Note:

This research describes the changes in cell morphology of Azotobacter vinelandii cells cultured in dialyzed soil medium. This particular culture medium was assumed to provide the bacteria with an environment similar to their natural habitat, the soil.

T cells are key players in adaptive immune response. Originating from the thymus, they seek and eliminate infected cells in various locations of our body. T cells are not anchorage-dependent in nature. However, in our body, cells are constantly under physiological stress. It is not yet known how natural changes in physical environment could affect T cell behaviors. This thesis focuses to study the role, pathway, and main mechanism of rigidity sensing in T cells. Most studies of T cell rigidity sensing have showed that T cell responses are sensitive to external forces. It is unclear whether T cells could generate forces, translate them to biochemical signaling, and regulate their function based on the physical sensing. We tested the idea by developing the use of substrate with varying modulus to analyze the impact of rigidity to T cell activation. We demonstrated that mouse naive CD4+ T cells were capable of sensing and transmitting information from substrate modulus, ultimately affecting the regulation of cytokine secretion, a key indicator of T cell activation. Interestingly, this cytokine secretion correlated with increasing substrate rigidity. This increased cytokine secretion diminished when T cells lost the ability to contract in sensing the underlying substrate rigidity. Contrary to the presumption that T cells are not able to regulate their function based on the forces applied to the environment, our study provides the first demonstration that substrate rigidity has a functional impact to naive CD4+ T cell activation. To understand the translation process from physical to biochemical signaling in T cells, we determined the signaling pathway that regulated T cell rigidity sensing. We found that T cell rigidity sensing was associated with the signaling molecules of the T cell receptor (TCR) complex, the central pathway of T cell response. Analysis of TCR signaling molecules revealed that T cell rigidity sensing was mediated downstream of the early signaling components in the TCR complex. Lastly, we developed a method of combining micron-scale patterning in elastic substrates to determine whether T cell mechanosensing was mediated from local adhestion sites or globally throughout the cell. Circular features of primary signal for naive CD4+ T cells were spatially segregated and patterned on elastic substrates to analyze T cell contractility in generating forces across the segregated primary signals, leading to sustained TCR triggering. We found out that T cell contractility failed to generate forces when the primary signals were arranged in equilateral triangle geometry, leading to loss of TCR triggering. This result shows that T cell rigidity sensing is mediated globally throughout the whole cell rather than locally from adhesion sites. Furthermore, the loss of TCR triggering by T cells when sensing the equilateral triangle geometry in elastic substrates opens up new ideas in characterizing force generation within the cell.

published_or_final_version / Anatomy / Master / Master of Philosophy

Following 1 hr incubation with ACTH, cytochalasin D or ACTH/cytochalasin, detergent-solubilized mouse adrenal tumor cells cytoskeletal changes were examined using scanning and transmission electron microscopy. Steroid production was also examined.

Embryo development is a highly coordinated process where genetic regulation and mechanical forces interplay to drive the transformation of a single cell into a complex, multicellular organism. It involves many fundamental processes such as cell division, cell differentiation, and morphogenesis. Morphogenesis, the shape changes of tissue, results from collective cell movement, growth, proliferation, and shape changes, guided by genetic and mechanical cues. Despite the comprehensive data obtained from experimental measurements and advanced imaging, the physical mechanisms underlying morphogenesis are poorly understood, a quantitative cell shape pattern that describes morphogenesis has yet to be discovered, and the coupling between cytoskeleton that generates stress and shape changes has not been quantitatively demonstrated. To address these unsolved questions, we utilized a powerful combination of first-principles modeling and empirical, data-driven approaches. Chapter 1 presents our mathematical model of Drosophila ventral furrow formation, which incorporates actomyosin contractile stress and viscous tissue responses. With all model parameters fitted from experiment, our model quantitatively explained numerous experimental observations in wild-type and genetically perturbed embryos, which were not fully explained by other models assuming elastic tissue responses. Our model revealed that the tissue-scale contraction in ventral furrow formation is driven by the curvature of the multicellular myosin profile. We also demonstrated that the pulsatile time-dependence of myosin acts as a protective mechanism for tissue contraction, suppressing cell-to-cell myosin fluctuations through a low-pass filter effect. This is crucial because tissue contraction is highly sensitive to even small myosin fluctuations, which would otherwise lead to significant inhomogeneous contractions. Chapter 2 details our data-driven approach to studying Drosophila ventral furrow formation, utilizing time-lapse 3D data from light sheet microscopy. We developed computational algorithms to systematically parameterize over 28,000 cell shapes, designed interpretable cell shape features, and employed unsupervised learning to classify cell shape evolution trajectories. By mapping these classes onto the embryo, we extracted the first quantitative cell shape pattern in the Drosophila embryo. This pattern unveiled key physical mechanisms underlying embryo development, including how mechanical stresses propagate, how cell packing is influenced by embryo curvature, and the stochastic nature of apical constriction during tissue contraction. Chapter 3 explores the coupling between actomyosin density and shape changes. We developed a mathematical model of the actomyosin cortex, using partial differential equations to describe the evolution of actomyosin density on a deformable surface, which is represented through differential geometry. Our model revealed that although under physiological conditions, the cell cortex is observed to maintain a homogeneous density and shape, this stability is challenged by two factors: increased cortical tension, which is mechanical in nature, and an elongated aspect ratio, which is a geometric feature. Higher cortical tension disrupts this homogeneity, leading to patterned actomyosin density and multiply furrowed shape. In contrast, an elongated aspect ratio drives constriction through a mechanism we named active Rayleigh instability, a modified form of the Plateau-Rayleigh instability. Furthermore, friction plays a crucial role in protecting the homogeneous state by preserving a large region of homogeneity in the state diagram of the cortex. When friction is reduced, this homogeneous region shrinks significantly, making the cortex more vulnerable to destabilization caused by increased tension and an elongated aspect ratio.

N^6,O^2' -Dibutyryl cyclic adenosine 3',5'-monophosphate (Bt_2cAMP) was investigated for its effects on various tissue culture cells infected with temperature-sensitive (ts) mutant, LA31 and Bratislava 77 (B77), a wild-type Rous sarcoma virus. Specifically, known parameters of transformation were investigated and a possible site of action has been tenably proposed. The drug Bt_2cAMP was found to have little effect on the transformation related properties of primary chick embryo fibroblasts (CEF) infected with either virus or normal rat kidney fibroblasts (NRK) infected with the wild-type B77-RSV. However, significant inhibition of the transforming properties in NRK infected with the ts mutant LA31 (LA31-NRK) were reported at the permissive temperature 33 degrees centigrade (33 C).

The basis of this investigation is to characterize growth property differences in normal versus virally transformed cells. Using a temperature-sensitive mutant of Rous sarcoma virus, the cells' transformation state is regulated by the growth temperature; at 33°C the cells are transformed, while at 39°C the cells have normal characteristics. The morphology of NRK cells is elongated and fibroblastic; when transformed the cells are rounded. Normal cells grow to a monolayer and stop, while transformed cells grow to saturation densities greater than just a monolayer amount. Transformed cells can form foci when grown in mixture with normal cells. Normal cells must be in contact with the culture vessel in order to grow, but transformed cells lack anchorage dependence for growth.

Using cultured Y-1 mouse adrenal tumor cells which produce the steroid 20(-hydroxypregn-4-en-3-one (20-DHP), it was found that 10-5 M corticosterone and deoxycorticosterone increased basal and inhibited ACTH-induced 20-DHP production. The steroid effects were concentration-dependent, reversible, and specific since six other steroids did not stimulate steroidogenesis and varied in their ability to inhibit ACTH-induced steroidogenesis. Cytochalasin D inhibited steroid-stimulated 20-DHP production, suggesting a mechanism of steroid stimulation similar to that of ACTH. Steroidogenesis stimulated by cholera toxin, (Bu) 2 cAMP, or pregnenolone was not inhibited by exogenous steroid; corticosterone increased basal and inhibited ACTH-induced intracellular cAMP production. Steroids altered cell surface morphology. These findings suggest that steroids alter adrenal steroidogenesis by acting within the plasma membrane.

This thesis presents an investigation into the factors affecting the morphology of hybrid inorganic/organic photoactive layers used in photovoltaic cells. Although optimisation of the organic (polymer) phase has received substantial attention, research into the morphology of the inorganic phase (semiconducting nanocrystals) remains limited. It is believed that there is a strong link between the morphology of the final photoactive film and the quality of the initial nanocrystal dispersion. To this end, two nanocrystal systems were investigated; zinc oxide (ZnO) and lead sulphide (PbS). ZnO nanocrystals were synthesised and found to possess reproducible characteristics. It was determined that colloid stability was initially dependent upon the presence of acetate groups bound to the surface, which in turn required a small quantity of methanol to be present in the organic dispersant. It was also discovered that while methanol evaporated readily from the surface of the nanocrystals, another molecule, 1-propylamine (1-PA), did not. Further investigations showed that while methanol only weakly physisorbed to the surface of ZnO nanocrystals, 1-PA formed strong, dative covalent bonds with Zn2+, preventing evaporation despite a low boiling point. Subsequent investigations into the effects of different ligands upon colloid stability found that amine-based groups typically possessed superior stabilising capabilities compared to alcohol-based analogues. The characteristics of nanocrystal / polymer blends were also investigated. It was determined that the nanocrystal dispersion became unstable at higher concentrations of polymer due to depletion aggregation. Films of nanocrystal / polymer blends were cast from dispersions containing either alcohol or amine-based ligands, and it was observed that dispersions stabilised with 1-PA possessed smooth morphologies on the micrometer scale. Investigations at the nanometer scale, however, revealed aggregates large enough to favour recombination.The latter half of this thesis regards the characterisation of PbS nanocrystals and investigations into triggered aggregation. It was determined that while PbS nanocrystals possessed reproducible characteristics, the stabilising molecule, oleic acid (OA) was insulating. The effects of exchanging the OA groups for a shorter ligand, butylamine (BA) were investigated.Finally, PbS nanocrystals were treated with a bidentate ligand, 1,2-ethanedithiol (EDT) to induce triggered aggregation. It was observed that the system was highly sensitive to the concentration of EDT in dispersion, forming small, relatively dispersed aggregates at low [EDT], and micrometer-sized crystalline structures at high [EDT]. The characterisation and entrapment of these nanocrystal structures within semi-conducting polymer films is also discussed.