No description available.

Digitized by Kansas Correctional Industries

A protocol adapted for the propagation of monolayer cultures from human villous placenta is described. Keratin immunofluorescent localization was utilized to characterize trophoblast cells in sub-cultivated cultures derived from first trimester placentae. The appearance of autofluorescent particles in long-term first trimester placental cultures is reported. The monolayer culture system was employed to study living mitochondria. Using the vital fluorescent dye rhodamine 123, fluorescence microscopy of living first trimester human placental cells and choriocarcinoma cells was carried out. The length and intracellular distribution of mitochondria are described for normal placental cells, somatic hybrids of normal cells, and choriocarcinoma cells. In normal first trimester placental cells, quantitative relationships between the projected cell surface areas and numbers/lengths of their mitochondria are described by regression equations. From the equations, it is deduced that the total (aggregate) length of mitochondria per cell is directly proportional to the 0.86 +/- 0.07 power of the cell's projected surface area. The number of mitochondria per cell is also a direct function of the cellular surface area, with an exponent of 0.82 +/- 0.07; whilst the total length of mitochondria per unit projected cell surface area is an inverse function (exponent = -0.14 + 0.07) of the cell surface area. The reaction of living mitochondria in cultured first trimester placental cells to a number of chemical agents including colchicine was investigated. In the presence of colchicine, it appears that the distribution of mitochondria becomes more restricted to the perinuclear cytoplasm and that the mean length of the organelles is reduced. However, the total length of all mitochondria in colchicine-treated cells was not significantly different from that in similar cells which were not exposed to the drug. Experiments investigating the recovery of cells from colchicine-mediated changes in their mitochondrial orientation and length is presented.

The use of protein microfluidic systems is of growing interest for a variety of applications, including but not limited to tissue engineering, drug delivery and biosensors. The means by which to control chemistries on substrates for biological and medical applications is therefore in high demand. Here the creation of a bio-functional gradient on silica and polymeric surfaces using a micro fluidic technique, for the guidance of cell adhesion and functionality, using AFM tools for protein imaging and force spectroscopy investigation is reported. Atomic force microscopy (AFM) is a high resolution microscopic technique highly used in biological investigations, allowing conformational elucidation of protein deposition on the substrate. In this work application of the techniques of AFM, fluorescence microscopy and cell adhesion studies were used to assess the protein deposition along the microfluidic system. From the fluorescence analysis, it was immediately observed that successful protein immobilization on both substrates was achieved. Differences in fluorescence intensity were also registered along the microfluidic channel (start and end point) suggesting a variation in protein adsorption along the channel. The AFM imaging analysis conducted on the same samples revealed a difference in surface coverage considering the injection and end point (from 70% to 14% respectively) of the protein pattern. The difference in protein density registered along the fibronectin pattern was tested using a functionalised probe AFM technique, allowing molecular resolution of ligands in a physiological environment. A difference in the percentage of observed adhesion events was registered considering the start and end point of the microfluidic pattern, from 90% to 37% respectively. This is likely due to the fact that at the higher surface concentration there is a higher probability of the functionalised tip interacting with multiple fibronectin molecules, as confirmed from the presence of multiple adhesions at start point with a higher adhesion force of 82 pN ± 7.4 pN. To complement the AFM force measurements, protein functionality was tested by investigating the cell adhesion, shape and migration on the protein pattern. The fibronectin protein gradient was shown to control cell adhesion and migration along the patterns, demonstrating that this system can be used for biological applications to monitor the cell behaviour using difference protein concentration and cell density all in the same microfluidic channel. The ability to control cell adhesion and migration on substrates could be of significant interest when researching possible applications in future tissue engineering and biological studies. The combination of AFM and fluorescence microscopy techniques for protein density investigation used in this work demonstrated that protein deposition and arrangement on substrate play an important role in cell adhesion and migration studies.

published_or_final_version / Anatomy / Master / Master of Philosophy

Previous studies on the mechanism of action of pyrethroids have confirmed that voltage-gated sodium channels (VGSC) in the axon membrane are the major target site of these compounds. The use of pyrethroids to control maize weevils, Sitophilus zeamais, a major pest of stored maize in Brazil, has led to the occurrence of resistance. The work described here seeks to establish whether changes in VGSC of S.zeamais can explain pyrethroid resistance. The S. zeamais homologue of the Drosophila para VGSC was identified using degenerate primers and sequenced. Resistance mutations were examined by sequencing the IIS4-IIS6 region of the gene from laboratory strains of susceptible and resistant insects, revealing one amino acid replacement (T929I). The T929I mutation has been identified in other insects but always associated with a second mutation together producing a highly resistant phenotype. The occurrence of T929I in isolation is rare. DNA-based diagnostic assays were designed to screen weevils for the T929I mutation and analyse Brazilian field populations revealing a low frequency of heterozygous individuals carrying the mutation. The effect of the T929I mutation on VGSC function was investigated using whole cell patch clamping on cultured neurons isolated from thoracic ganglia of wild-type and resistant weevils. Inward currents were recorded by depolarizing the neuron to test potentials in the range -70mV to +70mV in 10mV increments for 25ms from a holding potential of -80mV. Current amplitudes were similar in cells from resistant weevils however other changes were apparent, notably a significant depolarizing shift in the voltage-dependence of activation of sodium currents in the resistant animals (P<0.05). Mutant neurons are also less sensitive to deltamethrin than the wild types.

3, 11-difluoro-6, 8, 13-trimethyl-8H- quino [4, 3, 2-kl] acridinium methosulfate (RHPS4) is a member of a series of pentacyclic acridines developed at the University of Nottingham, which bind to, and stabilise the structure of G-quadruplex DNA and inhibit the action of telomerase at sub-micromolar concentrations in the cell free TRAP assay and limit cancer cell growth therefore leading to the conclusion that RHPS4 has potential anti-tumour activity. Previous biological studies, however, have suggested that the mechanism of action of RHPS4 may be much more complicated than previously anticipated. Exposure of human melanoma cell lines to low doses of RHPS4 reveal an irreversible cessation of growth and telomere erosion. When used at higher doses, RHPS4 elicits short-term apoptosis/senescence concurrent with an increased number of telomere fusions and this is not a result of telomere shortening suggesting that the mechanism of action of RHPS4 is more than that of a simple telomerase inhibitor. In this study, we have developed a systems biology approach to the analysis and integration of biological data from investigations of the activity of RHPS4. The aim of this approach was to link the emergent properties of the biological studies of RHPS4 to a potential molecular target. In the laboratory, we are able to quickly derive data with respect to the time- and concentration- effects of RHPS4 on cell cycle distribution, population doublings and senescence levels, however, the kinetics of the cell cycle are neglected. A mathematical model has been derived of adequate complexity to reproduce the biological results of the laboratory and yet sufficient simplicity to yield predictive information and biological insights into the action of this complex molecule has been developed allowing us to integrate the data we can derive. The mathematical model has five compartments, those being X (representative of G1/G0 phase), Y (representative of S phase), Z (representative of G2/M phase), Σ (representative of senescence) and A (representative of apoptosis) with rate and movement between the compartments denoted by the rate parameters kxy, kyz, kzx, kxΣ and kΣA, respectively. Parameterisation of the mathematical model requires robust data from a well-characterised cell lines and initially four colorectal cell lines were chosen, namely HCT116, HT-29, KM12 and HCC-2998 to determine the short- and long-term effects of RHPS4 on their cell cycle distributions, population doublings and senescence levels. The aim was to determine which of the cell lines displayed good sensitivity to RHPS4, good growth characteristics in the presence and absence of RHPS4 and tractability in a range of biological assays. Initial studies revealed the HCT116 cell line was the most relevant for the further study of RHPS4. Data regarding the effects of RHPS4 on cell cycle distribution, growth rate and senescence over 21 days was derived and fitted to the five-compartment mathematical model to reveal aspects of the action of RHPS4 that would be difficult to appreciate without it. The model suggests that RHPS4 increases the rate of cells moving into a senescent state (indicated by an increase in kxƩ relative to control) and there is inhibition of apoptosis (indicated by a reduction in kƩA relative to control). Data with respect to doxorubicin was also applied to the model and suggested that consistent with RHPS4 there was inhibition of apoptosis relative to control, however, unlike RHPS4, there were initial reductions followed by increases in the rates of cells moving between G1/G0 and S by kxy, S and G2/M by kyz and G2/M and G1/G0 by kzx relative to control in a concentration- and time-dependant manner. This study has highlighted the important role of mathematics in the understanding of biomolecular processes and this is not the end as we have simply allowed the data to speak for itself. This project is the starting point for further, more focussed studies of RHPS4, and other anti-tumour agents. This approach to the analysis of anti-tumour agents is general, however, allows us preliminary insight into the mode of action of RHPS4 and to relate changes observed to potential underlying molecular targets and further the understanding of this complex molecule.

The development of microspheres for the sustained delivery of protein and small drug delivery has been utilised in tissue engineering and drug delivery applications. However problems exist in obtaining a controlled and predictable release pattern of the encapsulated molecules from these materials. In this study, microspheres with a zero order release kinetic profile and no lag phase were developed from a novel PLGA based polymer blend. The novel PLGA based polymer blend was made from blending PLGA with varying compositions of the triblock co-polymer PLGA-PEG-PLGA. These blends were subsequently used in the fabrication of lysozyme and dexamethasone loaded microspheres. Blending of the triblock copolymer with PLGA resulted in a reduction of the glass transition temperature (36.1ºC against 59.7ºC) and an increased mechanical strength (25.25 ± 1.26MPa against 0.26 ± 0.05MPa) for PLGA and 30% triblock w/w microspheres respectively. An incremental increase in the triblock composition within the Triblock/PLGA blends resulted in a corresponding reduction in glass transition temperature of the microspheres. Varying the triblock composition within the microspheres showed no significant effect on entrapment efficiency (EE) of lysozyme (protein) and dexamethasone (drug) within fabricated microspheres (EE ~ 60% for and 75% for loading weight 5% w/w for lysozyme and dexamethasone microspheres respectively). Controlled release experiments showed incorporation of the triblock increased the burst release of the protein and drug molecules from the microspheres and improved their release kinetics, with zero-order release profile (post burst phase) observed at a triblock composition of 30% w/w. A positive correlation between the amount of triblock within the triblock / PLGA blend and the rate of protein and drug release was also observed. The induction of osteogenesis and chondrogenesis within stem cells seeded on dexamethasone and ascorbate phosphate, and TGF-β3 loaded scaffolds was successfully demonstrated. Zonal release of TGF-β3 and BMP4 proteins from a bilayered scaffold was also demonstrated. However experiments conducted to demonstrate the tissue zonation within a bone cartilage bilayered construct developed from embryonic stem cell seeded TGF-β3 and BMP4 loaded bilayered scaffolds yielded inconclusive data. These results suggests that protein and drug loaded injectable microspheres for tissue engineering applications can be formed from triblock/PLGA blends, and that by varying the triblock composition, the temperature at which the microspheres form scaffolds, the release kinetics and the mechanical strength of the resulting scaffolds can be controlled.

No description available.

No description available.