Aktive Filament-Systeme, wie zum Beispiel das Zellskelett, sind Beispiele einer interessanten Klasse neuartiger Materialien, die eine wichtige Rolle in der belebten Natur spielen. Viele wichtige Prozesse in lebenden Zellen wie zum Beispiel die Zellbewegung oder Zellteilung basieren auf dem Zellskelett. Das Zellskelett besteht aus Protein-Filamenten, molekularen Motoren und einer großen Zahl weiterer Proteine, die an die Filamente binden und diese zu einem Netz verbinden können. Die Filamente selber sind semifexible Polymere, typischerweise einige Mikrometer lang und bestehen aus einigen hundert bis tausend Untereinheiten, typischerweise Mono- oder Dimeren. Die Filamente sind strukturell polar, d.h. sie haben eine definierte Richtung, ähnlich einer Ratsche. Diese Polarität begründet unterschiedliche Polymerisierungs- und Depolymerisierungs-Eigenschaften der beiden Filamentenden und legt außerdem die Bewegungsrichtung molekularer Motoren fest. Die Polymerisation von Filamenten sowie Krafterzeugung und Bewegung molekularer Motoren sind aktive Prozesse, die kontinuierlich chemische Energie benötigen. Das Zellskelett ist somit ein aktives Gel, das sich fern vom thermodynamischen Gleichgewicht befindet. In dieser Arbeit präsentieren wir Beschreibungen solcher aktiven Filament-Systeme und wenden sie auf Strukturen an, die eine ähnliche Geometrie wie zellulare Strukturen haben. Beispiele solcher zellularer Strukturen sind Spannungsfasern, kontraktile Ringe oder mitotische Spindeln. Spannungsfasern sind für die Zellbewegung essentiell; sie können kontrahieren und so die Zelle vorwärts bewegen. Die mitotische Spindel trennt Kopien der Erbsubstanz DNS vor der eigentlichen Zellteilung. Der kontraktile Ring schließlich trennt die Zelle am Ende der Zellteilung. In unserer Theorie konzentrieren wir uns auf den Einfluß der Polymerisierung und Depolymerisierung von Filamenten auf die Dynamik dieser Strukturen. Wir zeigen, dass der kontinuierliche Umschlag (d.h. fortwährende Polymerisierung und Depolymerisierung) von Filamenten unabdingbar ist für die kontraktion eines Rings mit konstanter Geschwindigkeit, so wie in Experimenten mit Hefezellen beobachtet. Mit Hilfe einer mikroskopisch motivierten Beschreibung zeigen wir, wie "filament treadmilling", also Filament Polymerisierung an einem Ende mit der gleichen Rate wie Depolymerisierung am anderen Ende, zur Spannung in Filament Bündeln und Ringen beitragen kann. Ein zentrales Ergebnis ist, dass die Depolymerisierung von Filamenten in Anwesenheit von filamentverbindenden Proteinen das Zusammenziehen dieser Bündel sogar in Abwesenheit molekulare Motoren herbeiführen kann. Ferner entwickeln wir eine generische Kontinuumsbeschreibung aktiver Filament-Systeme, die ausschließlich auf Symmetrien der Systeme beruht und von mikroskopischen Details unabhängig ist. Diese Theorie erlaubt uns eine komplementäre Sichtweise auf solche aktiven Filament-Systeme. Sie stellt ein wichtiges Werkzeug dar, um die physikalischen Mechanismen z.B. in Filamentbündeln aber auch bei der Bildung von Filamentringen im Zellkortex zu untersuchen. Schließlich entwickeln wir eine auf einem Kräftegleichgewicht basierende Beschreibung für bipolare Strukturen aktiver Filamente und wenden diese auf die mitotische Spindel an. Wir diskutieren Bedingungen für die Bildung und Stabilität von Spindeln. / Active filament systems such as the cell cytoskeleton represent an intriguing class of novel materials that play an important role in nature. The cytoskeleton for example provides the mechanical basis for many central processes in living cells, such as cell locomotion or cell division. It consists of protein filaments, molecular motors and a host of related proteins that can bind to and cross-link the filaments. The filaments themselves are semiflexible polymers that are typically several micrometers long and made of several hundreds to thousands of subunits. The filaments are structurally polar, i.e. they possess a directionality. This polarity causes the two distinct filament ends to exhibit different properties regarding polymerization and depolymerization and also defines the direction of movement of molecular motors. Filament polymerization as well as force generation and motion of molecular motors are active processes, that constantly use chemical energy. The cytoskeleton is thus an active gel, far from equilibrium. We present theories of such active filament systems and apply them to geometries reminiscent of structures in living cells such as stress fibers, contractile rings or mitotic spindles. Stress fibers are involved in cell locomotion and propel the cell forward, the mitotic spindle mechanically separates the duplicated sets of chromosomes prior to cell division and the contractile ring cleaves the cell during the final stages of cell division. In our theory, we focus in particular on the role of filament polymerization and depolymerization for the dynamics of these structures. Using a mean field description of active filament systems that is based on the microscopic processes of filaments and motors, we show how filament polymerization and depolymerization contribute to the tension in filament bundles and rings. We especially study filament treadmilling, an ubiquitous process in cells, in which one filament end grows at the same rate as the other one shrinks. A key result is that depolymerization of filaments in the presence of linking proteins can induce bundle contraction even in the absence of molecular motors. We extend this description and apply it to the mitotic spindle. Starting from force balance considerations we discuss conditions for spindle formation and stability. We find that motor binding to filament ends is essential for spindle formation. Furthermore we develop a generic continuum description that is based on symmetry considerations and independent of microscopic details. This theory allows us to present a complementary view on filament bundles, as well as to investigate physical mechanisms behind cell cortex dynamics and ring formation in the two dimensional geometry of a cylinder surface. Finally we present a phenomenological description for the dynamics of contractile rings that is based on the balance of forces generated by active processes in the ring with forces necessary to deform the cell. We find that filament turnover is essential for ring contraction with constant velocities such as observed in experiments with fission yeast.

INTRODUÇÃO: o padrão-ouro para o diagnóstico histológico dos tumores corticais adrenais (TCAs) e sua diferenciação entre adenomas e carcinomas é o sistema de Weiss, cuja aplicação é limitada pela baixa reprodutibilidade de alguns dos critérios que o compõe. Recentemente foi proposto e validado um algoritmo diagnóstico para os TCAs baseado na integridade do arcabouço de reticulina e da membrana basal. Os carcinomas adrenais são tumores raros e apresentam prognóstico reservado, mesmo nos pacientes com doença aparentemente localizada. Além do estadiamento e da extensão da ressecção cirúrgica, outros dados foram reportados na literatura como tendo importância prognóstica, tais como idade ao diagnóstico, padrão funcional, tamanho tumoral, extensão local do tumor primário e alguns achados histológicos e imuno-histoquímicos, com destaque à taxa mitótica e ao índice de Ki-67. O sistema de Weiss, embora permita o diagnóstico diferencial entre adenomas e carcinomas, não foi testado completamente como uma ferramenta para distinguir os carcinomas com boa evolução clínica daqueles com desfecho desfavorável. OBJETIVOS: o presente estudo teve como objetivo primário construir um nomograma para estimar o risco de metástases e recorrência local em portadores de carcinoma adrenal, a partir de dados clínico-patológicos. O objetivo secundário foi avaliar o desempenho do algoritmo da reticulina no diagnóstico diferencial entre adenomas e carcinomas do córtex adrenal. MÉTODOS: para a construção do nomograma, foram analisados dados clínico-patológicos de 129 portadores de carcinomas adrenais atendidos no Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo entre 1976 e 2010. A avaliação do desempenho do algoritmo da reticulina para o diagnóstico histológico dos TCAs foi feita a partir do exame de 89 lâminas (45 adenomas e 44 carcinomas adrenais), que foram classificadas de acordo com o sistema de Weiss e pelo algoritmo da reticulina, pelo mesmo patologista e de forma cega. RESULTADOS: utilizando a análise de regressão logística binária, foi proposto um escore prognóstico com cinco covariáveis: padrão funcional outro que não o hiperandrogenismo isolado, tamanho tumoral > 7,5 cm, tumor primário classificado como T3 ou T4, presença de invasão venosa microscópica e índice mitótico > 5/50 campos de grande aumento. O escore prognóstico estava calibrado de acordo com o teste de aderência de Hosmer-Lemeshow (P =0,9329) e apresentou excelente desempenho global (escore de Brier =0,0738). Finalmente, a capacidade discriminatória do modelo, determinada pela área sob a curva ROC (AROC), foi considerada quase perfeita (AROC, 0,9611; IC95%, 0,92676-0,99552). O modelo preditivo foi validado internamente com 200 reamostragens por bootstrap. A partir destes resultados, desenvolvemos um aplicativo para iOS7 para estimar o risco de metástases e recorrência local em portadores de carcinomas adrenais (ACCPS - Adrenal Cortical Carcinoma Prognostic Score). O aplicativo está disponível para download gratuito na App Store. Em relação à avaliação do desempenho do algoritmo da reticulina, o mesmo apresentou sensibilidade =84,1% e especificidade =95,6% para o diagnóstico de carcinoma adrenal. Não se observou diferença estatisticamente significante entre os diagnósticos obtidos pelo escore de Weiss e pelo algoritmo da reticulina (P =0,1797; Teste exato de McNemar). A concordância dos dois sistemas, avaliada pelo coeficiente k, foi considerada substancial (k =0,7975; IC95%, 0,6728-0,9221). CONCLUSÕES: propusemos e validamos internamente um escore prognóstico baseado em informações clínicopatológicas que estão facilmente disponíveis ao clínico. O modelo proposto está disponível para uso como um aplicativo gratuito para iOS7 e é capaz de estimar o risco de desfechos desfavoráveis nos pacientes com carcinoma cortical adrenal. O escore pode ter grande utilidade na determinação do tipo e frequência do acompanhamento clínico-radiológico e na decisão de um tratamento profilático, após a retirada cirúrgica completa do carcinoma adrenal. O algoritmo da reticulina teve desempenho equivalente ao sistema de Weiss para o diagnóstico histológico dos tumores corticais adrenais, mas oferece a vantagem de ter aplicação mais fácil / INTRODUCTION: The gold standard for the histological diagnosis of adrenal cortical tumors (ACTs) and for the differentiation between adenomas and carcinomas is the Weiss system, whose application is limited by poor reproducibility of some of its criteria. Recently, a diagnostic algorithm for ACT diagnosis based on the integrity of the reticulin network and the basal membrane has been proposed and validated. Adrenal carcinomas are rare tumors and have a poor prognosis, even in patients with apparently localized disease. Besides tumor staging and extent of surgical resection, other data have been reported in the literature as having prognostic importance, such as age at diagnosis, the functional pattern, tumor size, local extension of the primary tumor and some histological and immunohistochemical findings, such as the mitotic rate and the Ki-67 index. The Weiss system, while allowing the differential diagnosis between adrenal cortical adenomas and carcinomas, has not been fully tested as a tool for distinguishing carcinomas with favorable clinical outcome from those with unfavorable outcome. OBJECTIVES: The primary objective of this study was to construct a nomogram for estimating the risk of metastasis and local recurrence in patients with adrenal cortical carcinoma, based on clinical and pathological data. The secondary objective was to evaluate the performance of the reticulina algorithm in the differential diagnosis between adenomas and carcinomas of the adrenal cortex. METHODS: For the construction of the nomogram, clinical and pathological data from 129 patients with adrenal cortical carcinomas treated at the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo between 1976 and 2010 were analyzed. The evaluation of the performance of the reticulin algorithm for the histological diagnosis of ACTs was made from the examination of 89 slides (45 adenomas and 44 adrenal carcinomas), which were classified according to the Weiss system and the reticulin algorithm by the same pathologist in a blinded fashion. RESULTS: Using binary logistic regression analysis, we proposed a prognostic score with five covariates: a functional pattern other than isolated hyperandrogenism, a tumor size > 7.5 cm, a primary tumor classified as T3 or T4, the presence of microscopic venous invasion and a mitotic index > 5/50 high-power fields. The prognostic score was calibrated according to the Hosmer-Lemeshow goodness-of-fit test (P =0.9329) and showed excellent overall performance (Brier score =0.0738). Finally, the discriminatory ability of the model, as determined by the area under the ROC curve (AROC), was considered near-perfect (AROC, 0.9611; 95%CI, 0.92676 to 0.99552). The predictive model was internally validated with 200 bootstrap resamples. Based on these results, we develop an app for iOS7 for estimating the risk of metastasis and local recurrence in patients with adrenal carcinomas (ACCPS - Adrenal Cortical Carcinoma Prognostic Score). The app is available for free download on the App Store. Regarding the assessment of the performance of the reticulin algorithm, it had a sensitivity of 84.1% and specificity of 95.6% for the diagnosis of adrenal cortical carcinoma. No statistically significant difference between the diagnoses obtained by Weiss score and the reticulin algorithm was observed (P =0.1797, McNemar\'s exact test). The concordance between the two systems, assessed by the k coefficient, was considered substantial (k =0.7975; 95%CI, 0.6728 to 0.9221). CONCLUSIONS: we proposed and internally validated a prognostic score based on clinical and pathological information that are readily available to the attending physician. The proposed model is available as a free app for iOS7 and can estimate the risk of adverse outcomes in patients with adrenal cortical carcinoma. The score may be useful in determining the type and frequency of clinical and radiological evaluation and the decision of adjuvant treatment after complete surgical removal of the adrenal carcinoma. The reticulin algorithm was equivalent to the Weiss system for the histological diagnosis of adrenal cortical tumors, but offers the advantage of an easier application

Made available in DSpace on 2014-07-29T15:16:29Z (GMT). No. of bitstreams: 1 MANUELA da rocha.pdf: 1137336 bytes, checksum: 8f429c6f06f0af7ebb98f1fc1b0badbe (MD5) Previous issue date: 2010-02-24 / Chemotherapy agents are increasingly being utilized due to their promising effects in cancer treatment. The desired actions of these drugs are obtained at the cost of frequent and severe side effects. It is essential that drugs utilized for cancer treatment are tested in relation to not only their cytotoxic, but also their clastogenic and genotoxic potentials to establish their clinical risk. Thus, there is increased interest in searching for new metallic compounds presenting antitumor activity with therapeutic potential and reduced side effects, such as those containing platinum (Pt), iron (Fe), titanium (Ti), rhodium (Rh), gold (Au) and ruthenium (Ru). In this work, the cytotoxic, clastogenic and genotoxic properties of cis-tetraamine(oxalato)ruthenium(III) dithionite, cis-[Ru(C2O4)(NH3)4]2(S2O6), were evaluated in peripheral human blood lymphocytes in vitro. Mitotic index (MI), chromosomal aberrations (CA) and DNA damage were analyzed by comet assay. The MI values of human peripheral blood lymphocyte cultures treated with 0.0075, 0.075, 0.75 and 7.5 μM cis-[Ru(C2O4)(NH3)4]2(S2O6) were 6.1, 3.9, 3.2 and 0.2%, respectively. The lowest concentration 0.0075 did not show cytotoxic activity compared to negative control. The CA values obtained for the 0.0075, 0.075 and 0.75 μM concentrations presenting low frequency (1.5, 1.6 and 2.3%, respectively) not expressing clastogenic activity compared to negative control and only at the highest concentration 7.5 μM, showed clastogenic activity, predominantly chromatid breaks and gaps. The data obtained by the comet assay, using cis-[Ru(C2O4)(NH3)4]2(S2O6), suggest that this compound does not show genotoxic activity at concentrations lower than 0.0075 μM. The results of these studies show that cis-[Ru(C2O4)(NH3)4]2(S2O6) has no potential cytotoxic, clastogenic and genotoxic in vitro at concentrations less than or equal to 0.0075 μM. / Estudos de citoxicidade e genotoxicidade têm sido realizados para o desenvolvimento de novos agentes anticâncer com maior seletividade e menores efeitos colaterais de agentes antitumorais convencionais. Neste trabalho, foram avaliadas as propriedades citotóxicas, clastogênicas e genotóxicas do composto ditionato de cis-tetraamino(oxalato)rutênio(III), {cis-[Ru(C2O4)(NH3)4]2(S2O6)}, em cultura de linfócitos de sangue periférico humano in vitro. Foram analisados os parâmetros índice mitótico (IM), aberrações cromossômicas (ACs) e danos induzidos no DNA detectados pelo ensaio cometa. O IM de cultura de linfócitos de sangue periférico humano tratados com 0,0075; 0,075; 0,75 e 7,5μM de {cis-[Ru(C2O4)(NH3)4]2(S2O6)} foram 6,1; 3,9; 3,2 e 0,2%, respectivamente. A menor concentração 0,0075 não apresentou atividade citotóxica comparada ao controle negativo. As ACs derivadas das concentrações 0,0075; 0,075 e 0,75, apresentaram frequência de 1,5; 1,6 e 2,3 %, respectivamente, não manifestando atividade clastogênica comparadas ao contole negativo e somente na concentração de 7,5μM, demonstrou atividade clastogênica, predominando quebras e falhas cromatídicas. Os dados obtidos por meio do ensaio cometa, utilizando {cis-[Ru(C2O4)(NH3)4]2(S2O6)}, sugerem que este composto não apresenta atividade genotóxica em concentrações menores que 0,0075 μM. Os resultados desses estudos demonstram que {cis-[Ru(C2O4)(NH3)4]2(S2O6)} não possui potencial citotóxico, clastogênico e genotóxica in vitro em concentrações menor ou igual a 0,0075 μM.

No description available.

Aktive Filament-Systeme, wie zum Beispiel das Zellskelett, sind Beispiele einer interessanten Klasse neuartiger Materialien, die eine wichtige Rolle in der belebten Natur spielen. Viele wichtige Prozesse in lebenden Zellen wie zum Beispiel die Zellbewegung oder Zellteilung basieren auf dem Zellskelett. Das Zellskelett besteht aus Protein-Filamenten, molekularen Motoren und einer großen Zahl weiterer Proteine, die an die Filamente binden und diese zu einem Netz verbinden können. Die Filamente selber sind semifexible Polymere, typischerweise einige Mikrometer lang und bestehen aus einigen hundert bis tausend Untereinheiten, typischerweise Mono- oder Dimeren. Die Filamente sind strukturell polar, d.h. sie haben eine definierte Richtung, ähnlich einer Ratsche. Diese Polarität begründet unterschiedliche Polymerisierungs- und Depolymerisierungs-Eigenschaften der beiden Filamentenden und legt außerdem die Bewegungsrichtung molekularer Motoren fest. Die Polymerisation von Filamenten sowie Krafterzeugung und Bewegung molekularer Motoren sind aktive Prozesse, die kontinuierlich chemische Energie benötigen. Das Zellskelett ist somit ein aktives Gel, das sich fern vom thermodynamischen Gleichgewicht befindet. In dieser Arbeit präsentieren wir Beschreibungen solcher aktiven Filament-Systeme und wenden sie auf Strukturen an, die eine ähnliche Geometrie wie zellulare Strukturen haben. Beispiele solcher zellularer Strukturen sind Spannungsfasern, kontraktile Ringe oder mitotische Spindeln. Spannungsfasern sind für die Zellbewegung essentiell; sie können kontrahieren und so die Zelle vorwärts bewegen. Die mitotische Spindel trennt Kopien der Erbsubstanz DNS vor der eigentlichen Zellteilung. Der kontraktile Ring schließlich trennt die Zelle am Ende der Zellteilung. In unserer Theorie konzentrieren wir uns auf den Einfluß der Polymerisierung und Depolymerisierung von Filamenten auf die Dynamik dieser Strukturen. Wir zeigen, dass der kontinuierliche Umschlag (d.h. fortwährende Polymerisierung und Depolymerisierung) von Filamenten unabdingbar ist für die kontraktion eines Rings mit konstanter Geschwindigkeit, so wie in Experimenten mit Hefezellen beobachtet. Mit Hilfe einer mikroskopisch motivierten Beschreibung zeigen wir, wie "filament treadmilling", also Filament Polymerisierung an einem Ende mit der gleichen Rate wie Depolymerisierung am anderen Ende, zur Spannung in Filament Bündeln und Ringen beitragen kann. Ein zentrales Ergebnis ist, dass die Depolymerisierung von Filamenten in Anwesenheit von filamentverbindenden Proteinen das Zusammenziehen dieser Bündel sogar in Abwesenheit molekulare Motoren herbeiführen kann. Ferner entwickeln wir eine generische Kontinuumsbeschreibung aktiver Filament-Systeme, die ausschließlich auf Symmetrien der Systeme beruht und von mikroskopischen Details unabhängig ist. Diese Theorie erlaubt uns eine komplementäre Sichtweise auf solche aktiven Filament-Systeme. Sie stellt ein wichtiges Werkzeug dar, um die physikalischen Mechanismen z.B. in Filamentbündeln aber auch bei der Bildung von Filamentringen im Zellkortex zu untersuchen. Schließlich entwickeln wir eine auf einem Kräftegleichgewicht basierende Beschreibung für bipolare Strukturen aktiver Filamente und wenden diese auf die mitotische Spindel an. Wir diskutieren Bedingungen für die Bildung und Stabilität von Spindeln. / Active filament systems such as the cell cytoskeleton represent an intriguing class of novel materials that play an important role in nature. The cytoskeleton for example provides the mechanical basis for many central processes in living cells, such as cell locomotion or cell division. It consists of protein filaments, molecular motors and a host of related proteins that can bind to and cross-link the filaments. The filaments themselves are semiflexible polymers that are typically several micrometers long and made of several hundreds to thousands of subunits. The filaments are structurally polar, i.e. they possess a directionality. This polarity causes the two distinct filament ends to exhibit different properties regarding polymerization and depolymerization and also defines the direction of movement of molecular motors. Filament polymerization as well as force generation and motion of molecular motors are active processes, that constantly use chemical energy. The cytoskeleton is thus an active gel, far from equilibrium. We present theories of such active filament systems and apply them to geometries reminiscent of structures in living cells such as stress fibers, contractile rings or mitotic spindles. Stress fibers are involved in cell locomotion and propel the cell forward, the mitotic spindle mechanically separates the duplicated sets of chromosomes prior to cell division and the contractile ring cleaves the cell during the final stages of cell division. In our theory, we focus in particular on the role of filament polymerization and depolymerization for the dynamics of these structures. Using a mean field description of active filament systems that is based on the microscopic processes of filaments and motors, we show how filament polymerization and depolymerization contribute to the tension in filament bundles and rings. We especially study filament treadmilling, an ubiquitous process in cells, in which one filament end grows at the same rate as the other one shrinks. A key result is that depolymerization of filaments in the presence of linking proteins can induce bundle contraction even in the absence of molecular motors. We extend this description and apply it to the mitotic spindle. Starting from force balance considerations we discuss conditions for spindle formation and stability. We find that motor binding to filament ends is essential for spindle formation. Furthermore we develop a generic continuum description that is based on symmetry considerations and independent of microscopic details. This theory allows us to present a complementary view on filament bundles, as well as to investigate physical mechanisms behind cell cortex dynamics and ring formation in the two dimensional geometry of a cylinder surface. Finally we present a phenomenological description for the dynamics of contractile rings that is based on the balance of forces generated by active processes in the ring with forces necessary to deform the cell. We find that filament turnover is essential for ring contraction with constant velocities such as observed in experiments with fission yeast.

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.

Indiana University-Purdue University Indianapolis (IUPUI) / Fanconi anemia (FA) is a heterogenous genetic syndrome characterized by progressive bone marrow failure, aneuploidy, and cancer predisposition. It is incompletely understood why FA-deficient cells develop gross aneuploidy leading to cancer. Since the mitotic spindle assembly checkpoint (SAC) prevents aneuploidy by ensuring proper chromosome segregation during mitosis, we hypothesized that the FA signaling network regulates the mitotic SAC. A genome-wide RNAi screen and studies in primary cells were performed to systematically evaluate SAC activity in FA-deficient cells. In these experiments, taxol was used to activate the mitotic SAC. Following taxol challenge, negative control siRNA-transfected cells appropriately arrested at the SAC. However, knockdown of fourteen FA gene products resulted in a weakened SAC, evidenced by increased formation of multinucleated, aneuploid cells. The screen was independently validated utilizing primary fibroblasts from patients with characterized mutations in twelve different FA genes. When treated with taxol, fibroblasts from healthy controls arrested at the mitotic SAC, while all FA patient fibroblasts tested exhibited weakened SAC activity, evidenced by increased multinucleated cells. Rescue of the SAC was achieved in FANCA patient fibroblasts by genetic correction. Importantly, SAC activity of FANCA was confirmed in primary CD34+ hematopoietic cells. Furthermore, analysis of untreated primary fibroblasts from FA patients revealed micronuclei and multinuclei, reflecting abnormal chromosome segregation. Next, microscopy-based studies revealed that many FA proteins localize to the mitotic spindle and centrosomes, and that disruption of the FA pathway results in supernumerary centrosomes, establishing a role for the FA signaling network in centrosome maintenance. A mass spectrometry-based screen quantifying the proteome and phospho-proteome was performed to identify candidates which may functionally interact with FANCA in the regulation of mitosis. Finally, video microscopy-based experiments were performed to further characterize the mitotic defects in FANCA-deficient cells, confirming weakened SAC activity in FANCA-deficient cells and revealing accelerated mitosis and abnormal spindle orientation in the absence of FANCA. These findings conclusively demonstrate that the FA signaling network regulates the mitotic SAC, providing a mechanistic explanation for the development of aneuploidy and cancer in FA patients. Thus, our study establishes a novel role for the FA signaling network as a guardian of genomic integrity.