Advances in computational modelling offer unprecedented potential for obtaining insights into the mechanics of cell-cell interactions. With the aid of such models, cell-level phenomena such as cell sorting and tissue self-organization are now being understood in terms of forces generated by specific sub-cellular structural components. Three-dimensional systems can behave differently from two-dimensional ones and since models cannot be validated without corresponding data, it is crucial to build accurate three-dimensional models of real cell aggregates. The lack of automated methods to determine which cell outlines in successive images of a confocal stack or time-lapse image set belong to the same cell is an important unsolved problem in the reconstruction process. This thesis addresses this problem through a contour grouping algorithm (CGA) designed to lead to unsupervised three-dimensional reconstructions of biological cells. The CGA associates contours obtained from fluorescently-labeled cell membranes in individual confocal slices using concepts from the fields of machine learning and combinatorics. The feature extraction step results in a set of association metrics. The algorithm then uses a probabilistic grouping step and a greedy-cost optimization step to produce grouped sets of contours. Groupings are representative of imaged cells and are manually evaluated for accuracy. The CGA presented here is able to produce accuracies greater than 96% when properly tuned. Parameter studies show that the algorithm is robust. That is, acceptable results are obtained under moderately varied probabilistic constraints and reasonable cost weightings. Image properties – such as slicing distance, image quality – affect the results. Sources of error are identified and enhancements based on fuzzy-logic and other optimization methods are considered. The successful grouping of cell contours, as realized here, is an important step toward the development of realistic, three-dimensional, cell-based finite element models.

Advances in computational modelling offer unprecedented potential for obtaining insights into the mechanics of cell-cell interactions. With the aid of such models, cell-level phenomena such as cell sorting and tissue self-organization are now being understood in terms of forces generated by specific sub-cellular structural components. Three-dimensional systems can behave differently from two-dimensional ones and since models cannot be validated without corresponding data, it is crucial to build accurate three-dimensional models of real cell aggregates. The lack of automated methods to determine which cell outlines in successive images of a confocal stack or time-lapse image set belong to the same cell is an important unsolved problem in the reconstruction process. This thesis addresses this problem through a contour grouping algorithm (CGA) designed to lead to unsupervised three-dimensional reconstructions of biological cells. The CGA associates contours obtained from fluorescently-labeled cell membranes in individual confocal slices using concepts from the fields of machine learning and combinatorics. The feature extraction step results in a set of association metrics. The algorithm then uses a probabilistic grouping step and a greedy-cost optimization step to produce grouped sets of contours. Groupings are representative of imaged cells and are manually evaluated for accuracy. The CGA presented here is able to produce accuracies greater than 96% when properly tuned. Parameter studies show that the algorithm is robust. That is, acceptable results are obtained under moderately varied probabilistic constraints and reasonable cost weightings. Image properties – such as slicing distance, image quality – affect the results. Sources of error are identified and enhancements based on fuzzy-logic and other optimization methods are considered. The successful grouping of cell contours, as realized here, is an important step toward the development of realistic, three-dimensional, cell-based finite element models.

Monothiolate protected gold nanoclusters with near IR luminescence underwent a five-to-ten fold enhancement of quantum efficiency by heating in the presence of excess thiols. Two monothiolate nanoclusters, mercaptosuccinic acid and tiopronin, were shown to benefit from this procedure. Emission maximum around 700-900 nm is favorable for bioimaging applications due to reduction of background signal from autofluorescence. Dithiolate lipoic acid protected gold nanoclusters with higher near IR quantum efficiency present an interesting candidate for biological imaging due to the difference in hydrophobicity, resistance to quenching by divalent cations and cell growth media, and retained quantum efficiency when coupled to agents such as polyethylene glycol. Intracellular and nuclear internalization of mercaptosuccinic gold nanoclusters demonstrate a potential vector for delivery of nuclear targeting agents. The small size, chemical stability, high luminescence, and potential for targeting various intracellular domains make gold nanoclusters worthwhile for further studies as potential bioimaging probes.

During inflammatory responses, macrophages make and secrete cytokines, including the proinflammatory cytokine TNF-alpha (TNF). TNF is a highly potent activator of immune responses with pleiomorphic effects throughout the body. TNF is a key causative agent of chronic inflammatory diseases and is of an intense clinical interest as a therapeutic target. At the outset of this thesis, little was known about how macrophages secrete TNF. Notably, the pathways, carriers and molecules that regulate TNF secretion had not been characterised. A main goal of this work was to identify compartment and molecules involved in the intracellular trafficking of TNF. Live cell imaging of GFP-TNF was established and this provided novel and important new insights into trafficking. Both endogenous and GFP-tagged TNF were followed in macrophages using fluorescence microscopy. The trafficking of other molecules in macrophages was also studied. The major findings of this work include the identification of a new two-step secretory pathway for TNF and other proteins from the trans-Golgi Network (TGN) to the cell surface. This pathway goes via the recycling endosome as an intermediate station. Pleiotrophic tubular-vesicular carriers containing TNF bud off the TGN for the post-Golgi trafficking of TNF and their characterization both in live cell imaging and in biochemical analysis of isolated vesicles constituted the main parts of this work. Functional studies, including endosome inactivation and overexpression of Rab11 mutants (proteins functioning at the level of the recycling endosome) revealed that recycling endosomes have indeed an essential role in the exocytic trafficking of TNF in macrophages. This thesis also provides further insight into recycling endosomes as a possible intermediate step in the exocytic trafficking of several other proteins including the adhesion protein E-Cadherin, that function at the cell surface. Finally, the last chapter of this thesis examines endocytic pathways in activated macrophages. Assays for fluid phase endocytosis and receptor-mediated endocytosis were established and the regulation of both pathways was compared. The results show that LPS has opposite effects on fluid phase and receptor mediated endocytosis, decreasing and increasing their activity respectively. Recycling of transferrin through the recycling endosome was also measured, providing a link with studies on TNF exocytosis. Overall, the work in this thesis has made a major contribution to our understanding of TNF trafficking in macrophages, of macrophage pathways more generally and of trafficking at a fundamental level. The findings herein set the stage for more in depth analysis at a single molecular level to explore TNF regulation in normal and disease cells.

No description available.

A new type of adaptive optics (AO) based on the principles of digital holography (DH) is proposed and developed for the use in wide-field and confocal retinal imaging. Digital holographic adaptive optics (DHAO) dispenses with the wavefront sensor and wavefront corrector of the conventional AO system. DH is an emergent imaging technology that gives direct numerical access to the phase of the optical field, thus allowing precise control and manipulation of the optical field. Incorporation of DH in an ophthalmic imaging system can lead to versatile imaging capabilities at substantially reduced complexity and cost of the instrument. A typical conventional AO system includes several critical hardware pieces: spatial light modulator, lenslet array, and a second CCD camera in addition to the camera for imaging. The proposed DHAO system replaces these hardware components with numerical processing for wavefront measurement and compensation of aberration through the principles of DH. We first design an image plane DHAO system which is basically simulating the process the conventional AO system and replacing the hardware pieces and complicated control procedures by DH and related numerical processing. In this original DHAO system, CCD is put at the image plane of the pupil plane of the eye lens. The image of the aberration is obtained by a digital hologram or guide star hologram. The full optical field is captured by a second digital hologram. Because CCD is not at the conjugate plane of the sample, a numerical propagation is necessary to find the image of the sample after the numerical aberration compensation at the CCD plane. The theory, simulations and experiments using an eye model have clearly demonstrated the effectiveness of the DHAO. This original DHAO system is described in Chapter 2. Different from the conventional AO system, DHAO is a coherent imaging modality which gives more access to the optical field and allows more freedom in the optical system design. In fact, CCD does not have to be put at the image plane of the CCD. This idea was first explored by testing a Fourier transform DHAO system (FTDHAO). In the FTDHAO, the CCD can directly record the amplitude point spread function (PSF) of the system, making it easier to determine the correct guide star hologram. CCD is also at the image plane of the target. The signal becomes stronger than the image plane DHAO system, especially for the phase aberration sensing. Also, the numerical propagation is not necessary. In the FTDHAO imaging system, the phase aberration at the eye pupil can be retrieved by an inverse Fourier transform (FT) of the guide star hologram and the complex amplitude of the full field optical field at the eye pupil can be obtained by an inverse FT of the full field hologram. The correction takes place at the eye pupil, instead of the CCD plane. Taking FT of the corrected field at the eye pupil, the corrected image can be obtained. The theory, simulations, and experiments on FTDHAO are detailed in chapter 3. The successful demonstration of FTDHAO encourages us to test the feasibility of putting CCD at an arbitrary diffraction plane in the DHAO system. Through theoretical formulation by use of paraxial optical theory, we developed a correction method by correlation for the general optical system to perform the DHAO. In this method, a global quadratic phase term has to be removed before the correction operation. In the formulation, it is quite surprising to find that the defocus term can be eliminated in the correlation operation. The detailed formulations, related simulations, and experimental demonstrations are presented in Chapter 4. To apply the DHAO to the confocal retinal imaging system, we first transformed the conventional line-scanning confocal imaging system into a digital form. That means each line scan is turned into a digital hologram. The complex amplitude of the optical field from each slice of the sample and aberration of the optical system can be retrieved by digital holographic process. In Chapter 5, we report our experiments on this digital line-scanning confocal imaging system. This digital line-scanning confocal image absorbs the merits of the conventional line-scanning confocal imaging system and DH. High-contrast intensity images with low coherent noise, and the optical sectioning capability are made available due to the confocality. Phase profiles of the samples become accessible thanks to DH. The quantitative phase map is even better than that from the wide field DH. We then explore the possibility of applying DHAO to this newly developed digital line-scanning confocal imaging system. Since optical field of each line scan can be achieved by the DH, the aberration contained in this field can be eliminated if we are able to obtain the phase aberration. We have demonstrated that the phase aberration can be obtained by a guide star hologram in the wide field DHAO systems. We then apply this technique to acquire the aberration at the eye pupil, remove this aberration from the optical fields of the line scans and recover the confocal image. To circumvent the effect of phase aberration on the line illumination, a small collimated laser beam is shone on the cylindrical lens. Thus the image is solely blurred by the second passage through the aberrator. This way, we can clearly demonstrate the effect of DHAO on the digital line-scanning confocal image system. Simulations and experiments are presented in chapter 6, which clearly demonstrates the validity of this idea. Since line-scanning confocal imaging system using spatially coherent light sources has proven an effective imaging tool for retinal imaging, the presented digital adaptive optics line-scanning confocal imaging system is quite promising to become a compact digital adaptive optics laser scanning confocal ophthalmoscope.

No description available.

Colorectal cancer is the third most common cancer in the United States and there is currently a lot of research going into new antitumor agents to kill the cancer. One method for replicating the tumor response to a drug in vivo is by creating an in vitro drug testing model to replicate the in vivo condition. This research project was conducted to determine the efficacy of testing tumor cultures in a microfluidic device as a way to provide accurate drug responses in vitro instead of using in vivo subjects in clinical trials. A total of four experiments were conducted with each experiment increasing the complexity of the culture model. The first experiment was a 2-dimensional tumor culture that was seeded in a well plate to study how 5-fluorouracil treatments affected the tumor cell viability. The second experiment was a 2-dimensional tumor culture that was seeded on top of a fibrin extracellular matrix (ECM) gel to determine how the tumor cells would respond to the 5-luorouracil treatments while growing on the fibrin. The third experiment was to create a 3-dimensional tumor culture that was seeded inside the fibrin ECM gel. This experiment was conducted to determine if tumor cells cultured within the fibrin gel could receive nutrients from the medium diffusing through the gel. Once the tumors responded as expected in the fibrin gel, the gel could be injected into a microfluidic device for the fourth experiment. The fourth experiment was a proof of concept to determine if the tumor cells could survive in the microfluidic device and be properly treated with 5-fluorouracil. The experiment with the cells seeded in the well plates showed that an increase in 5-fluorouracil concentration caused a significant decrease in cell viability. Both fibrin gel experiments showed that the average tumor size, total tumor area, and tumor count decreased as the 5-fluorouracil concentration increased. The tumor cells were successfully able to be cultured in the microfluidic device and the average tumor size decreased significantly when the culture was exposed to the 5-fluorouracil treatment.

Current high-throughput screening (HTS) tools (i.e., single-use 96-well filter plate) are limited to the few membrane types that are sold commercially, restricting the ability to screen membrane materials for targeted applications. In this thesis, a multi-well device capable of screening any flat-sheet membrane was designed, where multiple devices can be stacked for extensive HTS (>32 experiments). Confocal imaging of a Natrix Q cross-section – a membrane type not sold in a commercial filter plate – was carried out after 24 h in contact with green fluorescent protein to visually confirm protein-membrane interactions. The static binding capacity (SBC) of bovine serum albumin (BSA) and Herring testes DNA was found for specific parameters: membrane type (Mustang Q, Sartobind Q, Natrix Q, Durapore), salt concentration (0, 50, 100 mM NaCl), and contact time (1 min, 4 h, 8 h, 24 h). Considering solution conditions, the highest BSA SBC was observed with Natrix Q at 0 M NaCl with a contact time of 24 h. The DNA and BSA SBC values for Natrix Q were the highest among the membrane types evaluated, demonstrating consistency with literature trends. These findings suggest that SBC experiments can predict promising membrane materials for scaled-up applications. Finally, the chromatography process was replicated in this multi-well device (Natrix Q), showing 50% BSA elution from the membrane. The results of this thesis confirmed this ability to accommodate any membrane adsorber, simultaneously compare different membrane materials, and extract the membrane for post-experimental analysis. This work’s significance was emphasized in its future potential to aid with membrane material selection, particularly with exploring the properties of next-generation membrane materials (e.g., 3D-printed membranes). Three future areas for optimization with this multi-well device were highlighted: biotherapeutic purification, sequencing of membrane materials within a process, and applying it as a tool to understand ion selectivity. / Thesis / Master of Applied Science (MASc) / Membranes are used in many industries, such as water treatment, environmental remediation, and biopharmaceuticals. In the biopharmaceutical industry, high-throughput screening (HTS) tools (e.g., filter plates), which allow for miniaturized experiments, are used to perform extensive experimental analysis to determine optimal solution conditions (e.g., pH) for biomolecule binding. Unfortunately, commercial filter plates are limited in customizability for HTS of membrane materials. To address these limitations, this thesis focuses on designing and validating a multi-well device capable of incorporating any membrane adsorber. Different biomolecules (proteins, DNA), solution conditions, and membrane materials were evaluated. The results of this thesis confirmed this ability to accommodate any membrane adsorber, simultaneously compare different membrane materials, and extract the membrane for post-experimental analysis. This work also discussed using this device for future rapid membrane material selection in multiple industries (e.g., biotherapeutics, ion extraction).

Yes / Effective suppression of JAK–STAT signalling by the inducible inhibitor “suppressor of cytokine signalling 3” (SOCS3) is essential for limiting signalling from cytokine receptors. Here we show that cavin-1, a component of caveolae, is a functionally significant SOCS3- interacting protein. Biochemical and confocal imaging demonstrate that SOCS3 localisation to the plasma membrane requires cavin-1. SOCS3 is also critical for cavin-1 stabilisation, such that deletion of SOCS3 reduces the expression of cavin-1 and caveolin-1 proteins, thereby reducing caveola abundance in endothelial cells. Moreover, the interaction of cavin-1 and SOCS3 is essential for SOCS3 function, as loss of cavin-1 enhances cytokine-stimulated STAT3 phosphorylation and abolishes SOCS3-dependent inhibition of IL-6 signalling by cyclic AMP. Together, these findings reveal a new functionally important mechanism linking SOCS3-mediated inhibition of cytokine signalling to localisation at the plasma membrane via interaction with and stabilisation of cavin-1. / This work was supported by project grants to T.M.P. from the Chief Scientist Office (ETM/226), British Heart Foundation (PG12/1/ 29276, PG 14/32/30812), and a National Health Service Greater Glasgow and Clyde Research Endowment Fund (2011REFCH08). P.F.P. was supported by the National Institutes of Health grant DK097708. J.J.L.W. was supported by a doctoral training studentship from the Biotechnology and Biological Sciences Research Council Doctoral Training Programme in Biochemistry and Molecular Biology at the University of Glasgow (BB/F016735/1). N.A. was supported by a Saudi Government PhD Scholarship. This work was also supported in part by equipment grants to T.M.P. from Diabetes UK (BDA 11/0004309) and Alzheimer’s Research UK (ARUK-EG2016A-3).