE), namely dextran and hydroxyethylstarch (HES). Specificity of the assay was improved, since interfering matrix compounds were removed by size exclusion chromatography (SEC). ISCID produced three characteristic ions with an excellent mean mass accuracy of 0.82 mDa at physiological concentration levels. In summary, by combining TOFMS with a proper sample preparation and chromatographic separation, the technique can be utilized extensively in doping control laboratories for comprehensive screening of chemically different low and high molecular weight compounds, for quantification of threshold substances and even for confirmation. LC-TOFMS rationalized the work flow in doping control laboratories by simplifying the screening scheme, expediting reporting and minimizing the analysis costs. Therefore LC-TOFMS can be exploited widely in doping control, and the need for several separate analysis techniques is reduced.

Maturation of a transient outward potassium current in mouse fetal hypothalamic neurons in culture

The whole-cell voltage clamp technique was used to record potassium currents in mouse fetal hypothalamic neurons developing in culture medium from days 1 to 17. The neurons were derived from fetuses of IOPS/OF1 mice on the 14th day of gestation. The mature neurons (>six days in culture) showed both a transient potassium current and a non-inactivating delayed rectifier potassium current. These were identified pharmacologically by using the potassium channel blockers tetraethyl ammonium chloride and 4-aminopyridine, and on the basis of their kinetics and voltage sensitivities. The delayed rectifier potassium current had a threshold of −20 mV, a slow time-course of activation, and was sustained during the voltage pulse. The 4-aminopyridine-sensitive current was transient, and was activated from a holding potential more negative (−80 mV) than that required for evoking the delayed rectifier potassium current (−40 mV). The delayed rectifier potassium current was detectable from day 1 onwards, while the transient potassium current showed a distinct developmental trend. The time-constant of inactivation became faster with age in culture. The half steady-state inactivation potential showed a shift towards less negative membrane potentials with age, and the relationship was best described by a logarithmic regression equation.The developmental trend of the transient potassium current may relate functionally to the progressive morphological changes, and the appearance of synaptic connections during ontogenesis.

Development of tissue culture and virus-induced gene silencing for Calendula officinalis

Calendula officinalis is grown widely as an ornamental plant across Europe. It belongs to the large. Asteraceae family. In this study, the aim was to explore the possibilities to use Calendula officinalis as a new model organism for flower development and secondary mechanism studies in Asteraceae. Tissue culture of Calendula officinalis was established using nine different cultivars. Murashige & Skoog (MS) medium with four different combinations of plant growth regulators were tested. Of all these combinations, the medium containing 1mg/l BAP, 0.1 mg/l IAA, and 1mg/l Zeatin achieved highest frequency of adventitious shoot regeneration from hypocotyl and cotyledon explants. Virus-induced gene silencing is a recent developed genetic tool for charactering the gene functions in plants, and extends the range of host plants that are not accessible for Agrobacterium transformation. Here, tobacco rattle virus (TRV)-based VIGS technique was tested in calendula (cv. Single Orange). We used TRV carrying Gerbera hybrid phytoene desaturase (PDS) gene fragment to induce PDS silencing in calendula. Vacuum infiltration and syringe infiltration methods both resulted in photo-bleaching phenotypes in leaves, bracts and petals. Loss-of-function phenotypes occurred on calendula 13 days post-infiltration. In conclusion, the data indicates that calendula explants can be regenerated through tissue culture which is a prerequisite for development of stable transformation methods. However, further optimization is still needed to improve the frequency. In addition, VIGS was applied to silence PDS marker gene expression indicating that this method has potential for gene functional studies in future.

Supermacroporous Polymer-Based Cryogel Bioreactor for Monoclonal Antibody Production in Continuous Culture Using Hybridoma Cells

Cryogel matrices composed of different polymeric blends were synthesized, yielding a unique combination of hydrophilicity and hydrophobicity with the presence or absence of charged surface. Four such cryogel matrices composed of polyacrylamide-chitosan (PAAC), poly(N-isopropylacrylamide)-chitosan, polyacrylonitrile (PAN), and poly(N-isopropylacrylamide) were tested for growth of different hybridoma cell lines and production of antibody in static culture. All the matrices were capable for the adherence of hybridoma cell lines 6A4D7, B7B10, and H9E10 to the polymeric surfaces as well as for the efficient monoclonal antibody (mAb) production. PAAC proved to be relatively better in terms of both mAb production and cell growth. Further, PAAC cryogel was designed into three different formats, monolith, disks, and beads, and used as packing material for packed-bed bioreactor. Longterm cultivation of 6A4D7 cell line on PAAC cryogel scaffold in all the three formats could be successfully done for a period of 6 weeks under static conditions. Continuous packed-bed bioreactor was setup using 6A4D7 hybridoma cell line in the three reactor formats. The reactors ran continuously for a period of 60 days during which mAb production and metabolism of cells in the bioreactors were monitored periodically. The monolith bioreactor performed most efficiently over a period of 60 days and produced a total of 57.5 mg of antibody in the first 30 days (in 500 mL) with a highest concentration of 115 mu g mL(-1), which is fourfold higher than t-flask culture. The results demonstrate that appropriate chemistry and geometry of the bioreactor matrix for cell growth and immobilization can enhance the reactor productivity. (C) 2010 American Institute of Chemical Engineers Biotechnol. Prog., 27: 170-180, 2011

A model for tool-use traditions in primates: implications for the coevolution of culture and cognition

Inspired by the demonstration that tool-use variants among wild chimpanzees and orangutans qualify as traditions (or cultures), we developed a formal model to predict the incidence of these acquired specializations among wild primates and to examine the evolution of their underlying abilities. We assumed that the acquisition of the skill by an individual in a social unit is crucially controlled by three main factors, namely probability of innovation, probability of socially biased learning, and the prevailing social conditions (sociability, or number of potential experts at close proximity). The model reconfirms the restriction of customary tool use in wild primates to the most intelligent radiation, great apes; the greater incidence of tool use in more sociable populations of orangutans and chimpanzees; and tendencies toward tool manufacture among the most sociable monkeys. However, it also indicates that sociable gregariousness is far more likely to produce the maintenance of invented skills in a population than solitary life, where the mother is the only accessible expert. We therefore used the model to explore the evolution of the three key parameters. The most likely evolutionary scenario is that where complex skills contribute to fitness, sociability and/or the capacity for socially biased learning increase, whereas innovative abilities (i.e., intelligence) follow indirectly. We suggest that the evolution of high intelligence will often be a byproduct of selection on abilities for socially biased learning that are needed to acquire important skills, and hence that high intelligence should be most common in sociable rather than solitary organisms. Evidence for increased sociability during hominin evolution is consistent with this new hypothesis. (C) 2003 Elsevier Science Ltd. All rights reserved.