Dissecting the Mechanical Control of Mitotic Entry Using a Cell Confinement Setup.

Proliferating cells need to cope with extensive cytoskeletal and nuclear remodeling as they prepare to divide. These events are tightly regulated by the nuclear translocation of the cyclin B1-CDK1 complex, that is partly dependent on nuclear tension. Standard experimental approaches do not allow the manipulation of forces acting on cells in a time-resolved manner. Here, we describe a protocol that enables dynamic mechanical manipulation of single cells with high spatial and temporal resolution and its application in the context of cell division. In addition, we also outline a method for the manipulation of substrate stiffness using polyacrylamide hydrogels. Finally, we describe a static cell confinement setup, which can be used to study the impact of prolonged mechanical stimulation in populations of cells. Key features • Protocol for microfabrication of confinement devices. • Single-cell dynamic confinement coupled with high-resolution microscopy. • Static cell confinement protocol that can be combined with super-resolution STED microscopy. • Analysis of the mechanical control of mitotic entry in a time-resolved manner.

Nuclear tension controls mitotic entry by regulating cyclin B1 nuclear translocation.

As cells prepare to divide, they must ensure that enough space is available to assemble the mitotic machinery without perturbing tissue homeostasis. To do so, cells undergo a series of biochemical reactions regulated by cyclin B1-CDK1 that trigger cytoskeletal reorganization and ensure the coordination of cytoplasmic and nuclear events. Along with the biochemical events that control mitotic entry, mechanical forces have recently emerged as important players in cell-cycle regulation. However, the exact link between mechanical forces and the biochemical pathways that control mitotic progression remains unknown. Here, we identify a tension-dependent signal on the nucleus that sets the time for nuclear envelope permeabilization (NEP) and mitotic entry. This signal relies on actomyosin contractility, which unfolds the nucleus during the G2-M transition, activating the stretch-sensitive cPLA2 on the nuclear envelope and regulating the nuclear translocation of cyclin B1. Our data demonstrate how nuclear tension during the G2-M transition contributes to timely and efficient mitotic spindle assembly and prevents chromosomal instability.

High-Resolution Analysis of Centrosome Behavior During Mitosis.

Cell division requires a dynamic reorganization of cytoskeletal and nuclear components. One essential step is the separation of centrosomes, which allows the assembly of a microtubule-based mitotic spindle. This has to be spatially and temporally coordinated with other events such as adhesion complex disengagement, assembly of an actin-rich cell cortex and nuclear envelope breakdown (NEB), to ensure chromosome segregation fidelity. Previous methodologies often focused on a single event and failed to provide an integrated view of the process. In this chapter, we describe a method to study mitosis with high resolution, by analyzing the dynamic interplay between centrosomes, nucleus, and cell membrane, using a combination of live-cell imaging and micromanipulation with custom-designed computational tools.

Nucleus-Cytoskeleton Crosstalk During Mitotic Entry.

In preparation for mitosis, cells undergo extensive reorganization of the cytoskeleton and nucleus, so that chromosomes can be efficiently segregated into two daughter cells. Coordination of these cytoskeletal and nuclear events occurs through biochemical regulatory pathways, orchestrated by Cyclin-CDK activity. However, recent studies provide evidence that physical forces are also involved in the early steps of spindle assembly. Here, we will review how the crosstalk of physical forces and biochemical signals coordinates nuclear and cytoplasmic events during the G2-M transition, to ensure efficient spindle assembly and faithful chromosome segregation.

Centrosome-nuclear axis repositioning drives the assembly of a bipolar spindle scaffold to ensure mitotic fidelity.

During the initial stages of cell division, the cytoskeleton is extensively reorganized so that a bipolar mitotic spindle can be correctly assembled. This process occurs through the action of molecular motors, cytoskeletal networks, and the nucleus. How the combined activity of these different components is spatiotemporally regulated to ensure efficient spindle assembly remains unclear. To investigate how cell shape, cytoskeletal organization, and molecular motors cross-talk to regulate initial spindle assembly, we use a combination of micropatterning with high-resolution imaging and 3D cellular reconstruction. We show that during prophase, centrosomes and nucleus reorient so that centrosomes are positioned on the shortest nuclear axis at nuclear envelope (NE) breakdown. We also find that this orientation depends on a combination of centrosome movement controlled by Arp2/3-mediated regulation of microtubule dynamics and Dynein-generated forces on the NE that regulate nuclear reorientation. Finally, we observe this centrosome configuration favors the establishment of an initial bipolar spindle scaffold, facilitating chromosome capture and accurate segregation, without compromising division plane orientation.

Southwestern Oncology Group pretreatment risk criteria as predictive or prognostic factors in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a clonal hematological malignant condition and the implications of pretreatment risk criteria as predictive or prognostic factors are constantly under evaluation. With this study, the authors' intent was to characterize AML patients and to evaluate the clinical outcome associated with Southwestern Oncology Group (SWOG) coding pretreatment risk criteria/cytogenetic score. Between 2002 and 2010, 225 patients were diagnosed with AML at the Portuguese Institute of Oncology (Porto, Portugal). From this patient group, 128 patients aged <65 years were selected. The patients were treated using a combination of cytarabine and anthracycline, with the addition of cyclosporine when bone marrow dysplasia was observed. A median survival of 24 months was observed in this group. The patients were divided in subgroups according to the SWOG pretreatment risk criteria. We observed a statistically significant association of non-favorable SWOG coding with female gender [P=0.025; risk ratio (RR)=3.632, 95% confidence interval (CI): 1.113-11.852], indication for allogeneic bone marrow transplantation (P=0.023, RR=1.317, 95% CI: 1.184-1.465), complete response achievement (P=0.013, RR=1.385, 95% CI: 11.232-1.556) and relapse (P=0.048, RR=3.181, 95% CI: 10.966-10.478). Furthermore, SWOG pretreatment risk criteria also significantly affected global overall survival (OS; P=0.003) and OS at 5 years (P=0.001). A multivariate Cox regression analysis supported response to induction therapy (3-year OS: P=0.011, RR=0.385, 95% CI: 10.184-0.806; 5-year OS: P=0.012, RR=0.388, 95% CI: 10.597-1.994), consolidation (3-year OS: P=0.005, RR=0.328, 95% CI: 0.150-0.720; 5-year OS: P=0.002, RR=0.308, 95% CI: 0.144-0.657) and the diagnosis of therapy-related aml (3-year OS: P=0.016, RR=2.756, 95% CI: 0.486-1.281; 5-year OS: P=0.031, RR=2.369, 95% CI: 1.081-5.189) as prognostic factors, but this was not confirmed for SWOG pretreatment risk criteria. Therefore, we concluded that the reproducibility of the application of the SWOG pretreatment risk criteria may not be available as a prognostic factor in every acute leukemia population. However, its application as a predictive factor of response has been confirmed in our population.

Conjugalidade e HIV/AIDs: a desmesura do amor e a delicadeza da dor / Conjugality and HIV/AIDS: on disproportionality of love and tenderness of pain / Conyugalidad y el VIH/SIDA: el carácter excesivo del amor y la delicadeza del dolor

Este trabalho objetiva aprofundar o entendimento sobre os impactos subjetivos do diagnóstico de soropositividade para HIV/Aids, tanto para o sujeito, ao se descobrir portador do vírus,quanto para o seu parceiro. Participou desta pesquisa um casal homossexual masculino, que mantinha relacionamento afetivo-sexual consensualmente aberto e não preventivo, que descobriu que um de seus membros tinha HIV/Aids. Os instrumentos utilizados para a coleta de dados foram a entrevista semidirigida, a observação participante e o diário de campo. O discurso do casal foi compreendido a partir da analítica descritiva, na perspectiva foucaultiana. Os resultados evidenciaram que o diagnóstico de infecção pelo HIV foi um momento doloroso, de agonia e profunda tristeza, mas que, por outro lado, permitiu a ressignificação e construção de um novo estilo de vida. Procurou-se pensar o HIV/Aids para além de uma doença do corpo, mas como uma prática discursiva produtora de subjetividade, entrelaçada às relações sociais e atravessamentos históricos e culturais.

This paper aims to get further on understanding subjective impact on an individual and his partner when diagnosed HIV positive. Participants were a male homosexual couple who was dealing with the discovery of HIV/AIDS in one of the subjects in that relationship. They have kept a love and sexual relationship which was consensually an opened one (by means of sexual encounters) and without any use of sexual protection. Instruments used to collect data were semi-directed interviews, participant observation and field diary. Couple ́s discourse was understood using a descriptive-analytic approach in a Foucauldian perspective. Results pointed out a painful, full of agony and deep sadness moment when HIV diagnose came out, and nevertheless it allowed a construction of a new life style. We consider HIV/AIDS to be much more than a disease of the body; we also take it as a discursive practice producing subjectivity related to social relations as much as historical and cultural crossovers.

Este estudio tiene como objetivo profundizar en la comprensión del impacto subjetivo del diagnóstico de seropositividad para VIH/SIDA, tanto para el sujeto que se descubre portador del virus cuanto para su pareja amorosa. Los participantes fueran una pareja homosexual masculina que mantuvo relación afectivo-sexual consensuada abierta y no preventiva y descubrió que uno de los miembros tenía el VIH/SIDA. Los instrumentos utilizados para la recolección de datos fueron entrevistas semiestructuradas, observación participante y el diario de campo. El discurso de la pareja se analizó desde el punto de vista analítico descriptivo en la perspectiva de Foucault. Los resultados mostraron que el diagnóstico de la infección por el VIH fue un momento doloroso, de agonía y tristeza profunda, pero, por otro lado, permitió la construcción de un nuevo estilo de vida. Buscamos pensar el síndrome del VIH/SIDA más allá de una enfermedad del cuerpo, sino como una práctica discursiva que produce subjetividad, entrelazada a las relaciones sociales y los cambios históricos y culturales.

The Illness Experience: Palliative Care Given the impossibility of Healing.

This paper presents a reflection about being terminally ill and the various ways that the subject has at its disposal to deal with this event. The objective is to understand the experience of palliation for patients undergoing no therapeutic possibilities of cure. The methodology of this study has the instruments to semi-structured interview, the participant observation and the field diary, and the Descriptive Analysis of Foucault's inspiration how the narratives of the subjects were perceived. The Results of paper there was the possibility of looking at the experience of illness through the eyes of a subject position assumed by the very sick. As conclusion we have than when choosing palliative care, the terminally ill opts for a way to feel more comfortable and resists the impositions of the medical model of prolonging life. O presente trabalho traz uma reflexão a respeito do ser um doente terminal e das várias maneiras que o sujeito tem a seu dispor para lidar com esse acontecimento. Nosso objetivo foi compreender a experiência da paliação por sujeitos doentes sem possibilidades terapêuticas de cura. A metodologia deste estudo teve como instrumentos a Entrevista Narrativa, a Observação Participante e o Diário de Campo, sendo a Análise Descritiva de inspiração foucaultiana o modo como as narrativas dos sujeitos foram percebidas. O resultado do estudo mostrou a possibilidade de olhar a experiência do adoecer através da ótica de uma posição de sujeito assumida pelo próprio enfermo. E concluímos que ao escolher os cuidados paliativos, o doente terminal opta por um modo de se sentir mais confortável e resiste às imposições do modelo médico de prolongamento da vida.

The Illness Experience: Palliative Care Given the impossibility of Healing / La experiencia de la enfermedad: Cuidados Paliativos Dada la imposibilidad de curación / A Experiência do Adoecer: Os Cuidados Paliativos diante da Impossibilidade da Cura

This paper presents a reflection about being terminally ill and the various ways that the subject has at its disposal to deal with this event. The objective is to understand the experience of palliation for patients undergoing no therapeutic possibilities of cure. The methodology of this study has the instruments to semi-structured interview, the participant observation and the field diary, and the Descriptive Analysis of Foucault’s inspiration how the narratives of the subjects were perceived. The Results of paper there was the possibility of looking at the experience of illness through the eyes of a subject position assumed by the very sick. As conclusion we have than when choosing palliative care, the terminally ill opts for a way to feel more comfortable and resists the impositions of the medical model of prolonging life.

Este artículo presenta una reflexión acerca de ser un enfermo terminal y las diversas formas que el sujeto tiene a su disposición para hacer frente a este evento. Nuestro objetivo fue comprender la experiencia de pacientes sin posibilidad terapéuticas de cura, sometidos a los cuidados paliativos. La metodología de este estudio cuenta con los instrumentos de entrevista semiestructurada, la observación participante y el diario de campo. Las narrativas de los sujetos han sido analizadas bajo la analítica descriptiva de inspiración foucaultiana. Os Resultados nos dicen que la posibilidad de ver la experiencia de la enfermedad a través de la óptica de una posición de sujeto asumida por el propio enfermo. La conclusión es que es posible el sujeto enfermo terminal elegir los cuidados paliativos como una manera de sentirse más cómodo y para resistir a las imposiciones del modelo médico de la prolongación de la vida.

O presente trabalho traz uma reflexão a respeito do ser um doente terminal e das várias maneiras que o sujeito tem a seu dispor para lidar com esse acontecimento. Nosso objetivo foi compreender a experiência da paliação por sujeitos doentes sem possibilidades terapêuticas de cura. A metodologia deste estudo teve como instrumentos a Entrevista Narrativa, a Observação Participante e o Diário de Campo, sendo a Análise Descritiva de inspiração foucaultiana o modo como as narrativas dos sujeitos foram percebidas. O resultado do estudo mostrou a possibilidade de olhar a experiência do adoecer através da ótica de uma posição de sujeito assumida pelo próprio enfermo. E concluímos que ao escolher os cuidados paliativos, o doente terminal opta por um modo de se sentir mais confortável e resiste às imposições do modelo médico de prolongamento da vida.