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Indiana University-Purdue University Indianapolis (IUPUI) / Meningioma represent more than one-third of all primary central nervous system (CNS) tumors, and it can be classified into three grades according to WHO (World Health Organization) in terms of clinical aggressiveness and risk of recurrence. A key component of meningioma grades is the mitotic count, which is defined as quantifying the number of cells in the process of dividing (i.e., undergoing mitosis) at a specific point in time. Currently, mitosis counting is done manually by a pathologist looking at 10 consecutive high-power fields (HPF) on a glass slide under a microscope, which is an extremely laborious and time-consuming process. The goal of this thesis is to investigate the use of computerized methods to automate the detection of mitotic nuclei with limited labeled data. We built computational methods to detect and quantify the histological features of mitotic cells on a whole slides image which mimic the exact process of pathologist workflow. Since we do not have enough training data from meningioma slide, we learned the mitotic cell features through public available breast cancer datasets, and predicted on meingioma slide for accuracy. We use either handcrafted features that capture certain morphological, statistical, or textural attributes of mitoses or features learned with convolutional neural networks (CNN). Hand crafted features are inspired by the domain knowledge, while the data-driven VGG16 models tend to be domain agnostic and attempt to learn additional feature bases that cannot be represented through any of the handcrafted features. Our work on detection of mitotic cells shows 100% recall , 9% precision and 0.17 F1 score. The detection using VGG16 performs with 71% recall, 73% precision, and 0.77 F1 score. Finally, this research of automated image analysis could drastically increase diagnostic efficiency and reduce inter-observer variability and errors in pathology diagnosis, which would allow fewer pathologists to serve more patients while maintaining diagnostic accuracy and precision. And all these methodologies will increasingly transform practice of pathology, allowing it to mature toward a quantitative science.

In cell culture, entry into mitosis of many adherent mammalian cells is accompanied by substantial changes in cellular architecture. Flat, spread-out interphase cells detach from the extracellular matrix and become more spherical. These changes in cell shape are mediated by rearrangements in the actin cytoskeleton, a dynamic network of actin filaments that are organized by actin-binding proteins. Filamin A (FLNa) is a 280 kD actin-binding protein that crosslinks actin filaments into parallel bundles or three-dimensional orthogonal networks. We previously identified FLNa as an in vitro substrate of cyclin-dependent kinase 1 (Cdk1), a kinase that regulates entry into mitosis, and hypothesized that Cdk1 phosphorylation of FLNa regulates mitotic actin remodelling. Using mass spectrometry and a p-FLNa antibody, we show that FLNa is phosphorylated in vivo in HeLa cells on multiple Cdk1 sites, including serines 1084, 1459 and 1533. All three sites match the phosphorylation consensus sequence of Cdk1. We further show that p-FLNa is almost fully dephosphorylated by anaphase, consistent with it being a cell cycle-regulated substrate. Using a phospho-specific antibody, we find that p-FLNa has decreased cortical actin localization compared to total FLNa in mitotic cells. To investigate the functional role of mitotic FLNa phosphorylation, we mutated serines 1084, 1459 and 1533 to nonphosphorylatable alanine and expressed this FLNa mutant (FLNa-S1084A, S1459A, S1533A, referred to as “FLNa-AAA GFP”) in FLNa-deficient human M2 melanoma cells. FLNa-AAA GFP-expressing cells have enhanced FLNa-AAA GFP localization at sites of contact between daughter cells and this correlates with defects in cell division and impaired cell migration. Therefore, mitotic delocalization of cortical FLNa is critical for successful cell division and interphase cell behaviour.

yes / Coinciding with advances in whole-slide imaging scanners, it is become essential to automate the conventional image-processing techniques to assist pathologists with some tasks such as mitotic-cells detection. In histopathological images analysing, the mitotic-cells counting is a significant biomarker in the prognosis of the breast cancer grade and its aggressiveness. However, counting task of mitotic-cells is tiresome, tedious and time-consuming due to difficulty distinguishing between mitotic cells and normal cells. To tackle this challenge, several deep learning-based approaches of Computer-Aided Diagnosis (CAD) have been lately advanced to perform counting task of mitotic-cells in the histopathological images. Such CAD systems achieve outstanding performance, hence histopathologists can utilise them as a second-opinion system. However, improvement of CAD systems is an important with the progress of deep learning networks architectures. In this work, we investigate deep YOLO (You Only Look Once) v2 network for mitotic-cells detection on ICPR (International Conference on Pattern Recognition) 2012 dataset of breast cancer histopathology. The obtained results showed that proposed architecture achieves good result of 0.839 F1-measure.

During mitosis, adherent animal cells undergo a drastic shape change, from essentially flat to round, in a process known as mitotic cell rounding (MCR). The aim of this thesis was to critically examine the physical and biological basis of MCR. The experimental part of this thesis employed a combined optical microscope-atomic force microscope (AFM) setup in conjunction with flat tipless cantilevers to analyze cell mechanics, shape and volume. To this end, two AFM assays were developed: the constant force assay (CFA), which applies constant force to cells and measures the resultant height, and the constant height assay (CHA), which confines cell height and measures the resultant force. These assays were deployed to analyze the shape and mechanical properties of single cells trans-mitosis. The CFA results showed that cells progressing through mitosis could increase their height against forces as high as 50 nN, and that higher forces can delay mitosis in HeLa cells. The CHA results showed that mitotic cells confined to ~50% of their normal height can generate forces around 50-100 nN without disturbing mitotic progression. Such forces represent intracellular pressures of at least 200 Pascals and cell surface tensions of around 10 nN/µm. Using the CHA to compare mitotic cell rounding with induced cell rounding, it was observed that the intracellular pressure of mitotic cells is at least 3-fold higher than rounded interphase cells. To investigate the molecular basis of the mechanical changes inherent in mitotic cell rounding, inhibitors and toxins were used to pharmacologically dissect the role of candidate cellular processes. These results implicated the actomyosin cortex and osmolyte transporters, the most prominent of which is the Na+/H+ exchanger, in the maintenance of mechanical properties and intracellular hydrostatic pressure. Observations on blebbing cells under the cantilever supported the idea that the actomyosin cortex is required to sustain hydrostatic pressure and direct this pressure into cell shape changes. To gain further insight into the relationship between actomyosin activity and intracellular pressure, dynamic perturbation experiments were conducted. To this end, the CHA was used to evaluate the pressure and volume of mitotic cells before, during and after dynamic perturbations that included tonic shocks, influx of specific inhibitors, and exposure to pore-forming toxins. When osmotic pressure gradients were depleted, pressure and volume decreased. When the actomyosin cytoskeleton was abolished, cell volume increased while rounding pressure decreased. Conversely, stimulation of actomyosin cortex contraction triggered an increase in rounding pressure and a decrease in volume. Taken together, the dynamic perturbation results demonstrated that the actomyosin cortex contracts against an opposing intracellular pressure and that this relationship sets the surface tension, pressure and volume of the cell. The discussion section of this thesis provides a comprehensive overview of the physical basis of MCR by amalgamating the experimental results of this thesis with the literature. Additionally, the biochemal signaling pathways and proteins that drive MCR are collated and discussed. An exhaustive and unprecedented synthesis of the literature on cell rounding (approx. 750 papers as pubmed search hits on “cell rounding”, April 2012) reveals that the spread-to-round transition can be thought of in terms of a surface tension versus adhesion paradigm, and that cell rounding can be physically classified into four main modes, of which one is an MCR-like category characterized by increased actomyosin cortex tension and diminution of focal adhesions. The biochemical pathways and signaling patterns that correspond with these four rounding modes are catalogued and expounded upon in the context of the relevant physiology. This analysis reveals cell rounding as a pertinent topic that can be leveraged to yield insight into core principles of cell biophysics and tissue organization. It furthermore highlights MCR as a model problem to understand the adhesion versus cell surface tension paradigm in cells and its fundamentality to cell shape, mechanics and physiology.

During mitosis, adherent animal cells undergo a drastic shape change, from essentially flat to round, in a process known as mitotic cell rounding (MCR). The aim of this thesis was to critically examine the physical and biological basis of MCR. The experimental part of this thesis employed a combined optical microscope-atomic force microscope (AFM) setup in conjunction with flat tipless cantilevers to analyze cell mechanics, shape and volume. To this end, two AFM assays were developed: the constant force assay (CFA), which applies constant force to cells and measures the resultant height, and the constant height assay (CHA), which confines cell height and measures the resultant force. These assays were deployed to analyze the shape and mechanical properties of single cells trans-mitosis. The CFA results showed that cells progressing through mitosis could increase their height against forces as high as 50 nN, and that higher forces can delay mitosis in HeLa cells. The CHA results showed that mitotic cells confined to ~50% of their normal height can generate forces around 50-100 nN without disturbing mitotic progression. Such forces represent intracellular pressures of at least 200 Pascals and cell surface tensions of around 10 nN/µm. Using the CHA to compare mitotic cell rounding with induced cell rounding, it was observed that the intracellular pressure of mitotic cells is at least 3-fold higher than rounded interphase cells. To investigate the molecular basis of the mechanical changes inherent in mitotic cell rounding, inhibitors and toxins were used to pharmacologically dissect the role of candidate cellular processes. These results implicated the actomyosin cortex and osmolyte transporters, the most prominent of which is the Na+/H+ exchanger, in the maintenance of mechanical properties and intracellular hydrostatic pressure. Observations on blebbing cells under the cantilever supported the idea that the actomyosin cortex is required to sustain hydrostatic pressure and direct this pressure into cell shape changes. To gain further insight into the relationship between actomyosin activity and intracellular pressure, dynamic perturbation experiments were conducted. To this end, the CHA was used to evaluate the pressure and volume of mitotic cells before, during and after dynamic perturbations that included tonic shocks, influx of specific inhibitors, and exposure to pore-forming toxins. When osmotic pressure gradients were depleted, pressure and volume decreased. When the actomyosin cytoskeleton was abolished, cell volume increased while rounding pressure decreased. Conversely, stimulation of actomyosin cortex contraction triggered an increase in rounding pressure and a decrease in volume. Taken together, the dynamic perturbation results demonstrated that the actomyosin cortex contracts against an opposing intracellular pressure and that this relationship sets the surface tension, pressure and volume of the cell. The discussion section of this thesis provides a comprehensive overview of the physical basis of MCR by amalgamating the experimental results of this thesis with the literature. Additionally, the biochemal signaling pathways and proteins that drive MCR are collated and discussed. An exhaustive and unprecedented synthesis of the literature on cell rounding (approx. 750 papers as pubmed search hits on “cell rounding”, April 2012) reveals that the spread-to-round transition can be thought of in terms of a surface tension versus adhesion paradigm, and that cell rounding can be physically classified into four main modes, of which one is an MCR-like category characterized by increased actomyosin cortex tension and diminution of focal adhesions. The biochemical pathways and signaling patterns that correspond with these four rounding modes are catalogued and expounded upon in the context of the relevant physiology. This analysis reveals cell rounding as a pertinent topic that can be leveraged to yield insight into core principles of cell biophysics and tissue organization. It furthermore highlights MCR as a model problem to understand the adhesion versus cell surface tension paradigm in cells and its fundamentality to cell shape, mechanics and physiology.