<div><div><p>Important cellular processes such as migration, differentiation, and development often rely on precise timing. Yet, the molecular machinery that regulates timing is inherently noisy. How do cells achieve precise timing with noisy components? We investigate this question using a first-passage-time approach, for an event triggered by a molecule that crosses an abundance threshold. We investigate regulatory strategies that decrease the timing noise of molecular events. We look at several strategies which decrease the noise: i) Regulation performed by an accumulating activator, ii) Regulation dues to a degrading repressor, iii) Auto-regulation and the presence of feedback. We find that either activation or repression outperforms an unregulated strategy. The optimal regulation corresponds to a nonlinear increase in the amount of the target molecule over time, arises from a tradeoff between minimizing the timing noise of the regulator and that of the target molecule itself, and is robust to additional effects such as bursts and cell division. Our results are in quantitative agreement with the nonlinear increase and low noise of <i>mig-1</i> gene expression in migrating neuroblast cells during <i>Caenorhabditis elegans</i> development. These findings suggest that dynamic regulation may be a simple and powerful strategy for precise cellular timing.</p><p>Autoregulatory feedback increases noise. Yet, we find that in the presence of regulation by a second species, autoregulatory feedback decreases noise. To explain this finding, we develop a method to calculate the optimal regulation function that minimizes the timing noise. Our method reveals that the combination of feedback and regulation minimizes noise by maximizing the number of molecular events that must happen in sequence before a threshold is crossed. We compute the optimal timing precision for all two-node networks with regulation and feedback, derive a generic lower bound on timing noise, and compare our results with the neuroblast migration during <i>C. elegans</i> development, as well as two mutants. We finds that indeed our model is aligned with the experimental findings.</p></div></div><div><p>Furthermore, we apply our framework of temporal regulation to explain how the stopping point of the migrating cells in <i>C. elegans</i> depends on the body size. Considering temporal regulation, we find the termination point of the cell for various larval sizes. We discuss three possible mechanisms: i) No compensation; here the migration velocity is constant across the mutants of <i>C. elegans</i>, and this results in the migration distance to be constant but the relative position to be different across various sizes; ii) Total compensation; here the velocity is compensated with body size, hence resulting in the same relative position of cells across mutants; and iii) Partial compensation; here the velocity of migration is correlated with body size to some degree, resulting in a non-linear relationship between termination point and body size. We find that our partial compensation model is consistent with experimental observations of cell termination.</p><p>Finally, we look at the detection of traveling waves by single-celled organisms. Cells must use temporal and spatial information to sense the direction of traveling waves, as seen in cAMP detection by the <i>amoeba </i><i>Dictyostelium</i>. If a cell only uses spatial information to sense the direction of the wave then the cell will move forward when the wave hits the front of the cell, and move backward when the wave hits the back of the cell, resulting in neutral movement. Cells must use temporal information along with spatial information to effectively move towards the source. Here we develop a mechanism by which cells are able to integrate the spatial and temporal information through a system of inhibitors. We find the optimal time to release the inhibitors for maximizing the precision of directional sensing.</p></div>

Directed cancer cell migration induced by the environmental signals is a critical process in cancer metastasis. Cancer cells are exposed to complex chemical and mechanical signals stimulating directed migration in the tumor microenvironment, where the physical nature is highly complex. It is still barely understood how cells sense and process the complex environmental signals through the complex intercellular signaling networks to execute the cell responses. This study explores the migratory response of cancer cells under a single and combined signal. The driving hypothesis is that the cell innate capability constraints the signal stimulations physically in inducing directed cell migration. We assess the hypothesis by engineering the microenvironment in the microfluidic platform, exposing a single or combined signal environment. The combined signal environment is established by 1) two different chemoattractants (TGF-β1 and EGF) and 2) the convection-driven signal environment (TGF-β1 and interstitial flow). The results show that the performance of cancer cell directed migration is physically constrained when the environmental stimulation meets the cell’s innate physical limit. We illustrate the results in a physical and quantitative manner. This approach provides a novel insight to understand the cellular process and eventually enables to predict the cellular response under the complex environmental signals. <br>

Cell-to-cell variations and intracellular processes such as cytoskeletal organization and organelle dynamics exhibit massive heterogeneity. Advances in imaging and optics have enabled researchers to access spatiotemporal information in living cells efficiently. Even though current imaging technologies allow us to acquire an unprecedented amount of cell images, it is challenging to extract valuable information from the massive and complex dataset to interpret heterogeneous biological processes. Machine learning (ML), referring to a set of computational tools to acquire knowledge from data, provides promising solutions to meet this challenge. In this dissertation, we developed ML pipelines for deconvolution of subcellular protrusion heterogeneity from live cell imaging and molecular diagnostic from lens-free digital in-line holography (LDIH) imaging. Cell protrusion is driven by spatiotemporally fluctuating actin assembly processes and is morphodynamically heterogeneous at the subcellular level. Elucidating the underlying molecular dynamics associated with subcellular protrusion heterogeneity is crucial to understanding the biology of cellular movement. Traditional ensemble averaging methods without characterizing the heterogeneity could mask important activities. Therefore, we established an ACF (auto-correlation function) based time series clustering pipeline called HACKS (deconvolution of heterogeneous activities in coordination of cytoskeleton at the subcellular level) to identify distinct subcellular lamellipodial protrusion phenotypes with their underlying actin regulator dynamics from live cell imaging. Using our method, we discover “accelerating protrusion”, which is driven by the temporally ordered coordination of Arp2/3 and VASP activities. Furthermore, deriving the merits of ML, especially Deep Learning (DL) to learn features automatically, we advanced our pipeline to learn fine-grained temporal features by integrating the prior ML analysis results with bi-LSTM (bi-direction long-short term memory) autoencoders to dissect variable-length time series protrusion heterogeneity. By applying it to subcellular protrusion dynamics in pharmacologically and metabolically perturbed epithelial cells, we discovered fine differential response of protrusion dynamics specific to each perturbation. This provides an analytical framework for detailed and quantitative understanding of molecular mechanisms hidden in their heterogeneity. Lens-free digital in-line holography (LDIH) is a promising microscopic tool that overcomes several drawbacks (e.g., limited field of view) of traditional lens-based microscopy. Numerical reconstruction for hologram images from large-field-of-view LDIH is extremely time-consuming. Until now, there are no effective manual-design features to interpret the lateral and depth information from complex diffraction patterns in hologram images directly, which limits LDIH utility for point-of-care applications. Inherited from advantages of DL to learn generalized features automatically, we proposed a deep transfer learning (DTL)-based approach to process LDIH images without reconstruction in the context of cellular analysis. Specifically, using the raw holograms as input, the features extracted from a well-trained network were able to classify cell categories according to the number of cell-bounded microbeads, which performance was comparable with that of object images as input. Combined with the developed DTL approach, LDIH could be realized as a low-cost, portable tool for point-of-care diagnostics. In summary, this dissertation demonstrate that ML applied to cell imaging can successfully dissect subcellular heterogeneity and perform cell-based diagnosis. We expect that our study will be able to make significant contributions to data-driven cell biological research.

Capillary Morphogenesis Gene 2 protein (CMG2) is a type 1 transmembrane receptor known as the anthrax toxin receptor 2 (ANTXR2). While it is documented that the cell surface receptor CMG2 mediates anthrax toxin entry into the cell via endocytosis, the physiological role of CMG2 is not well understood. Others have suggested that CMG2 may have a role in mediating ECM homeostasis and angiogenesis. Additionally, both anthrax protective antigen (PA) and a furin protease-resistant mutant, PASSSR, inhibit corneal neovascularization in a mouse model, and interestingly PASSSR has a greater affinity to CMG2 receptor. PASSSRalso has a more potent antiangiogenic effect than wild-type PA. However, a mechanism for PASSSR inhibition of the putative CMG2 role in angiogenesis is not yet elucidated. The experimental results in this thesis provide evidence that CMG2 is the key receptor for regulating adhesion, migration, and actin dynamics in cells, and 200-pM PASSSR inhibits cell adhesion, migration, and actin localization at the cell leading edge. Furthermore, we observed that PASSSR remains bound to CMG2 under acidic conditions similar to the lysosome (pH 4). This observation suggests that the PASSSR-CMG2 complex remains intact following internalization and traffic to lysosomes, different from previous reports for PA, which likely results in CMG2 recycling. Together, these results suggest that following PASSSR treatment, CMG2 traffics to the lysosome for degradation; hence, we predict fewer CMG2 receptors are available at the cell surface to function in their native role in signaling angiogenic processes such as adhesion and chemotaxis towards vascular growth factors.

Epithelial wound healing is essential for maintaining the function and clarity of the cornea. Successful repair after injury involves the coordinated movements of cell sheets over the wounded region. While collective migration has been the focus of many studies, the effects that environmental changes have on this form of movement are poorly understood. In certain pathologies where the cornea exists in a chronic hypoxic state, wound healing is delayed. The goal of this work is to examine the changes in corneal structure in hypoxic corneas that may affect migration and to determine the effects that changes in basement membrane stiffness and focal adhesion turnover have on epithelial cell migration. We analyzed migration after injury in organ cultures and determined that hypoxic corneas exhibited alterations in leading edge morphology. Under hypoxia, fibronectin localization to the apical epithelium and anterior stroma was reduced. Investigators have suggested that alterations in basal lamina composition may increase the stiffness of the membrane. To examine the effect that increased stiffness has on collective migration we performed traction force microscopy. Using multi-layered corneal epithelial sheets, we developed a novel method to analyze the generation of cellular traction forces and the directionality of sheet movement on polyacrylamide gels. We determined that the leading edges of corneal epithelial sheets undergo contraction prior to migration. Alterations in stiffness affected the amount of force exerted by cells at the leading edge. On stiffer surfaces, individual cells within sheets exhibited greater movement compared to cells on softer substrates. To further assess sheet dynamics, we examined the activation of focal adhesion proteins in hypoxic corneas and in human corneal limbal epithelial (HCLE) cells seeded onto soft and rigid substrates. Wounded hypoxic corneas displayed alterations in the localization of the focal adhesion proteins paxillin and vinculin. In HCLE cells cultured on stiff substrates, there was an elevation in vinculin pY1065 phosphorylation after injury, a reduction in vinculin-positive focal adhesions, and decreased actin bundle thickness. Our results demonstrate that changes in membrane stiffness may affect cellular tractions and vinculin dynamics, possibly contributing to the delayed healing response associated with certain corneal pathologies.

A dairy-based whipped topping consisting of 22.0 percent milk fat, 7.5 percent milk solids-not-fat, 12.0 percent sucrose, 10.0 percent corn syrup solids, 0.60 percent gum arabic, 0.06 percent carrageenin, 0.19 percent polyoxyethylene sorbitan rnonostearate, 0.19 percent polyoxythylene sorbitan tristearate, and 0.12 percent sodium stearoyl-2-lactylate was developed that would withstand the rigors of frozen storage. The best products were obtained when the topping was whipped on a Creamery Package 3M-30 continuous type ice cream freezer. Toppings whipped in the laboratory at temperatures higher or lower than -2.2 to -1.0 centigrade were weak and slightly wet. After 18 days frozen storage the toppings whipped at -2.2 centigrade had the best body and texture characteristics. Refrigerated storage after 18 days frozen storage resulted in an enlargement of the air cells and after about 10 days a stale flavor was detected. Commercial application of the formulation was considered feasible.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第18267号 / 工博第3859号 / 新制||工||1592(附属図書館) / 31125 / 京都大学大学院工学研究科機械理工学専攻 / (主査)教授 富田 直秀, 教授 安達 泰治, 教授 井手 亜里 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(人間健康科学) / 甲第18196号 / 人健博第13号 / 新制||人健||1(附属図書館) / 31054 / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 齋藤 邦明, 教授 足立 壯一, 教授 長田 重一 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

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Gewebe sind nicht nur durch ihre biochemische Zusammensetzung definiert, sondern auch durch ihre individuellen mechanischen Eigenschaften. Inzwischen ist es weithin akzeptiert, dass Zellen ihre mechanische Umgebung spüren und darauf reagieren. Zum Beispiel werden Zellmigration und die Differenzierung von Stammzellen durch die Umgebungssteifigkeit beeinflusst. Um diese Effekte in vitro zu untersuchen, wurden viele Zellkulturstudien auf 2D Hydrogelsubstraten durchgeführt. Zusätzlich dazu steigt die Anzahl von Studien an, die hydrogelbasierte 3D-Scaffolds nutzen, um 2D Studien zu validieren und die experimentellen Bedingungen der Situation in vivo anzunähern. Jedoch erweist es sich weiterhin als schwierig den Effekt von Mechanik in 3D in vitro zu untersuchen, da in den gemeinhin genutzten 3D Hydrogelsystemen immer eine Kopplung zwischen Gelporosität und Steifigkeit besteht. Zusätzlich hängt die Konzentration der biologisch aktiven Bindungsstellen für Zellen oft ebenfalls von der Steifigkeit ab. Diese Arbeit präsentiert die Entwicklung und Optimierung neuer 3D Hydrogelkugel-Scaffolds, in denen die Steifigkeit von der Porosität schließlich entkoppelt wird. Mit Hydrogelkugeln als Scaffold-Bausteine ist es nun möglich 3D Scaffolds mit definierten mechanischen Eigenschaften und konstanter Porengröße zu generieren. Während der Methodenentwicklung wurden verschiedene Prinzipien und Kultivierungskammern konstruiert und überarbeitet, gefolgt von der theoretischen Betrachtung der Sauerstoffdiffusion, um die Eignung der gewählten Kammer hinsichtlich Zellvitalität und Zellwachstum zu überprüfen. Eine Kombination aus mehreren getesteten Filtern wurde ausgewählt um HydrogelkugelScaffolds erfolgreich in der ausgewählten Kammer zu generieren. Im Weiteren wurden verschiedene Hydrogelmaterialien untersucht hinsichtlich der erfolgreichen Produktion monodisperser Hydrogelkugeln und der Erzeugung stabiler Scaffolds. Hydrogelkugeln aus Polyacrylamid (PAAm) wurden als Scaffold-Bausteine ausgewählt um damit die Eignung des entwickelten Systems zu demonstrieren lebende Zellen zu mikroskopieren. Außerdem wurde das Überleben von Fibroblasten über vier Tage in unterschiedlich steifen HydrogelkugelScaffolds erfolgreich gezeigt. Weiterhin war es möglich erste Zellmigrationsexperimente durchzuführen. Dafür wurden sowohl einfache PAAm-Hydrogelkugeln als auch mit Adhäsionsmolekülen funktionalisierte Hydrogelkugeln genutzt, um unterschiedlich steife Schichten in einem Scaffold zu erzeugen. Dadurch war es möglich nicht nur Zellmigration anhand von Zelladhäsion in 3D Scaffolds mit Steifigkeitsgradienten zu beobachten, sondern auch Zellmigration ohne Zelladhäsion.:1 Introduction 1.1 Mechanics play a role in biology 1.2 3D cultures and scaffolds 1.3 3D hydrogel systems to study effects of mechanics 1.4 Decoupling stiffness and porosity in 3D scaffolds 2 Materials 3 Methods 3.1 Laser scanning microscopy and microscopy data processing 3.2 Atomic force microscopy (AFM) 3.3 Refractive index matching of PMMA beads 3.4 Regular PMMA bead scaffolds for developing analysis algorithm 3.5 Cell culture standards 3.6 Fluorescent labelling of ULGP agarose 3.7 Production of polydisperse ULGP agarose beads 3.8 Hydrogel bead production via microfluidics 3.9 PAAm bead functionalization 3.10 Real-time fluorescence and deformability cytometry (RT-fDC) 3.11 3D scaffolds made from hydrogel beads 3.12 Statistics 4 Results 4.1 Design of a suitable scaffold device 4.2 Theoretical oxygen supply in 3D culture system is sufficient for cell survival and proliferation 4.3 Further optimization of 3D scaffold device 4.3.1 PMMA beads can be arranged in stable scaffolds 4.3.2 Regular PMMA bead scaffolds can be achieved and analysed 4.3.3 PMMA bead scaffolds and agarose bead scaffolds act as combined filter to stack up hydrogel beads 4.4 PAAm hydrogel beads produced by microfluidics are suitable to create compliant 3D scaffolds 4.5 Reproducible, regular and stable 3D scaffolds made of hydrogel beads 4.6 NIH-3T3/GFP cell migration within 3D hydrogel bead scaffolds 5 Discussion and Concluding Remarks 6 Bibliography List of Figures List of Tables Eigenständigkeitserklärung Appendix A Appendix B FIJI macro for FFT analysis maxima Python script to determine regularity of PMMA bead scaffolds Excel macro to determine number of peaks for regularity analysis / Tissues are defined not only by their biochemical composition, but also by their distinct mechanical properties. It is now widely accepted that cells sense their mechanical environment and respond to it. For example, cell migration and stem cell differentiation is affected by stiffness. To study these effects in vitro, many cell culture studies have been performed on 2D hydrogel substrates. Additionally, the amount of 3D studies based on hydrogels as 3D scaffold is increasing to validate 2D in vitro studies and adjust experimental conditions closer to the situation in vivo. However, studying the effects of mechanics in vitro in 3D is still challenging as commonly used 3D hydrogel assays always link gel porosity with stiffness. Additionally, the concentration of biologically active adhesion sides often also depends on the stiffness. This work presents the development and optimization of novel 3D hydrogel bead scaffolds where the stiffness is finally decoupled from porosity. With hydrogel beads as scaffold building blocks it was possible to generate 3D scaffolds with defined mechanical properties and a constant pore size. During the method development, different culture devices were constructed and revised, followed by oxygen diffusion simulations to proof the suitability of the chosen device for cell survival and growth. A combination of different filter approaches was selected to generate hydrogel bead scaffolds in the culture device. Furthermore, different hydrogel materials were investigated regarding successful production of monodisperse beads and stable scaffold generation. Polyacrylamide (PAAm) hydrogel beads were chosen as scaffold building blocks to demonstrate live-cell imaging and successful cell survival over four days in differently compliant hydrogel bead scaffolds. Moreover, first cell migration experiments were performed by using plain PAAm hydrogel beads as well as PAAm hydrogel beads functionalized with adhesion molecules with differently stiff layers in one scaffold. Thereby fibroblast migration was observed not only in adhesion-dependent migration manner, but also in an adhesion-independent mode .:1 Introduction 1.1 Mechanics play a role in biology 1.2 3D cultures and scaffolds 1.3 3D hydrogel systems to study effects of mechanics 1.4 Decoupling stiffness and porosity in 3D scaffolds 2 Materials 3 Methods 3.1 Laser scanning microscopy and microscopy data processing 3.2 Atomic force microscopy (AFM) 3.3 Refractive index matching of PMMA beads 3.4 Regular PMMA bead scaffolds for developing analysis algorithm 3.5 Cell culture standards 3.6 Fluorescent labelling of ULGP agarose 3.7 Production of polydisperse ULGP agarose beads 3.8 Hydrogel bead production via microfluidics 3.9 PAAm bead functionalization 3.10 Real-time fluorescence and deformability cytometry (RT-fDC) 3.11 3D scaffolds made from hydrogel beads 3.12 Statistics 4 Results 4.1 Design of a suitable scaffold device 4.2 Theoretical oxygen supply in 3D culture system is sufficient for cell survival and proliferation 4.3 Further optimization of 3D scaffold device 4.3.1 PMMA beads can be arranged in stable scaffolds 4.3.2 Regular PMMA bead scaffolds can be achieved and analysed 4.3.3 PMMA bead scaffolds and agarose bead scaffolds act as combined filter to stack up hydrogel beads 4.4 PAAm hydrogel beads produced by microfluidics are suitable to create compliant 3D scaffolds 4.5 Reproducible, regular and stable 3D scaffolds made of hydrogel beads 4.6 NIH-3T3/GFP cell migration within 3D hydrogel bead scaffolds 5 Discussion and Concluding Remarks 6 Bibliography List of Figures List of Tables Eigenständigkeitserklärung Appendix A Appendix B FIJI macro for FFT analysis maxima Python script to determine regularity of PMMA bead scaffolds Excel macro to determine number of peaks for regularity analysis