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Dry eye syndrome (DES) is disease of the eye surface, where tear film is ruptured and lubrication or moistness of the corneal and conjunctival epithelia do not work properly. DES is most common in older people, but also people with rheumatoid diseases, damaged corneal epithelia and certain medications are more likely to have DES. There are lubricating and moisturizing eye drops to relieve the symptoms of DES, administered at least twice a day depending on the severity, but they are only palliative. Eye drops for DES are composed of natural and synthetic origin polymers and their blends. Retaining methods are mainly viscosity and adherence to surfaces by weak interactions. In situ gelation by temperature, pH and osmotic change have been under research in recent years. Many studies have concentrated on ambiphilic polymer conjugates, which fit to the oily and aqueous layer of the tear film and have a better ability to carry a hydrophobic therapeutic drug molecule. Interest has also been pointed to mucoadhesive polymers, which are adhering to mucin by covalent bond. Thiolation of the polymer has been seen as a very promising method to achieve better muchoadhesion and thus has been extensively studied. A chitosan and hyaluronan are natural origin polymers, which have been thiolated and these polymer matrixes can be loaded with trehalose or albumin as therapeutic agent. In this thesis the anatomy of the eye, and especially the corneal and precorneal area and the conjunctiva is discussed. Ethiology of DES and commercial available therapies are covered in this thesis, but also therapies under research and corneal simulation methods in vitro. Kuivasilmäisyys on sairaus, jossa silmän sarveiskalvon pinnalla oleva kyynelkalvo rikkoutuu. Sarveis- ja sidekalvon kosteutus ja voitelu häiriintyvät kyynelkalvo epätasapainosta. Kuivasilmäisyys on ikääntyvien ihmisten sairaus, mutta sitä tavataan myös sarveiskalvovaurio- ja reumapotilailla. Joidenkin lääkkeiden käyttöön liittyy sivuoireina kuivasilmäisyyttä. Kuivasilmäisyyteen on saatavilla kaupallisia oireita lievittäviä silmätippoja, jotka kosteuttavat ja voitelevat silmän pintaa, mutta eivät hoida sairautta. Kuivan silmän tipat on valmistettu luonnon polymeereistä tai synteettisistä polymeereistä, joita annostellaan vähintään kahdesti päivässä. Terapeuttisen vaikutuksen kesto perustuu viskositeettiin tai tarttumiseen silmän pintaan heikoilla sidoksilla. In situ geeliytyminen lämpötilan, pH:n tai suolapitoisuuden muutoksesta on ollut tutkijoiden mielenkiinnon kohteena viime vuosina. Myös polymeerien tarttumista limakalvon pintaan (mukoadheesio) heikoilla vuorovaikutuksilla tai vahvemmilla kovalenttisilla sidoksilla on tutkittu. Amfifiliset polymeerit ovat myös vallanneet alaa, sillä niiden oletetaan sopivan kyynelkalvon pinnan öljykerrokseen ja edelleen sen alla olevaan vesikerrokseen. Amfifilisella polymeerillä on myös kyky kantaa hydrofobista lääkemolekyyliä. Polymeerien tiolointiin ja sen tutkimiseen on panostettu viime aikoina paljon. Tiolointi on menetelmä, jolla parannetaan polymeerin mukoadheesiota. Erityistä mielenkiintoa on herättänyt kitosaanin ja hyaluronihapon tiolointi, jolloin myös terapeuttinen molekyyli, kuten trehaloosi tai albumiini voitaisiin ladata polymeerimatriisiin. Tässä kirjallisuusselvityksessä käsitellään silmän anatomiaa erityisesti kyynelkalvon, sarveiskalvon ja sidekalvon osalta. Tässä opinnäytetyössä on käsitelty kuivasilmäisyyteen johtavia syitä ja nykyisiä sekä kehityksen kohteena olevia hoitomenetelmiä. Lopuksi esitellään sarveiskalvon simulointimenetelmiä, joita on käytetty laboratorio-olosuhteissa.

The number of people affected by degenerative diseases of the retina increases notably due to the longer life expectancy. The weakening of the sight concerns especially older people, who suffer from degeneration of the retina and its retinal pigment epithelium (RPE) monolayer. Currently, these degenerative diseases of the retina are incurable. Cell transplantation of healthy RPE cells is considered to be a potential treatment for these diseases. Due to the unlimited self-renewal capacity and pluripotency human pluripotent stem cells (hPSCs) are considered as possible cell sources for transplantation purposes. However, the current differentiation protocols for RPE cells from hPSCs are inefficient and result in low yields. The aim of this thesis was to study whether biodegradable poly(L-lactide) (PLLA) microparticles and commercial biostable cellulose anion exchange diethylaminoethyl (DE) 53 microparticles could be exploited in increasing the efficacy of differentiation of hPSCs into RPE cells. In addition, the potential of the microparticle culture system for stem cell maintenance in feeder-free conditions was evaluated. The attachment and proliferation of hPSCs on microparticles were analyzed by immunofluorescence analysis and microscopy. The success of differentiation of RPE from stem cells in microparticle cultures was evaluated by inspecting the appearance of pigmentation in cultures as well as by investigating the expression of RPE maturation markers on gene expression levels by RT-PCR. The PLLA microparticles did not support the attachment and proliferation of hPSCs in feeder-free conditions. The poor performance of PLLA particles in cell culture studies might be due to the hydrophobic nature of the particle surfaces. In contrast, hPSCs attached, proliferated and maintained their pluripotency on DE 53 particles. Furthermore, the hPSCs were successfully differentiated into RPE cells on these DE 53 particles. However, the differentiation efficacy did not outperform the conventional differentiation protocol. This was the first study in which the hPSCs were successfully differentiated into RPE cells on microparticles. Microparticles are far from being routinely used in hPSC maintenance and differentiation to mature cell types. More long-term and large-scale studies need to be conducted in the future. Nevertheless, at the moment they seem appealing and promising with a possibility to grow stem cells in large-scale and cost effectively. The microparticle culture systems set challenges for the cell analysis due to their 3-dimensional structure and autofluorescence property. Therefore, novel methods for characterization, analysis and imaging of microparticle culture systems should be developed. /Kir12

Escherichia coli is one of the most studied microorganisms and has found many uses in biotechnological applications. In order to make these applications economically feasible, it is necessary to get sufficiently high yields of the product molecules. Due to its popularity in bacteriological studies, a lot of knowledge about factors that are important to consider when optimizing E. coli bioprocesses has accumulated. This knowledge has been used to construct models about genetics and metabolism of this bacterium making genetic modification to improve bioprocess yields easier. Factors important in bioprocesses using E. coli are discussed in this work. While most of the work is concentrated on basic properties common to most bioprocesses, special emphasis is put on bioprocesses related to protein production as it was considered to be one of the most important bioprocess application. First, a very brief introduction to biology of E. coli is given. Then the basic parameters’ effects on bioprocesses are discussed, and finally some aspects of product formation and collection are discussed. It was shown in this work that, although a lot of progress has been made in optimization of bioprocesses where E. coli is used, in many aspects these processes still remain to be far from optimal. Therefore, it is clear that great amount of research on bioprocesses using this bacterium remains to be done.

Polttoaineiden hiilivetyjen on tiedetty ylläpitävän mikrobien kasvua jo yli 70 vuoden ajan. Tämän diplomityön yhtenä tavoitteena oli tehdä kirjallisuuskatsaus erilaisten polttoaineiden mikrobiologisesta kontaminaatiosta. Kokeellisen osuuden tavoitteena oli tutkia polttoaineiden soveltuvuutta mikrobien ravinnoksi, mikrobien määrää ja diversiteettiä eri polttoaineissa, polttoaineiden pilaantumisen etenemistä sekä mikrobikasvun hillitsemistä ja estoa. Diplomityön kokeellisessa osuudessa tutkittiin kahta polttoainetta. Polttoaineiden soveltuvuutta mikrobien ravinnoksi tutkittiin kolmella eri mikrobien kasvutavalla: kiinteällä kasvatusalustalla, liukoisessa vesifaasissa ravistelupullokokein sekä biofilmissä. Polttoainetta 1 ei voida käyttää sellaisenaan hiilenlähteenä kiinteässä kasvatusalustassa, sillä se ei absorboitunut alustaan. Ravistelupullokokeissa saatiin rikastettua polttoaineessa 1 esiintyviä mikro-organismeja ja molempien tutkittujen polttoaineiden havaittiin soveltuvan mikrobien ravinnoksi. Mineraalisuolaliuoksen havaittiin edistävän ja matalan pH:n rajoittavan mikrobikasvua polttoaineissa. Biofilmin mikrobikasvua havaittiin polttoaineen 1 varastosäiliöiden pinnoitemateriaalilla päällystetyissä mikroskoopin objektilaseissa sekä polttoaineen 1 että vesifaasien rajapinnoilla. Veden saatavuuden ja matalan pH:n havaittiin rajoittavan biofilmin muodostusta. Lämpötilan vaikutusta mikrobikasvun kinetiikkaan polttoaineessa 1 tutkittiin ravistelupullokokein kolmessa lämpötilassa. Mikrobikasvun havaittiin olevan nopeinta 27°C lämpötilassa sekä tapahtuvan viiveellä ja hitaammin 37°C lämpötilassa. 6°C lämpötilassa ei havaittu mikrobikasvua tutkimusjakson aikana. Mikrobikasvun hillintää tutkittiin anaerobisissa olosuhteissa polttoaineessa 1. Mikrobikasvua ei havaittu tutkimusjakson aikana. Mikrobikasvua polttoaineessa voidaan hillitä veden määrää vähentämällä, matalalla pH:lla ja lämpötilalla sekä anaerobisilla olosuhteilla. Veden saatavuuden rajoitus polttoaineen varastosäiliössä on toteutettavissa säännöllisellä ja huolellisella vedenpoistolla. Anaerobisten olosuhteiden teknisen toteutuksen mahdollisuutta ja taloudellisuutta tulisi tutkia täydessä mittakaavassa todellisessa polttoaineen varastointiympäristössä. Mikrobien lukumäärää polttoaineessa 1 tutkittiin standardin IP 385/99 mukaisen maljakasvatusmenetelmän ja mikroskopoinnin avulla. Sekä pesäkkeitä muodostavien yksiköiden määrä (11 – 15 PMY/ml) että kokonaissolumäärä (453 – 1271 solua/ml) oli tutkituissa näytteissä hyvin pieni. Mikrobien diversiteettiä polttoaineessa 1 tutkittiin molekulaaristen menetelmien avulla. Näytteiden prokarioottidiversiteetti oli laaja, mutta eukarioottidiversiteetti oli pieni. Mikro-organismien selektiivinen rikastuminen ravistelupullokokeissa havaittiin rikastetun biomassan mikrobidiversiteetin suppeudesta alkuperäiseen näytteeseen verrattuna. Maljakasvatusmenetelmän avulla eristettiin vain viisi todennäköisesti erilaista pesäkettä, joka havainnollistaa selkeästi menetelmän valikoivuuden ja sen antaman puutteellisen kuvan mikrobiston monimuotoisuudesta. /Kir11

Most severe degenerative diseases of retina are often due to malfunctions of retinal pigment epithelium (RPE). Absence of effective treatments has led to development of cell-biomaterial constructs with the aim of creating RPE equivalents for transplantation. Presently, the poor biocompatibility of allologous and xenologous culture substrata in addition with limited amount of source tissue poses the major issues. Well-defined synthetic substrata together with utilization of human embryonic stem cell-derived RPE cells (hESC RPE) are suggested to be potential solutions. In addition, need exists for an effective method to determine the developmental status of cells during the culturing period. This need could be addressed with automated image analysis. The aim of this thesis was to examine the capability of a few specific cell culture substrata to enable attachment, proliferation and maturation of hESC RPE cells. Study included total of 17 xeno-free synthetic materials including 12 BioMaDe Gelators, Purecoat amine and carboxyl, poly(D,L-lactic-co-glycolic acid) (75:25), poly(D,L-lactic acid) (96:4) and poly(L-lactic acid-co-?-caprolactone) (70:30). In addition five materials with natural-origin were studied including chitosan, type I collagen, Matrigel and Substrate X. Type IV collagen was used as control. Growth and maturation were monitored by taking images with specific time intervals. At the end point cellular developmental status was determined by assessing the expression of maturation specific mRNAs by PCR techniques and proteins by immunofluorescence microscopy. In addition, images were used to determine the potential of ImageJ-software as user-friendly image analysis tool for RPE cell analysis. Study demonstrated poor attachment and cell survival on every xeno-free synthetic substrate with cells retaining their initial developmental phase throughout the culturing period, which was supported by gene expression analysis. On the contrary, cells on natural materials attached and proliferated readily. Maturity was further confirmed with immunofluorescence labeling. Image analysis with ImageJ, in turn, confronted many problems mainly arising from heterogeneity of the images. As a conclusion, xeno-free synthetic materials tested in this study show low potential as RPE cell substrata. However, means to enhance their performance are suggested. Despite the good results obtained with natural materials, their ill-defined structure prone to alterations in physiological conditions remains an obstacle for entering clinical experiments. Further experiments should concentrate on combining the strengths of both approaches, that is, incorporation of attachment-related functional groups into well-defined xeno-free synthetic body. In order to increase image homogeneity imaging conditions should be more carefully considered. This way the benefits of automated image analysis could be more effectively exploited. /Kir11

During the development of the neural tissue of the nervous system, supporting neuroglial cells and the extracellular matrix (ECM) guide the migration of the immature functional neurons, provide them with a scaffold to grow on and aid in the formation of synapses. ECM provides the neurons with various guidance cues that guide the migration of the neurons and the extension of the neurites. The micrometre and even nanometre guidance cues can also be incorporated into neuronal cultures in vitro to study the effect of the guidance cues and to develop small neuronal networks with a desired architecture. Three-dimensional neurocage structures were fabricated from Ormocomp®, a polymer-ceramic hybrid material, by two-photon polymerisation for this study. In this study these structures were tested with neuronal cells differentiated from human embryonic stem cells (hESC). The neurocages were attached to microscope glass slide samples, each sample containing approximately ten neurocages. The neurocages were first coated with laminin, an ECM protein, to enable the adhesion of the cells to the glass surface. The cell suspension was then applied and the cells were cultured to observe their growth and to study the guidance effects of the neurocages. During the study eight individual experiments were carried out to optimise both the application methods for the laminin solution and the cell suspension and the growth of the cells. This study utilised both simple manual methods and two different micromanipulator set-ups in the application of the laminin solution and the cell suspension. It was concluded that the best method to apply the laminin solution was the micromanipulator set-up SU2, which utilised automated micromanipulator placement and pressure regulation. The cell suspension, on the other hand, could be easily applied onto the samples by placing droplets of the solution onto the medium covering each sample. As the cell population in the culture was small, conditioned medium taken from another neuronal cell culture was tested to increase the viability of the cell cultures of this study. The conditioned medium had a clear positive effect and the use of conditioned medium in future studies is therefore recommended. During the study it was found that the cells initially inside the neurocages did not attach to the glass bottom even when the insides of the neurocages were accurately coated with the laminin solution. In contrast, the cells outside the neurocages generally attached to the glass bottom well and had a tendency to migrate towards the neurocages and over the structure walls into them. Therefore, it was concluded that the material Ormocomp® was not harmful to neuronal cells and it even seemed to attract them. Furthermore, the cells that had migrated into the neurocages readily stayed inside them and extended neurites along the edges of the neurocages. This indicated that the neurite guidance properties of the structures were very promising. /Kir11

Acinetobacter baylyi sp. ADP1 is a nutritionally versatile bacterial strain capable of natural transformation and homologous recombination. It has a unique metabolism, as it is capable of using a vast collection of compounds as a sole carbon source. It is non-pathogenic and easy to work with in the laboratory. The great potential of ADP1 lies in its usage as a laboratory model organism, as well as in bioremediation. The strain is well characterized, as full genomic sequence and metabolic model exist already. However, no metabolomic analyses have been performed so far. Metabolomics has recently developed into an important field in biotechnology with many potential new applications. As a new area of interest, it is only in early stages of development, but nevertheless many powerful techniques already exist. However, lots remain to be discovered. As every bacterial species can be very different, the methods of metabolomics need to be designed for every study independently. This is why it is of great importance to optimize the metabolite extraction protocol specifically for each project. The purpose of this work was to optimize a new extraction protocol of intracellular metabolites from ADP1, for the purpose of metabolomic analysis with high resolution mass spectrometry. An initial protocol, set up and Genoscope, and based on literature and some previous testing, was used as a basis for the optimization. From this protocol, the culture conditions, quenching procedure, sample storage and data analysis were reassessed. In the end, the final protocol was tested with the cells cultivated in two different carbon sources. The final protocol improved the quality of acquired data significantly, providing a better efficiency in quenching of the metabolism and metabolite extraction, leading to more detected metabolites in the final data. In addition, the extraction process was made more organized and faster to perform. /Kir11