Packed bed bioreactor for the isolation and expansion of placental-derived Mesenchymal Stromal Cells

Large numbers of Mesenchymal stem/stromal cells (MSCs) are required for clinical relevant doses to treat a number of diseases. To economically manufacture these MSCs, an automated bioreactor system will be required. Herein we describe the development of a scalable closed-system, packed bed bioreactor suitable for large-scale MSCs expansion. The packed bed was formed from fused polystyrene pellets that were air plasma treated to endow them with a surface chemistry similar to traditional tissue culture plastic. The packed bed was encased within a gas permeable shell to decouple the medium nutrient supply and gas exchange. This enabled a significant reduction in medium flow rates, thus reducing shear and even facilitating single pass medium exchange. The system was optimised in a small-scale bioreactor format (160 cm2) with murine-derived green fluorescent protein-expressing MSCs, and then scaled-up to a 2800 cm2 format. We demonstrated that placental derived MSCs could be isolated directly within the bioreactor and subsequently expanded. Our results demonstrate that the closed system large-scale packed bed bioreactor is an effective and scalable tool for large-scale isolation and expansion of MSCs.

Fabrication and characterization of plasma polymer thin films from monoterpene alcohols for applications in organic electronics and biotechnology

After more than twenty years of basic and applied research, the use of nanotechnology in the design and manufacture of nanoscale materials is rapidly increasing, particularly in commercial applications that span from electronics across renewable energy areas, and biomedical devices. Novel polymers are attracting significant attention for they promise to provide a low−cost high−performance alternative to existing materials. Furthermore, these polymers have the potential to overcome limitations imposed by currently available materials thus enabling the development of new technologies and applications that are currently beyond our reach. This work focuses on the development of a range of new low−cost environmentally−friendly polymer materials for applications in areas of organic (flexible) electronics, optics, and biomaterials. The choice of the monomer reflects the environmentally−conscious focus of this project. Terpinen−4−ol is a major constituent of Australian grown Melaleuca alternifolia (tea tree) oil, attributed with the oil's antimicrobial and anti−inflammatory properties. Plasma polymerisation was chosen as a deposition technique for it requires minimal use of harmful chemicals and produces no hazardous by−products. Polymer thin films were fabricated under varied process conditions to attain materials with distinct physico−chemical, optoelectrical, biological and degradation characteristics. The resultant materials, named polyterpenol, were extensively characterised using a number of well−accepted and novel techniques, and their fundamental properties were defined. Polyterpenol films were demonstrated to be hydrocarbon rich, with variable content of oxygen moieties, primarily in the form of hydroxyl and carboxyl functionalities. The level of preservation of original monomer functionality was shown to be strongly dependent on the deposition energy, with higher applied power increasing the molecular fragmentation and substrate temperature. Polyterpenol water contact angle contact angle increased from 62.7° for the 10 W samples to 76.3° for the films deposited at 100 W. Polymers were determined to resist solubilisation by water, due to the extensive intermolecular and intramolecular hydrogen bonds present, and other solvents commonly employed in electronics and biomedical processing. Independent of deposition power, the surface topography of the polymers was shown to be smooth (Rq <0.5 nm), uniform and defect free. Hardness of polyterpenol coatings increased from 0.33 GPa for 10 W to 0.51 GPa for 100 W (at 500 μN load). Coatings deposited at higher input RF powers showed less mechanical deformation during nanoscratch testing, with no considerable damage, cracking or delamination observed. Independent of the substrate, the quality of film adhesion improved with RF power, suggesting these coatings are likely to be more stable and less susceptible to wear. Independent of fabrication conditions, polyterpenol thin films were optically transparent, with refractive index approximating that of glass. Refractive index increased slightly with deposition power, from 1.54 (10 W) to 1.56 (100 W) at 500 nm. The optical band gap values declined with increasing power, from 2.95 eV to 2.64 eV, placing the material within the range for semiconductors. Introduction of iodine impurity reduced the band gap of polyterpenol, from 2.8 eV to 1.64 eV, by extending the density of states more into the visible region of the electromagnetic spectrum. Doping decreased the transparency and increased the refractive index from 1.54 to 1.70 (at 500 nm). At optical frequencies, the real part of permittivity (k) was determined to be between 2.34 and 2.65, indicating a potential low-k material. These permittivity values were confirmed at microwave frequencies, where permittivity increased with input RF energy – from 2.32 to 2.53 (at 10 GHz ) and from 2.65 to 2.83 (at 20 GHz). At low frequencies, the dielectric constant was determined from current−voltage characteristics of Al−polyterpenol−Al devices. At frequencies below 100 kHz, the dielectric constant varied with RF power, from 3.86 to 4.42 at 1 kHz. For all samples, the resistivity was in order of 10⁸−10⁹ _m (at 6 V), confirming the insulating nature of polyterpenol material. In situ iodine doping was demonstrated to increase the conductivity of polyterpenol, from 5.05 × 10⁻⁸ S/cm to 1.20 × 10⁻⁶ S/cm (at 20 V). Exposed to ambient conditions over extended period of time, polyterpenol thin films were demonstrated to be optically, physically and chemically stable. The bulk of ageing occurred within first 150 h after deposition and was attributed to oxidation and volumetric relaxation. Thermal ageing studies indicated thermal stability increased for the films manufactured at higher RF powers, with degradation onset temperature associated with weight loss shifting from 150 ºC to 205 ºC for 10 W and 100 W polyterpenol, respectively. Annealing the films to 405 °C resulted in full dissociation of the polymer, with minimal residue. Given the outcomes of the fundamental characterisation, a number of potential applications for polyterpenol have been identified. Flexibility, tunable permittivity and loss tangent properties of polyterpenol suggest the material can be used as an insulating layer in plastic electronics. Implementation of polyterpenol as a surface modification of the gate insulator in pentacene-based Field Effect Transistor resulted in significant improvements, shifting the threshold voltage from + 20 V to –3 V, enhancing the effective mobility from 0.012 to 0.021 cm²/Vs, and improving the switching property of the device from 10⁷ to 10⁴. Polyterpenol was demonstrated to have a hole transport electron blocking property, with potential applications in many organic devices, such as organic light emitting diodes. Encapsulation of biomedical devices is also proposed, given that under favourable conditions, the original chemical and biological functionality of terpinen−4−ol molecule can be preserved. Films deposited at low RF power were shown to successfully prevent adhesion and retention of several important human pathogens, including P. aeruginosa, S. aureus, and S. epidermidis, whereas films deposited at higher RF power promoted bacterial cell adhesion and biofilm formation. Preliminary investigations into in vitro biocompatibility of polyterpenol demonstrated the coating to be non−toxic for several types of eukaryotic cells, including Balb/c mice macrophage and human monocyte type (HTP−1 non-adherent) cells. Applied to magnesium substrates, polyterpenol encapsulating layer significantly slowed down in vitro biodegradation of the metal, thus increasing the viability and growth of HTP−1 cells. Recently, applied to varied nanostructured titanium surfaces, polyterpenol thin films successfully reduced attachment, growth, and viability of P. aeruginosa and S. aureus.

Intracellular effects of atmospheric-pressure plasmas on melanoma cancer cells

Gas discharge plasmas formed at atmospheric pressure and near room temperature have recently been shown as a promising tool for cancer treatment. The mechanism of the plasma action is attributed to generation of reactive oxygen and nitrogen species, electric fields, charges, and photons. The relative importance of different modes of action of atmospheric-pressure plasmas depends on the process parameters and specific treatment objects. Hence, an in-depth understanding of biological mechanisms that underpin plasma-induced death in cancer cells is required to optimise plasma processing conditions. Here, the intracellular factors involved in the observed anti-cancer activity in melanoma Mel007 cells are studied, focusing on the effect of the plasma treatment dose on the expression of tumour suppressor protein TP73. Over-expression of TP73 causes cell growth arrest and/or apoptosis, and hence can potentially be targeted to enhance killing efficacy and selectivity of the plasma treatment. It is shown that the plasma treatment induces dose-dependent up-regulation of TP73 gene expression, resulting in significantly elevated levels of TP73 RNA and protein in plasma-treated melanoma cells. Silencing of TP73 expression by means of RNA interference inhibited the anticancer effects of the plasma, similar to the effect of caspase inhibitor z-VAD or ROS scavenger N-acetyl cysteine. These results confirm the role of TP73 protein in dose-dependent regulation of anticancer activity of atmospheric-pressure plasmas.

Pro-apoptotic NOXA is implicated in atmospheric-pressure plasma-induced melanoma cell death

Atmospheric-pressure plasma (APP) has been successfully used to treat several types of cancers in vivo and in vitro, with the effect being primarily attributed to the generation of reactive oxygen species (ROS). However, the mechanisms by which APP induces apoptosis in cancer cells require further elucidation. In this study, the effects of APP on the expression of 500 genes in melanoma Mel007 cancer cells were examined. Pro-apoptotic phorbol-12-myristate-13-acetate-induced protein (PMAIP1), also known as NOXA, was highly expressed as a result of APP treatment in a dose-dependent manner. Blocking of ROS using scavenger NAC or silencing of NOXA gene by RNA interference inhibited the APP-induced NOXA genes upregulation and impaired caspases 3/7 mediated apoptosis, confirming the important role plasma-generated ROS species and pro-apoptotic NOXA play in APP-induced cancer cell death.

Missing-In-Metastasis (MIM) regulates cell morphology by promoting plasma membrane and actin cytoskeleton dynamics

The cells of multicellular organisms have differentiated to carry out specific functions that are often accompanied by distinct cell morphology. The actin cytoskeleton is one of the key regulators of cell shape subsequently controlling multiple cellular events including cell migration, cell division, endo- and exocytosis. A large set of actin regulating proteins has evolved to achieve and tightly coordinate this wide range of functions. Some actin regulator proteins have so-called house keeping roles and are essential for all eukaryotic cells, but some have evolved to meet the requirements of more specialized cell-types found in higher organisms enabling complex functions of differentiated organs, such as liver, kidney and brain. Often processes mediated by the actin cytoskeleton, like formation of cellular protrusions during cell migration, are intimately linked to plasma membrane remodeling. Thus, a close cooperation between these two cellular compartments is necessary, yet not much is known about the underlying molecular mechanisms. This study focused on a vertebrate-specific protein called missing-in-metastasis (MIM), which was originally characterized as a metastasis suppressor of bladder cancer. We demonstrated that MIM regulates the dynamics of actin cytoskeleton via its WH2 domain, and is expressed in a cell-type specific manner. Interestingly, further examination showed that the IM-domain of MIM displays a novel membrane tubulation activity, which induces formation of filopodia in cells. Following studies demonstrated that this membrane deformation activity is crucial for cell protrusions driven by MIM. In mammals, there are five members of IM-domain protein family. Functions and expression patterns of these family members have remained poorly characterized. To understand the physiological functions of MIM, we generated MIM knockout mice. MIM-deficient mice display no apparent developmental defects, but instead suffer from progressive renal disease and increased susceptibility to tumors. This indicates that MIM plays a role in the maintenance of specific physiological functions associated with distinct cell morphologies. Taken together, these studies implicate MIM both in the regulation of the actin cytoskeleton and the plasma membrane. Our results thus suggest that members of MIM/IRSp53 protein family coordinate the actin cytoskeleton:plasma membrane interface to control cell and tissue morphogenesis in multicellular organisms.

Role of HMGB1 in cells of the circulation

The matrix of blood is a liquid plasma that transports molecules and blood cells within vessels lined by endothelial cells. High-mobility group B1 (HMGB1) is a protein expressed in blood cells. Under normal circumstances, HMGB1 is virtually absent from plasma, but during inflammation or trauma its level in plasma is increased. In resting and quiescent cells, HMGB1 is usually localized in the intracellular compartment, with the exception of motile cells that express HMGB1 on their outer surface to mediate cell migration. During cell transformation or immune cell activation HMGB1 can be actively secreted outside of the cell. Further, when a cell is damaged, HMGB1 can passively leak into extracellular environment. Extracellular HMGB1 can then participate in regulation of the immune response and under some conditions it can mediate lethality in systemic inflammatory response. The aim of this study was to evaluate the expression and functions of HMGB1 in cells of the vascular system and to investigate the prognostic value of circulating HMGB1 in severe sepsis and septic shock. HMGB1 was detected in platelets, leukocytes, and endothelial cells. HMGB1 was released from platelets and leukocytes, and it was found to mediate their adhesive and migratory functions. During severe infections the plasma levels of HMGB1 were elevated; however, no direct correlation with lethality was found. Further, the analysis of proinflammatory mechanisms suggested that HMGB1 forms complexes with other molecules to activate the immune system. In conclusion, HMGB1 is expressed in the cells of the vascular system, and it participates in inflammatory mechanisms by activating platelets and leukocytes and by mediating monocyte migration.

The high- and low-activity forms of human plasma phospholipid transfer protein (PLTP)

Plasma phospholipid transfer protein (PLTP) plays a crucial role in high-density lipoprotein (HDL) metabolism and reverse cholesterol transport (RCT). It mediates the generation of pre-beta-HDL particles, enhances the cholesterol efflux from peripheral cells to pre-beta-HDL, and metabolically maintains the plasma HDL levels by facilitating the transfer of post-lipolytic surface remnants of triglyceride-rich lipoproteins to HDL. In addition to the antiatherogenic properties, recent findings indicate that PLTP has also proatherogenic characteristics, and that these opposite characteristics of PLTP are dependent on the site of PLTP expression and action. In human plasma, PLTP exists in a high-activity (HA-PLTP) and a low-activity form (LA-PLTP), which are associated with macromolecular complexes of different size and composition. The aims of this thesis were to isolate the two PLTP forms from human plasma, to characterize the molecular complexes in which the HA- and LA-PLTP reside, and to study the interactions of the PLTP forms with apolipoproteins (apo) and the ability of apolipoproteins to regulate PLTP activity. In addition, we aimed to study the distribution of the two PLTP forms in a Finnish population sample as well as to find possible regulatory factors for PLTP by investigating the influence of lipid and glucose metabolism on the balance between the HA- and LA-PLTP. For these purposes, an enzyme-linked immunosorbent assay (ELISA) capable of determining the serum total PLTP concentration and quantitating the two PLTP forms separately was developed. In this thesis, it was demonstrated that the HA-PLTP isolated from human plasma copurified with apoE, whereas the LA-PLTP formed a complex with apoA-I. The separation of these two PLTP forms was carried out by a dextran sulfate (DxSO4)-CaCl2 precipitation of plasma samples before the mass determination. A similar immunoreactivity of the two PLTP forms in the ELISA could be reached after a partial sample denaturation by SDS. Among normolipidemic Finnish individuals, the mean PLTP mass was 6.6 +/- 1.5 mg/l and the mean PLTP activity 6.6 +/- 1.7 umol/ml/h. Of the serum PLTP concentration, almost 50% represented HA-PLTP. The results indicate that plasma HDL levels could regulate PLTP concentration, while PLTP activity could be regulated by plasma triglyceride-rich very low-density lipoprotein (VLDL) concentration. Furthermore, new evidence is presented that PLTP could also play a role in glucose metabolism. Finally, both PLTP forms were found to interact with apoA-I, apoA-IV, and apoE. In addition, both apoE and apoA-IV, but not apoA-I, were capable of activating the LA-PLTP. These findings suggest that the distribution of the HA- and LA-PLTP in human plasma is subject to dynamic regulation by apolipoproteins.

Sticking and erosion at carbon-containing plasma-facing materials in fusion reactors

Controlled nuclear fusion is one of the most promising sources of energy for the future. Before this goal can be achieved, one must be able to control the enormous energy densities which are present in the core plasma in a fusion reactor. In order to be able to predict the evolution and thereby the lifetime of different plasma facing materials under reactor-relevant conditions, the interaction of atoms and molecules with plasma first wall surfaces have to be studied in detail. In this thesis, the fundamental sticking and erosion processes of carbon-based materials, the nature of hydrocarbon species released from plasma-facing surfaces, and the evolution of the components under cumulative bombardment by atoms and molecules have been investigated by means of molecular dynamics simulations using both analytic potentials and a semi-empirical tight-binding method. The sticking cross-section of CH3 radicals at unsaturated carbon sites at diamond (111) surfaces is observed to decrease with increasing angle of incidence, a dependence which can be described by a simple geometrical model. The simulations furthermore show the sticking cross-section of CH3 radicals to be strongly dependent on the local neighborhood of the unsaturated carbon site. The erosion of amorphous hydrogenated carbon surfaces by helium, neon, and argon ions in combination with hydrogen at energies ranging from 2 to 10 eV is studied using both non-cumulative and cumulative bombardment simulations. The results show no significant differences between sputtering yields obtained from bombardment simulations with different noble gas ions. The final simulation cells from the 5 and 10 eV ion bombardment simulations, however, show marked differences in surface morphology. In further simulations the behavior of amorphous hydrogenated carbon surfaces under bombardment with D^+, D^+2, and D^+3 ions in the energy range from 2 to 30 eV has been investigated. The total chemical sputtering yields indicate that molecular projectiles lead to larger sputtering yields than atomic projectiles. Finally, the effect of hydrogen ion bombardment of both crystalline and amorphous tungsten carbide surfaces is studied. Prolonged bombardment is found to lead to the formation of an amorphous tungsten carbide layer, regardless of the initial structure of the sample. In agreement with experiment, preferential sputtering of carbon is observed in both the cumulative and non-cumulative simulations

Expression of lactate transporters MCT1, MCT2, MCT4 and the ancillary protein CD147 in horse muscle and red blood cells

Monocarboxylate transporters (MCTs) transport lactate and protons across cell membranes. During intense exercise, lactate and protons accumulate in the exercising muscle and are transported to the plasma. In the horse, MCTs are responsible for the majority of lactate and proton removal from exercising muscle, and are therefore also the main mechanism to hinder the decline in pH in muscle cells. Two isoforms, MCT1 and MCT4, which need an ancillary protein CD147, are expressed in equine muscle. In the horse, as in other species, MCT1 is predominantly expressed in oxidative fibres, where its likely role is to transport lactate into the fibre to be used as a fuel at rest and during light work, and to remove lactate during intensive exercise when anaerobic energy production is needed. The expression of CD147 follows the fibre type distribution of MCT1. These proteins were detected in both the cytoplasm and sarcolemma of muscle cells in the horse breeds studied: Standardbred and Coldblood trotters. In humans, training increases the expression of both MCT1 and MCT4. In this study, the proportion of oxidative fibres in the muscle of Norwegian-Swedish Coldblood trotters increased with training. Simultaneously, the expression of MCT1 and CD147, measured immunohistochemically, seemed to increase more in the cytoplasm of oxidative fibres than in the fast fibre type IIB. Horse MCT4 antibody failed to work in immunohistochemistry. In the future, a quantitative method should be introduced to examine the effect of training on muscle MCT expression in the horse. Lactate can be taken up from plasma by red blood cells (RBCs). In horses, two isoforms, MCT1 and MCT2, and the ancillary protein CD147 are expressed in RBC membranes. The horse is the only species studied in which RBCs have been found to express MCT2, and the physiological role of this protein in RBCs is unknown. The majority of horses express all three proteins, but 10-20% of horses express little or no MCT1 or CD147. This leads to large interindividual variation in the capacity to transport lactate into RBCs. Here, the expression level of MCT1 and CD147 was bimodally distributed in three studied horse breeds: Finnhorse, Standardbred and Thoroughbred. The level of MCT2 expression was distributed unimodally. The expression level of lactate transporters could not be linked to performance markers in Thoroughbred racehorses. In the future, better performance indexes should be developed to better enable the assessment of whether the level of MCT expression affects athletic performance. In human subjects, several mutations in MCT1 have been shown to cause decreased lactate transport activity in muscle and signs of myopathy. In the horse, two amino acid sequence variations, one of which was novel, were detected in MCT1 (V432I and K457Q). The mutations found in horses were in different areas compared to mutations found in humans. One mutation (M125V) was detected in CD147. The mutations found could not be linked with exercise-induced myopathy. MCT4 cDNA was sequenced for the first time in the horse, but no mutations could be detected in this protein.

USING FLUORESCENT AND NON-FLUORESCENT STEROLS TO STUDY RAFTS AND INTRACELLULAR CHOLESTEROL TRANSPORT IN MAMMALIAN CELLS

Cholesterol is an essential component in the membranes of most eukaryotic cells, in which it mediates many functions including membrane fluidity, permeability and the formation of ordered membrane domains. In this work a fluorescent and a non-fluorescent cholesterol analog were characterized as tools to study cholesterol. Next, these analogs were used to study two specific cell biological processes that involve cholesterol, i.e. the structure and function of ordered membrane domains/rafts and intracellular cholesterol transport. The most common method for studying ordered membrane domains is by disrupting them by cholesterol depletion. Because cholesterol depletion affects many cellular functions besides those mediated by membrane domains, this procedure is highly unspecific. The cellular exchange of cholesterol by desmosterol as a tool to study ordered membrane domains was characterized. It turned out that the ability of desmosterol to form and stabilize membrane domains in vitro was weaker compared to cholesterol. This result was reinforced by atomistic scale simulations that indicated that desmosterol has a lower ordering effect on phospholipid acyl chains. Three procedures were established for exchanging cellular cholesterol by desmosterol. In cells in which desmosterol was the main sterol, insulin signaling was attenuated. The results suggest that this was caused by desmosterol destabilizing membrane rafts. Contrary to its effect on ordered membrane domains it was found that replacing cholesterol by desmosterol does not change cell growth/viability, subcellular sterol distribution, Golgi integrity, secretory pathway, phospholipid composition and membrane fluidity. Together these results suggest that exchanging cellular cholesterol by desmosterol provides a selective tool for perturbing rafts. Next, the importance of cholesterol for the structure and function of caveolae was analyzed by exchanging the cellular cholesterol by desmosterol. The sterol exchange reduced the stability of caveolae as determined by detergent resistance of caveolin-1 and heat resistance of caveolin-1 oligomers. Also the sterol exchange led to aberrations in the caveolar structure; the morphology of caveolae was altered and there was a larger variation in the amount of caveolin-1 molecules per caveola. These results demonstrate that cholesterol is important for caveolar stability and structural homogeneity. In the second part of this work a fluorescent cholesterol analog was characterized as a tool to study cholesterol transport. Tight control of the intracellular cholesterol distribution is essential for many cellular processes. An important mechanism by which cells regulate their membrane cholesterol content is by cholesterol traffic, mostly from the plasma membrane to lipid droplets. The fluorescent sterol probe BODIPY-cholesterol was characterized as a tool to analyze cholesterol transport between the plasma membrane, the endoplasmic reticulum (ER) and lipid droplets. The behavior of BODIPY-cholesterol was compared to that of natural sterols, using both biochemical and live-cell microcopy assays. The results show that the transport kinetics of BODIPY-cholesterol between the plasma membrane, the ER and lipid droplets is similar to that of unesterified cholesterol. Next, BODIPY-cholesterol was utilized to analyze the importance of oxysterol binding protein related proteins (ORPs) for cholesterol transport between the plasma membrane, the ER, and lipid droplets in mammalian cells. By overexpressing all human ORPs it turned out that especially ORP1S and ORP2 enhanced sterol transport from the plasma membrane to lipid droplets. Our results suggest that the increased sterol transport takes place between the plasma membrane and ER and not between the ER and lipid droplets. Simultaneous knockdown of ORP1S and ORP2 resulted in a moderate but significant inhibition of sterol traffic from the plasma membrane to ER and lipid droplets, suggesting a physiological role for these ORPs in this process. The two phenylalanines in an acidic tract (FFAT) motif in ORPs, which mediates interaction with vesicle associated membrane protein associated proteins (VAPs) in the ER, was not necessary for mediating sterol transport. However, VAP silencing slowed down sterol transport, most likely by destabilizing ORPs containing a FFAT motif.

Two levels of MCT1 and CD147 expression in the equine red blood cells and muscle

Monocarboxylate transporters (MCTs), especially the isoforms MCT1 - MCT4, cotransport lactate and protons across the cell membranes. They are thus essential for pH regulation and homeostasis in glycolytic cells such as red blood cells (RBCs), and skeletal muscle cells during intense exercise. In 70% of the Standardbred horses the lactate transport activity (TA) in RBCs is high and transport is mediated mainly by MCTs. In the rest 30% of the Standardbreds MCT mediated transport route is not active and the TA is low. MCTs need an ancillary protein for their proper localization and functioning in the plasma membrane. The ancillary protein for MCT1 and MCT4 is a member of immunoglobulin superfamily, CD147. Here we determined the expression of MCT isoforms and CD147 in equine RBCs and gluteal muscle. We sequenced the cDNA of horse MCT1 and CD147 to achieve horse-specific antibodies and to reveal sequence variations that may affect the TA of RBCs. The amount of MCT1 and CD147 mRNA in muscle were also studied. ---- In all, 73 horses representing different breeds were used. Blood samples were drawn from the jugular vein and muscle samples were taken either from gluteal muscle using biopsy needle or during castration from expendable cremaster muscle. The TA of RBCs was studied using radiolabeled lactate and the amount of MCT isoforms and CD147 in the plasma membranes using Western blotting. The level of mRNA in muscle cells was determined using qPCR. Isoforms MCT1 and MCT2 were found in the RBCs and isoforms MCT1 and MCT4 in the muscle cells of horses. The TA of RBCs was dependent on the expression of CD147 and MCT1 in the plasma membrane. Sequence variations were found in the cDNA of both MCT1 and CD147, but they did not explain the inactivity of MCT1 mediated transport route. The single nucleotide polymorphism (SNP) Met125Val in CD147 that existed parallel with an SNP in 3´-untranslated region explained, however, attenuation in CD147 expression in Standardbreds. A single mutation Ile51Val also decreased the expression of CD147 in one Warmblood. The MCT1 and CD147 mRNA concentrations in the gluteal muscle were higher in horses with higher MCT1 and CD147 expression in RBCs and lower in horses with minor expression of CD147 and MCT1. This suggests that the bimodal distribution of TA is due to differences in transcriptional regulation that is functioning in parallel in MCT1 and CD147 gene.

A metabolomic approach to studying mechanisms of polymeric gene delivery to retinal pigment epithelial cells

Tiivistelmä ReferatAbstract Metabolomics is a rapidly growing research field that studies the response of biological systems to environmental factors, disease states and genetic modifications. It aims at measuring the complete set of endogenous metabolites, i.e. the metabolome, in a biological sample such as plasma or cells. Because metabolites are the intermediates and end products of biochemical reactions, metabolite compositions and metabolite levels in biological samples can provide a wealth of information on on-going processes in a living system. Due to the complexity of the metabolome, metabolomic analysis poses a challenge to analytical chemistry. Adequate sample preparation is critical to accurate and reproducible analysis, and the analytical techniques must have high resolution and sensitivity to allow detection of as many metabolites as possible. Furthermore, as the information contained in the metabolome is immense, the data set collected from metabolomic studies is very large. In order to extract the relevant information from such large data sets, efficient data processing and multivariate data analysis methods are needed. In the research presented in this thesis, metabolomics was used to study mechanisms of polymeric gene delivery to retinal pigment epithelial (RPE) cells. The aim of the study was to detect differences in metabolomic fingerprints between transfected cells and non-transfected controls, and thereafter to identify metabolites responsible for the discrimination. The plasmid pCMV-β was introduced into RPE cells using the vector polyethyleneimine (PEI). The samples were analyzed using high performance liquid chromatography (HPLC) and ultra performance liquid chromatography (UPLC) coupled to a triple quadrupole (QqQ) mass spectrometer (MS). The software MZmine was used for raw data processing and principal component analysis (PCA) was used in statistical data analysis. The results revealed differences in metabolomic fingerprints between transfected cells and non-transfected controls. However, reliable fingerprinting data could not be obtained because of low analysis repeatability. Therefore, no attempts were made to identify metabolites responsible for discrimination between sample groups. Repeatability and accuracy of analyses can be influenced by protocol optimization. However, in this study, optimization of analytical methods was hindered by the very small number of samples available for analysis. In conclusion, this study demonstrates that obtaining reliable fingerprinting data is technically demanding, and the protocols need to be thoroughly optimized in order to approach the goals of gaining information on mechanisms of gene delivery.

Gonadal cell surface receptor for plasma retinol-binding protein A method for its radioassay and studies on its level during spermatogenesis

A specific membrane receptor for plasma retinol-binding protein has been demonstrated in testicular cells. Prealbumin-2 did not show any specific binding to the membrane. The affinity of retinol-binding protein for receptor drastically decreases upon delivery of retinol and the retinol-binding protein does not enter the cell. The mechanism of delivery of retinol to the target cell by plasma retinol-binding protein has been investigated. The process involves two steps: direct binding of retinol-binding protein to the receptor and uptake of retinol by the target cell with a concomitant drastic reduction in the affinity of the retinol-binding protein to the receptor. Probably the second step of the process needs a cytosolic factor, possibly the cellular retinol-binding protein or an enzyme.The binding of retinol-binding protein to the receptor is saturable and reverible. The interaction shows a Kd value of 2.1 · 10−10 M. The specific binding of a retinol-binding protein with great affinity has been employed in the development of a method for radioassay of the receptor. The receptor level of the gonadal cell has been found to vary with the stage of differentiation. The receptor concentrations in 11-week-old birds and adult birds are comparable. Testoterone treatment of 11-week-old birds produced a substantial increase in the receptor concentration over control, while the protein content increased marginally, indicating that, probably, synthesis of the receptor is specifically induced by testoterone during spermatogenesis, and the concentration of receptor is relatively higher before the formation of the acrosome.

Temperature-Sensitive Src-Transformed Mdck Cells nn 3d Cultures as a Model of Malignant Transformation of Epithelial Cells

The equilibrium between cell proliferation, differentiation, and apoptosis is crucial for maintaining homeostasis in epithelial tissues. In order for the epithelium to function properly, individual cells must gain normal structural and functional polarity. The junctional proteins have an important role both in binding the cells together and in taking part in cell signaling. Cadherins form adherens junctions. Cadherins initiate the polarization process by first recognizing and binding the neighboring cells together, and then guiding the formation of tight junctions. Tight junctions form a barrier in dividing the plasma membranes to apical and basolateral membrane domains. In glandular tissues, single layered and polarized epithelium is folded into tubes or spheres, in which the basal side of the epithelial layer faces the outer basal membrane, and the apical side the lumen. In carcinogenesis, the differentiated architecture of an epithelial layer is disrupted. Filling of the luminal space is a hallmark of early epithelial tumors in tubular and glandular structures. In order for the transformed tumor cells to populate the lumen, enhanced proliferation as well as inhibition of apoptosis is required. Most advances in cancer biology have been achieved by using two-dimensional (2D) cell culture models, in which the cells are cultured on flat surfaces as monolayers. However, the 2D cultures are limited in their capacity to recapitulate the structural and functional features of tubular structures and to represent cell growth and differentiation in vivo. The development of three-dimensional (3D) cell culture methods enables the cells to grow and to be studied in a more natural environment. Despite the wide use of 2D cell culture models and the development of novel 3D culture methods, it is not clear how the change of the dimensionality of culture conditions alters the polarization and transformation process and the molecular mechanisms behind them. Src is a well-known oncogene. It is found in focal and adherens junctions of cultured cells. Active src disrupts cell-cell junctions and interferes with cell-matrix binding. It promotes cell motility and survival. Src transformation in 2D disrupts adherens junctions and the fibroblastic phenotype of the cells. In 3D, the adherens junctions are weakened, and in glandular structures, the lumen is filled with nonpolarized vital cells. Madin-Darby canine kidney (MDCK) cells are an epithelial cell type commonly used as a model for cell polarization. Its-src-transformed variants are useful model systems for analyzing the changes in cell morphology, and they play a role in src-induced malignant transformation. This study investigates src-transformed cells in 3D cell cultures as a model for malignant transformation. The following questions were posed. Firstly: What is the role of the composition and stiffness of the extracellular matrix (ECM) on the polarization and transformation of ts v-src MDCK cells in 3D cell cultures? Secondly: How do the culture conditions affect gene expression? What is the effect of v-src transformation in 2D and in 3D cell models? How does the shift from 2D to 3D affect cell polarity and gene expression? Thirdly: What is the role of survivin and its regulator phosphatase and tensin homolog protein (PTEN) in cell polarization and transformation, and in determining cell fate? How does their expression correlate with impaired mitochondrial function in transformed cells? In order to answer the above questions, novel methods of culturing and monitoring cells had to be created: novel 3D methods of culturing epithelial cells were engineered, enabling real time monitoring of a polarization and transformation process, and functional testing of 3D cell cultures. Novel 3D cell culture models and imaging techniques were created for the study. Attention was focused especially on confocal microscopy and live-cell imaging. Src-transformation disturbed the polarization of the epithelium by disrupting cell adhesion, and sensitized the cells to their environment. With active src, the morphology of the cell cluster depended on the composition and stiffness of the matrix. Gene expression studies revealed a broader impact of src transformation than mere continuous activity of src-kinase. In 2D cultures, src transformation altered the expression of immunological, actin cytoskeleton and extracellular matrix (ECM). In 3D, the genes regulating cell division, inhibition of apoptosis, cell metabolism, mitochondrial function, actin cytoskeleton and mechano-sensing proteins were altered. Surprisingly, changing the culture conditions from 2D to 3D affected also gene expression considerably. The microarray hit survivin, an inhibitor of apoptosis, played a crucial role in the survival and proliferation of src-transformed cells.

Angelica archengelica extract induced perturbation of rat skin and tight junctional protein (ZO-1) of HaCaT cells

Background and purpose of the study: Herbal enhancers compared to the synthetic ones have shown less toxis effects. Coumarins have been shown at concentrations inhibiting phospoliphase C-Y (Phc-Y) are able to enhance tight junction (TJ) permeability due to hyperpoalation of Zonolous Occludense-1 (ZO-1) proteins. The purpose of this study was to evaluate the influence of ethanolic extract of Angelica archengelica (AA-E) which contain coumarin on permeation of repaglinide across rat epidermis and on the tight junction plaque protein ZO-1 in HaCaT cells. Methods: Transepidermal water loss (TEWL) from the rat skin treated with different concentrations of AA-E was assessed by Tewameter. Scanning and Transmission Electron Microscopy (TEM) on were performed on AA-E treated rat skin portions. The possibility of AA-E influence on the architecture of tight junctions by adverse effect on the cytoplasmic ZO-1 in HaCaT cells was investigated. Finally, the systemic delivery of repaglinide from the optimized transdermal formulation was investigated in rats. Results: The permeation of repaglinide across excised rat epidermis was 7-fold higher in the presence of AA-E (5% w/v) as compared to propylene glycol:ethanol (7:3) mixture. The extract was found to perturb the lipid microconstituents in both excised and viable rat skin, although, the effect was less intense in the later. The enhanced permeation of repaglinide across rat epidermis excised after treatment with AA-E (5% w/v) for different periods was in concordance with the high TEWL values of similarly treated viable rat skin. Further, the observed increase in intercellular space, disordering of lipid structure and corneocyte detachment indicated considerable effect on the ultrastructure of rat epidermis. Treatment of HaCaT cell line with AA-E (0.16% w/v) for 6 hrs influenced ZO-1 as evidenced by reduced immunofluorescence of anti-TJP1 (ZO-1) antibody in Confocal Laser Scanning Microscopy studies (CLSM) studies. The plasma concentration of repaglinide from transdermal formulation was maintained higher and for longer time as compared to oral administration of repaglinide. Major conclusion: Results suggest the overwhelming influence of Angelica archengelica in enhancing the percutaneous permeation of repaglinide to be mediated through perturbation of skin lipids and tight junction protein (ZO-1).