Airway remodeling characteristic of asthma involves structural changes altering the elasticity of the airway smooth muscle (ASM) microenvironment potentially leading to ASM dysfunction. This effect of matrix stiffness was investigated using a physiologically relevant 3D culture model. Characterisation of microtissue responses with regards to contractile function and gene expression were studied varying the ECM stiffness and through stimulation with epithelial cell (AEC) conditioned media. ASM microtissues were fabricated under four different loading conditions and the matrix stiffness was increased by crosslinking through non-enzymatic glycation and increasing the collagen density. Function was assessed through the use of pharmacological agents and by imaging microcantilever deflection, used to calculate force generation. Crosslinking microtissues enhanced contractile function in response to agonists; however, this effect disappeared in microtissues tethered to stiff microcantilevers suggesting a limit of contractility within this model. Remarkably, there was a differential response in ASM function where increasing the collagen density (stiffness) significantly attenuated function. Additionally, contractility was significantly enhanced when chronically stimulated with AEC media. ASM tissue in 3D culture is responsive to the microenvironment stiffness and increases contractility in the presence of a stiffer ECM. This could occur with thickening of the airway wall in asthma. Decreased contractility with increased collagen density is in agreement with previous studies where it was shown that type I collagen is pro-proliferative and attenuates the contractile phenotype. We show the models ability to quantitatively demonstrate the impact of biomechanical cues on ASM function providing provides new ways to elucidate the mechanisms of cellular remodeling.

1 ABSTRACT Charles University in Prague Faculty of pharmacy in Hradec Králové Department of pharmacognosy Student: Václav Předota Supervisor: PharmDr. Marie Kašparová, PhD. Title of diploma thesis: Plant tissue culture of Juniperus virginiana The derivation of callus cultures from leaves of Juniperus virginiana (varieties Hetzii, Glauca and Grey Owl) and determination of their growth curves were studied in this work. The cultures were cultivated at the temperature of 25 řC and light period of 16 hours light/8 hours dark on the Schenk and Hildebrandt medium with the addition of 3.0 mg.l-1 α-NAA and 0.2 mg. l-1 kinetin. It is clear from the growth curves, that all three varieties reached their maximum in growth on 35th day of the cultivation. The best results were achieved by variety Glauca.

Call number: LD2668 .T4 1957 D87 / Master of Science

by Xu, Jing Huai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 119-126). / ABSTRACT --- p.i / ACKNOWLEDGMENTS --- p.ii / DEDICATION --- p.iii / TABLE OF CONTENTS --- p.iv / LIST OF ABBREVIATIONS --- p.viii / LIST OF FIGURES --- p.ix / LIST OF TABLES --- p.xii / Chapter 1. --- Introduction / Chapter 1.1. --- General Introduction --- p.1 / Chapter 1.2. --- Purpose of Study --- p.2 / Chapter 1.3. --- General Consideration and Objectives --- p.3 / Chapter 2. --- Literature Review / Chapter 2.1. --- Rhododendrons in Hong Kong --- p.9 / Chapter 2.2. --- History of Tissue Culture --- p.23 / Chapter 2.3. --- Tissue Culture of Rhododendron --- p.26 / Chapter 3. --- Materials and Methods / Chapter 3.1 --- General Protocol --- p.31 / Chapter 3.2. --- Plant Materials --- p.31 / Chapter 3.3. --- Culture Media / Chapter 3.3.1. --- Basal medium --- p.32 / Chapter 3.3.2. --- Sterilization --- p.32 / Chapter 3.4. --- Growth Condition --- p.33 / Chapter 3.5. --- Establishment of the Aseptic Culture / Chapter 3.5.1. --- Explants --- p.33 / Chapter 3.5.2. --- Pretreatment --- p.33 / Chapter 3.5.3. --- Disinfection --- p.33 / Chapter 3.5.4. --- Prevention of Browning --- p.34 / Chapter 3.6. --- Multiplication of the Culture / Chapter 3.6.1. --- General Operation --- p.35 / Chapter 3.6.2. --- Basal Medium --- p.35 / Chapter 3.6.3. --- Combination of Various Plant Hormones --- p.36 / Chapter 3.6.4. --- Addition of Vitamins --- p.36 / Chapter 3.6.5. --- Filter Paper as Supporting Material --- p.36 / Chapter 3.7. --- In Vitro Rooting / Chapter 3.7.1. --- General Operation --- p.37 / Chapter 3.7.2. --- Basal Medium --- p.37 / Chapter 3.7.3. --- Sucrose --- p.37 / Chapter 3.7.4. --- Auxin Concentration --- p.38 / Chapter 3.7.5. --- Activated Charcoal --- p.38 / Chapter 3.7.6. --- Effect of Ethylene on Rooting --- p.38 / Chapter 3.8. --- Acclimatization --- p.39 / Chapter 4. --- Results / Chapter 4.1. --- Establishment of the Aseptic Culture / Chapter 4.1.1. --- Optimization of the Disinfection Conditions --- p.45 / Chapter 4.1.2. --- Effect of Different Excised Plant Parts on Establishment of the Aseptic Culture --- p.47 / Chapter 4.2. --- Multiplication / Chapter 4.2.1. --- Effect of Basal Medium on Multiplication --- p.57 / Chapter 4.2.2. --- Combination of Plant Hormones --- p.57 / Chapter 4.2.3. --- Effect of Vitamins on Multiplication --- p.58 / Chapter 4.2.4. --- Using Filter Papers as Supporting Materials --- p.59 / Chapter 4.3. --- Rooting Studies / Chapter 4.3.1. --- Effect of Basal Medium Strength --- p.71 / Chapter 4.3.2. --- Effect of Sucrose Concentrations --- p.71 / Chapter 4.3.3. --- Effect of Auxins --- p.71 / Chapter 4.3.4. --- Effect of Activated Charcoal --- p.72 / Chapter 4.3.5. --- Effect of Ethylene --- p.72 / Chapter 4.4. --- Acclimatization / Chapter 4.4.1. --- Effect of Different Kinds of Supporting Materials for Transplanting --- p.90 / Chapter 4.4.2. --- Effect of Different pH on Growth of Explant after Transplanting --- p.90 / Chapter 4.4.3. --- Microcutting --- p.90 / Chapter 5. --- Discussions / Chapter 5.1. --- Factors Involved in Establishment of Aseptic Culture / Chapter 5.1.1. --- Quality of Explant --- p.97 / Chapter 5.1.2. --- The Importance of Sterility --- p.97 / Chapter 5.1.3. --- Prevention of Browning --- p.99 / Chapter 5.2. --- Multiplication of the Tissue / Chapter 5.2.1. --- Nutrient Media Factors --- p.101 / Chapter 5.2.2. --- Growth regulators --- p.101 / Chapter 5.2.3. --- Vitamins --- p.104 / Chapter 5.2.4. --- Supporting Material --- p.106 / Chapter 5.3. --- In Vitro Rooting / Chapter 5.3.1. --- Medium Strength --- p.107 / Chapter 5.3.2. --- Effect of Sucrose --- p.108 / Chapter 5.3.3. --- Effect of Auxins --- p.109 / Chapter 5.3.4. --- Effect of Activated Charcoal --- p.110 / Chapter 5.3.5. --- Ethylene --- p.111 / Chapter 5.4. --- Acclimatization --- p.115 / Chapter 6. --- Summary --- p.117 / Chapter 7. --- References --- p.119

By serial subcultivation on media containing egg-white lysozyme a highly resistant variant of M. lysodeikticus bacteriophage was selected. The parent strain was sensitive to 0.1 pg per ml. lysozyme while the variant was resistant to 4000 pg per ml. The two strains were examined in detail and it was found that the resistant strain differed genotypicelly from the sensitive strain in its ability to acetylate certain cell wall hydroxyl groups. This was the sole reason for lysozyme resistance. It was found that deacetylation of the resistant strain by ample chemical treatment restored its sensitivity to lysozyme while acetylation of the sensitive strain rendered it resistant.

Experimental studies indicate that culturing chondrocytes on biodegradable polymeric scaffolds may yield“engineered" cartilage for the replacement of tissue lost to injury or diseases such as osteoarthritis. A method of estimating the outcome of cell-polymer cultures would aid in the design and evaluation of engineered tissue for therapeutic use. The goals of this project were to develop, validate, and apply first-generation mathematical models that describe the kinetics of extracellular matrix (ECM) deposition and scaffold degradation in cell-polymer constructs cultured in vitro. The ECM deposition model is based on a product-inhibition mechanism and predicts an asymptotic, exponential increase in the concentration of ECM molecules found in cartilage, including collagen and glycosaminoglycans (GAG). The scaffold degradation model uses first-order kinetics to describe the hydrolysis of biodegradable polyesters in systems not limited by diffusion. Each model was fit to published data describing the accumulation of GAG and collagen, as well as the degradation of poly glycolic acid (PGA) and poly lactic acid (PLA), respectively. As experimental validation, cell-polymer constructs (n = 24) and unseeded scaffolds (n = 24) were cultured in vitro, and biochemical assays for GAG and collagen content, as well as scaffold mass measurements, were performed at 1, 2, 4, 6, 8, or 10 weeks of culture (n = 8 per time point). The mathematical models demonstrate a moderate to strong goodness of fit with the previously published data and our experimental results (R2=0.75-0.99). These models were also combined to predict the temporal evolution of total construct mass with reasonable accuracy (30% RMS deviation). In ongoing work, estimates of biochemical composition derived from these models are being proposed to predict the mechanical properties and functionality of the constructs. This modeling scheme may be useful in elucidating more specific mechanisms governing ECM accumulation. Given their potential predictive power, these models may also reduce the cost of performing long-term culture experiments.

The propagation of wild-type virus and a transgenic viral vector was examined in cultured plant cells to identify factors affecting viral infection of and accumulation in cultured cells and to determine if viral vectors could be used to facilitate the expression of heterologous proteins in vitro. Tobacco mosaic virus (TMV) accumulation was examined in Nicotiana tabacum and N. benthamiana suspension and hairy root cultures. TMV accumulation was superior in N. benthamiana hairy roots. Hairy roots were infected by adding TMV to the liquid culture medium at the same time as root inoculation. Hairy root growth was unaffected by virus infection. The distribution of virus within root mats from shake-flask grown cultures was non-uniform and the concentration of virus accumulated in replicate cultures was variable. When N. benthamiana hairy roots were infected using 1.5 μg mL-1 TMV, the average maximum concentration of virus accumulated in the biomass was 1.6 ?? 0.25 mg g-1 dry weight, or 15-fold lower than in TMV infected N. tabacum leaves. Virus coat protein accumulated to a level of (26 ?? 10)% total soluble protein in the hairy roots. Inoculum virus concentration and the medium in which infection was performed affected the virus yield and the percentage of inoculated cultures that accumulated virus. When cultures were inoculated using 9.0 μg mL-1 TMV, virus accumulated in the biomass to 4.2 ?? 0.60 mg g-1 dry weight. Proportional scale-up of hairy root infection in shake flasks did not result in constant virus concentrations in the scaled cultures. TMV accumulation in bioreactor-infected and -grown hairy roots was poor. N. benthamiana hairy roots were infected with a TMV-based vector (30B-GFPC3) that encoded Cycle 3 green fluorescent protein (GFP). TMV-GFPC3 was (260 ?? 140)-fold less infectious than TMV as measured by local lesion assays. Propagation of TMV-GFPC3 could not be confirmed using mass balance. GFP was not detected in the infected hairy roots or the culture medium. Hairy roots represent a potentially viable culture-based system for the in vitro production of virus and virus products when field-grown agricultural systems do not adequately address issues of containment or product safety.

Leaf discs of Saintpaulia ionantha x confusa hybrids (African Violets) were cultured and transferred between hormonal free medium (MS basal medium) and shoot-inducing medium (SIM) to determine whether there is a window of competence in shoot regeneration. The results showed that cultured cells were not responsive to shoot-inducing signals (i.e. not competent) until 3-5 days after explant isolation but the ability to regenerate shoots was not lost in surviving cells/tissues cultured on basal medium. Light microscopic observations found that first periclinal divisions of epidermal cells occurred at 3-5 days on SIM. Meristemoids were then formed from the derivatives of the original epidermal cells. It is proposed that cellular competence for shoot regeneration is acquired in culture. The pre-competent period consists of a resting phase and a "dedifferentiation" phase in which epidermal cells divide periclinally to form "dedifferentiated" cells which are the true target cells for shoot induction. Mutagenic treatments and propagation of a chimeral African Violet cultivar were carried out to study cell origin of adventitious shoots in African Violet leaf culture. The results suggest that adventitious shoots may originate from either multiple cells or single cells. The multiple cell origin is in contrast to the hypothesis of exclusively single cell origin for adventitious shoots proposed in several studies. Several factors were studied to identify the source of variations in shoot regeneration among individual explants and to define conditions favourable for shoot regeneration in African Violet in vitro culture. These factors include donor plant growth temperature, the presence of light and the quality of light in culture, the role of chlorophyll in cultured tissues, position of explants in leaves, age of explant materials, source and type of explants, wounding and leaf disc orientation on media. It was found that the presence of light in culture, age of leaf explants, source (in vitro cultured vs pot plant) and type (leaf vs petiole) of explants, wounding and leaf disc orientation on media all had a statistically significant effect on shoot production.

Donor cells for nuclear transfer are usually prepared by the culture of fresh tissue. However, animal carcasses are sometimes frozen without cryoprotectants and if it were possible to obtain live cells from carcasses (tissue) preserved in this manner, it could be very beneficial in nuclear transfer cloning of trophy or extinct animals. This study tested the hypothesis that tissue samples of skin, muscle, and oral mucosa could be cryopreserved without cryoprotection. The tissue samples were taken from euthanized goats and placed into a -20°C freezer for varying lengths of time. The samples were thawed by two different methods. One method was in 37°C water bath and the other was on ice, thawing to room temperature from 1°C to 25°C. The samples were then processed and placed into an incubator to evaluate cell growth. Skin samples frozen for up to 34 days obtained cell growth to confluency and the cells were then cryopreserved with cryoprotectant. The cells were able to tolerate the potentially lethal effects of ice nucleation and dehydration brought about by ice formation and colligative factors. Although this method of cryopreservation has been shown to yield growth of cells that might be useful for nuclear transfer cloning, it is not the recommended method to cryopreserve tissues if cryoprotectants are available or if only short term storage is needed. These procedures would be especially useful when a precious animal dies unexpectedly and cryoprotectant is not available and the sample can not be processed before 10 days.

The responses of the tepary bean (Phaseolus acutifolius) to in vitro tissue culture systems were documented. Tests were conducted to identify the optimal auxin and cytokinin combinations required for optimal callus growth. Regeneration experiments were conducted to: (1) determine the effect of explant source and age on regeneration, (2) effect of callus age on regeneration, (3) the cultivation status of the explant source, and (4) the effects of nutritional additives on somatic embryogenesis. The callus was easily induced and maintained in all hormonal medias except those containing IAA and 6BA. The results for regeneration were most promising from cultures derived from immature cotyledon tissue. Ammonium Chloride, glutamine and Absisic acid appeared to have little affect on embryogenesis, however the addition of kinetin enabled the embryos to develop to the torpedo stage. Callus age and cultivative status of explant source had no effects on plantlet regeneration.