Doctor of Philosophy (PhD) / The toxicity of platinum anticancer drugs presents a major obstacle in the effective treatment of tumours. Much of the toxicity stems from a lack of specificity of the drugs for the sites at which they are able to exert maximum anticancer activity. An improved understanding of the behaviour of the drugs in the tumour environment may assist in the rational design of future platinum anticancer agents with enhanced specificity and reduced toxicity. In the work presented herein, the specificity of two classes of platinum anticancer agents was assessed (platinum(IV) cisplatin analogues and platinum(II) anthraquinone complexes). The interaction of the platinum(IV) agents with DNA, believed to be their main cellular target, was examined using XANES spectroscopy. This experiment was designed to assess the ability of the drugs to interact with DNA and thus exert their anticancer activity. It was shown that the platinum(IV) complexes were not reduced by DNA during 48 hr incubation. It was not possible to conclusively determine whether the interaction of the complexes with DNA was direct or platinum(II) catalysed, or whether interaction had occurred at all. The distribution of platinum(II) anthraquinone complexes and their corresponding anthraquinone ligands in tumour cells (A2780 ovarian and DLD-1 colon cancer cell lines) was investigated. The cytotoxicity of the compounds in DLD-1 cells was also assessed. It was found that the compounds were efficiently taken up into the cells and entered the lysosomal compartments almost exclusively. This suggested that the cytotoxicity of the drugs was caused by lysosomal disruption, or that the platinum complexes were degraded, leaving a platinum species to enter the cell nuclei and interact with DNA. Alternatively, the complexes may bind to proteins and transport into the nuclei of the cells, though with their fluorescence quenched by the protein. The penetration and distribution of platinum(IV) complexes was assessed in DLD-1 multicellular tumour spheroids (established models of solid tumours) using a number of synchrotron techniques, including micro-tomography, micro-SRIXE, and micro-XANES. The complexes were found to be capable of penetrating throughout the entire volume of the spheroids. Micro-XANES indicated that in central and peripheral spheroidal regions, bound platinum species were present largely as platinum(II).

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第17557号 / 工博第3716号 / 新制||工||1566(附属図書館) / 30323 / 京都大学大学院工学研究科マイクロエンジニアリング専攻 / (主査)教授 安達 泰治, 教授 楠見 明弘, 准教授 井上 康博, 教授 琵琶 志朗 / 学位規則第4条第1項該当

Master of Science / Division of Biology / Bradley J. Olson / Unicellular organisms alter their behavior and morphology in response to environmental stresses, particularly in response to immediate threats to their survival. A common tactic of predator avoidance for unicellular green algae is to aggregate to form groups. We have found that the model unicellular green algae Chlamydomonas reinhardtii forms aggregates in response to the presence of the filter feeding zooplanktonic predator, Daphnia magna. Chalmydomonas is a member of the volvocine algae, a morphologically diverse group of closely related green algae that is often used to study multicellular development. We have characterized aggregation in Chlamydomonas reinhardtii and found that it is rapid, transient and induced by signals originating from the Daphnia predators. To understand the genetic basis of cooperative aggregation we used an RNA-seq approach. RNA-seq characterized the transcriptomic response by Chlamydomonas during aggregation, and we identified 131 genes are significantly differentially expressed between predated and unpredated cultures of Chlamydomonas. Several candidate genes were characterized based on existing annotations, evolutionary history and expression profile. Evolutionary relationships between candidate aggregation genes in Chlamydomonas and their orthologs in multicellular Volvocales suggest a possible role of aggregation genes in multicellular development. Our results demonstrate that Chlamydomonas dynamically alters its morphology based on its environment and identify several candidate genes for aggregation and multicellular development.

Tumour necrosis has long been associated with poor prognosis and reduced survival in cancer. Hypotheses to explain this include the idea that as aggressive tumours tend to grow rapidly, they outgrow their blood supply leading to areas of hypoxia and subsequently necrosis. However whilst this and similar hypotheses have been put forward to explain the association, the biological significance of the cells which make up necrotic tissue has been largely ignored. This stems from the belief that because a tumour is more aggressive and fast growing it develops areas of necrosis, rather than, the tumour is more aggressive because it contains areas of necrosis. Which came first like the egg and chicken is yet to be determined, however to date most research has only considered the possibility of the former. Viable cells were found in the necrotic core of Multicellular Tumour Spheroids. When examined these cells were found to be different to the original cell line in terms of proliferation, migration, and chemosensitivity. A proteomic analysis showed that these phenotypical changes were accompanied by changes in a large number of proteins within the cells, some of which could be potential therapeutic targets. Furthermore this has led to a new hypothesis for tumour necrosis and its association with poor prognosis. Necrotic tissue provides a microenvironemental niche for cells with increased survival capabilities. Protected from many chemotherapeutics by their non-proliferative status once conditions improve these cells can return to proliferation and repopulate the tumour with an increasingly aggressive population of cells. / Yorkshire Cancer Research

A wide variety of adherent mammalian cells can be induced into a reversible state of metabolic arrest (quiescence) by conversion to non-adherent multicellular aggregates. These "spheroids" can be maintained at room temperature under oxygen- and nutrient-deprived conditions for extended periods of time (weeks) as well as converted back to viable proliferating monolayers. Herein it is shown that HEK293 spheroid arrest and recovery requires the co-activation of both NF-kB and JNK, and chemical inhibition of either NF-κB nuclear translocation or JNK phosphorylation leads to cell death. Cytokine profiling within the aggregates during the arrest and recovery process is suggestive that a cyclical cascade was in operation, leading to endogenous cytokine production of TNF-Alpha, IL-1Beta, and IL-8, thereby propagating the cellular stress signal within cells as well as throughout the aggregate. Cytokines exist <i>in vivo</i> as mixtures, yet tissue culture studies delineating how cells respond to these molecules are often performed using individual effectors added exogenously. Are the results obtained in these studies true representations of physiological responses? As HEK293 multicellular aggregates (spheroids) survive long term arrest by endogenous cytokine (TNF-α and IL-1β) and chemokine (IL-8) signaling, adherent monolayer cells were evaluated for their ability to provide a "spheroid signal response" when exposed to TNF-α, IL-1β and IL-8 individually, and in combination, at concentrations observed in the aggregates. The spheroid signal transduction response was only observed when all three cytokines were present, demonstrating that signal transduction cascade mechanisms are cytokine-profile dependent. To determine if similar processes were involved in the arrest and recovery of multicellular aggregates derived from other cell types, the responses of primary human foreskin fibroblasts (HFF-2) and a glioblastoma cell line (T98G) were characterized, utilizing the procedures developed in the HEK293 study. Both the T98G and HFF-2 cell lines entered and exited from the long term arrest utilizing an autocrine response. However, while the carcinoma cell line was dependent upon NF-κB for survival, its signaling partner was Gadd45α and signaling occurred through the p38 pathway. Primary fibroblast arrest and recovery proceeded through the p38 pathway as well, but was independent of NF-κB. Thus, three different cell types and transformation states (HEK293, HFF-2, and T98G) provided three different routes to survival, all with cyclical cytokine production and signaling. These routes cannot be measured or modulated effectively in adherent monolayers. Multicellular aggregates provide higher ordered systems that can be used to describe signaling pathways within a cell, highlighting the role of autocrine responses and the synergistic relationships between cytokines and neighboring cells. / Ph. D.

Progress in the biological sciences relies on the availability of appropriate tools and strategies; in the areas of understanding and constructing multicellular systems, better methods of gene expression control are needed. Here, we work towards two approaches to controlling gene expression for applications related to multicellular coordination and pattern formation. First, we characterize the use of 2A self-cleaving peptides for polycistronic expression in Dictyostelium discoideum, a powerful model system for investigating the onset of coordinated behavior in a cell population. We also make novel observations about various factors that affect gene expression levels, which would inform future decisions on the use of 2A peptides in this system. Second, we examine the use of antisense RNA in mammalian cells for gene repression from an input that simultaneously activates other genes. We show that in transient overexpression systems, antisense RNA produced in trans can be a more effective repressor than convergent promoters and other well-established RNA-based methods for gene repression, and we explore ways in which this system could be improved for stable gene circuits. Overall, our results advance our understanding of available tools for gene expression control and the conditions under which they are appropriate to use.

As pontes de madeira com tabuleiro multicelular protendido são uma das mais recentes tecnologias usadas na construção das modernas pontes de madeira. Nesta tese é realizado o estudo teórico e experimental do comportamento estrutural destas pontes. Os principais métodos de cálculo são apresentados e discutidos. A investigação experimental foi realizada em dois modelos reduzidos em escala 1:3 com as mesmas dimensões externas mas diferente quantidade de nervuras. Os modelos foram ensaiados com diferentes posições de carregamento enquanto os deslocamentos, deformações e forças nas barras, eram monitorados. Os resultados obtidos demonstraram que os modelos de Placa Ortotrópica Equivalente e Elementos Finitos podem ser empregados para o dimensionamento das pontes de madeira com tabuleiro multicelular protendido. O método de Viga Equivalente pode ser empregado desde que utilizado o correto Fator de Distribuição de Carga. Os estudos realizados neste trabalho, indicam a viabilidade da utilização deste sistema estrutural para pontes com vãos de 12 a 25 m / Timber bridges with multicellular prestressed decks is one of the most recent technology for modern timber bridges construction. In this thesis the theoretical and experimental study of the structural behavior of these bridges is accomplished. The main calculation methods are introduced and discussed. Two reduced models on scale 1:3, with the same external dimensions but different number of webs, were used for the experimental investigation. The models were tested with different load positions meanwhile displacements, strains and bar forces were measured. The obtained results have show that either model of Equivalent Orthotropic Plate or Finite Elements can be used for the design of this type of bridge. The Equivalent Beam model can also be employed as long as the correct Load Distribution Factor is chosen. The accomplished studies demostrate that this structural system is viable for bridges with span from 12 to 25 m

Most embryonic development and tissue self-assembly requires the integration of cell movements within multiple cell layers composed of different cell types, which are integrated with the signaling networks in these 3D environments. Although the role of cell mechanics in tissue self-assembly has been demonstrated, little is known about the mechanical responses of 3D multi-layer tissues to chemical cues. To investigate the collective movements within multilayered tissues, I developed a novel microfluidic technique capable of removing desired height or width of tissue from a composite tissue. I call this technique "3D tissue-etching" because it is analogous to techniques used in the microelectromechanics (MEMS) field where complex 3D structures are built by successively removing material from a monolithic solid through subtractive manufacturing. I used a custom-designed microfluidic control system to deliver a range of tissue etching reagents (detergents, chelators, proteases, etc.) to specific regions of multilayered tissues microsurgically isolated from embryos of the African Clawtoed frog, Xenopus laevis. Xenopus embryos and explanted tissues have long been used to elucidate signaling and other cellular processes during development and here provide an ideal model 3D tissue etching. Long exposure to a narrow etchant stream cuts completely through cell-cell layers to expose the substrate. By reducing the exposure time a single layer may be removed. By controlling the width of the etchant and the exposure time a broader swath of the surface layer may be removed. For more refined etching, after removal of a broad swath the resistance circuits can be switched and a second narrow stream can remove only a single narrow band within the swath exposed cells. I developed tissue-etching techniques that allow me to shape complex multi-layered embryonic tissues. The ability to control 3D stimulation and the form of multicellular tissues will provide extend the tools of tissue engineering to synthesize highly complex 3D integrated multicellular biosystems. Integration of tissue etching in my custom microfluidic system provides a "test-bed" where a range of hypotheses concerning the control and regulation of development and cell differentiation can be implemented and tested.

abstract: This dissertation features a compilation of studies concerning the biophysics of multicellular systems. I explore eukaryotic systems across length scales of the cell cytoskeleton to macroscopic scales of tissues. I begin with a general overview of the natural phenomena of life and a philosophy of investigating developmental systems in biology. The topics covered throughout this dissertation require a background in eukaryotic cell physiology, viscoelasticity, and processes of embryonic tissue morphogenesis. Following a brief background on these topics, I present an overview of the Subcellular Element Model (ScEM). This is a modeling framework which allows one to compute the dynamics of large numbers of three-dimensional deformable cells in multi-cellular systems. A primary focus of the work presented here is implementing cellular function within the framework of this model to produce biologically meaningful phenotypes. In this way, it is hoped that this modeling may inform biological understanding of the underlying mechanisms which manifest into a given cell or tissue scale phenomenon. Thus, all theoretical investigations presented here are motivated by and compared to experimental observations. With the ScEM modeling framework I first explore the passive properties of viscoelastic networks. Then as a direct extension of this work, I consider the active properties of cells, which result in biological behavior and the emergence of non-trivial biological phenotypes in cells and tissues. I then explore the possible role of chemotaxis as a mechanism of orchestrating large scale tissue morphogenesis in the early embryonic stages of amniotes. Finally I discuss the cross-sectional topology of proliferating epithelial tissues. I show how the Subcellular Element Model (ScEM) is a phenomenological model of finite elements whose interactions can be calibrated to describe the viscoelastic properties of biological materials. I further show that implementing mechanisms of cytoskeletal remodeling yields cellular and tissue phenotypes that are more and more biologically realistic. Particularly I show that structural remodeling of the cell cytoskeleton is crucial for large scale cell deformations. I provide supporting evidence that a chemotactic dipole mechanism is able to orchestrate the type of large scale collective cell movement observed in the chick epiblast during gastrulation and primitive streak formation. Finally, I show that cell neighbor histograms provide a potentially unique signature measurement of tissue topology; such measurements may find use in identifying cellular level phenotypes from a single snapshot micrograph. / Dissertation/Thesis / Ph.D. Physics 2011

As pontes de madeira com tabuleiro multicelular protendido são uma das mais recentes tecnologias usadas na construção das modernas pontes de madeira. Nesta tese é realizado o estudo teórico e experimental do comportamento estrutural destas pontes. Os principais métodos de cálculo são apresentados e discutidos. A investigação experimental foi realizada em dois modelos reduzidos em escala 1:3 com as mesmas dimensões externas mas diferente quantidade de nervuras. Os modelos foram ensaiados com diferentes posições de carregamento enquanto os deslocamentos, deformações e forças nas barras, eram monitorados. Os resultados obtidos demonstraram que os modelos de Placa Ortotrópica Equivalente e Elementos Finitos podem ser empregados para o dimensionamento das pontes de madeira com tabuleiro multicelular protendido. O método de Viga Equivalente pode ser empregado desde que utilizado o correto Fator de Distribuição de Carga. Os estudos realizados neste trabalho, indicam a viabilidade da utilização deste sistema estrutural para pontes com vãos de 12 a 25 m / Timber bridges with multicellular prestressed decks is one of the most recent technology for modern timber bridges construction. In this thesis the theoretical and experimental study of the structural behavior of these bridges is accomplished. The main calculation methods are introduced and discussed. Two reduced models on scale 1:3, with the same external dimensions but different number of webs, were used for the experimental investigation. The models were tested with different load positions meanwhile displacements, strains and bar forces were measured. The obtained results have show that either model of Equivalent Orthotropic Plate or Finite Elements can be used for the design of this type of bridge. The Equivalent Beam model can also be employed as long as the correct Load Distribution Factor is chosen. The accomplished studies demostrate that this structural system is viable for bridges with span from 12 to 25 m