DNA Damage Responses of Mitotic Cells Following Laser Micro-irradiation
DNA Damage Responses of Mitotic Cells Following Laser Micro-irradiation

Author: Veronica Gomez Godinez

Publisher:

Published: 2012

Total Pages: 180

ISBN-13: 9781267711366

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The natural occurrence of DNA damage has resulted in the evolution of processes that maintain cellular genomic integrity. These include (a) recognition of damaged DNA, (b) cell cycle control that halts progression through mitosis and thus block passage of compromised DNA to the next generation of cells, and (c) various mechanisms of DNA damage repair. Defects associated with DNA repair are associated with developmental disorders, cancer and accelerated aging. Double strand breaks (DSBs) are amongst the most serious forms of DNA lesions. A single double strand break can lead to cell death if left unrepaired. Further, double strand breaks can lead to chromosomal translocations that are highly associated with tumorigenesis. Thus, interphase DSB responses are the focus of several investigations. Few studies, however, have investigated the DSB responses of cells in mitosis. My studies have focused on the use of laser microsurgery to induce DSBs on mitotic chromosomes, and to examine the subsequent DNA damage response mechanisms. These studies have led to the determination of laser parameters that result in consistent double strand break and pyrimidine dimer production as well as characterization of the change in chromatin ultrastructure following laser irradiation. In addition, a relationship between mitotic DNA damage and retention of the mitotic marker, phosho-histone H3 Serine 10, has been identified. My results show that contrary to other studies, proteins downstream of DSB recognition (Ubiquitin, BRCA1 and 53BP1) accumulate at laser-induced mitotic DNA damage sites. Furthermore, both homologous recombination and non-homologous end joining (NHEJ) repair pathways are activated. However, downstream activation is only observed for NHEJ. Additionally, cells deficient in DNA-PKcs have demonstrated the ability to repair via EdU incorporation, thus, suggesting the activity of alternative-NHEJ. In addition, activation of the nucleotide excision repair, single strand break repair, and Fanconi Anemia pathways have been detected in mitotic cells. The results also demonstrate that DNA damage in mitosis is repaired less effectively than DNA damage induced in G1. For the purposes of this thesis, "effective repair" means that a cell can undergo a subsequent cell division after it has repaired the damage produced in the previous mitosis.

Laser Ablation of Single Telomeres in Mitosis
Laser Ablation of Single Telomeres in Mitosis

Author: Barbara Alcaraz Silva

Publisher:

Published: 2014

Total Pages: 127

ISBN-13: 9781321093551

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Telomeres are essential for protecting chromosome ends not only from the replication-associated DNA loss, but also from unwanted DNA damage response (DDR) and repair by the damage machinery. Uncapping of telomeres during interphase elicits a DDR mechanism that results in cell cycle arrest. However, it is unclear how chromosome ends are normally protected from the DNA damage machinery, and how DDR is regulated at telomeres during mitosis. With that in mind, the goal of my thesis is to investigate the consequences of DNA damage occurring at specific chromosomal domains. Laser microirradiation was used in combination with dual fluorescent labeling to monitor the co-localization of DDR factors in PtK2 (Potorous tridactylus) chromosomes. The results of my thesis show that laser-induced DNA break in chromosome ends as well as in chromosome arms of anaphase cells result in recruitment of the following: poly (ADP-ribose) polymerase 1 (PARP1), checkpoint sensors (p-Chk1, p-Chk2), DNA repair protein Ku70/Ku80, and proliferating cell nuclear antigen (PCNA). However, p53 phosphorylated at serine 15 was detected only at chromosome ends, and not at chromosome arms of anaphase cells. Furthermore, my experiments show that damage to a single mitotic chromosome end, but not on a chromosome arm, results in specific DDR factor recruitment, damage and spindle checkpoint-dependent mitotic delay, and subsequent micronuclei formation in G1. Together these findings reveal mitosis-specific DDR uniquely associated with chromosome ends. Furthermore, we found the laser parameters used to induce telomeric TRF2 (repeat-binding factor 2) recruitment. We introduced double-strand breaks (DSBs) and assayed for the recruitment of TRF2. We found that a laser dose of 2.43e+11W/cm2 is sufficient to form DSBs, based on the recruitment of repair factor 53BP1. Nevertheless, at this laser dose, TRF2 fails to accumulate at damage sites. In contrast, at an irradiance of 2.65 e+11 W/cm2 or higher, TRF2 accumulates at damage sites, which is independent of ATM. We also found that phosphorylation of TRF2 on threonine 188 occurs at both low and high irradiance laser-induced DSBs in both interphase and mitotic cells in an ATM-dependent manner. By contrast, Phosphorylated TRF2 on threonine 188 did not form foci by using y-irradiation.

The Retinoblastoma Protein
The Retinoblastoma Protein

Author: Pedro G. Santiago-Cardona

Publisher: Humana Press

Published: 2018-02-22

Total Pages: 200

ISBN-13: 9781493975648

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This volume covers the mechanisms of pRb inactivation detailing repressive mechanisms commonly associated to cancer, and representative of the experimentally relevant tests used in the establishment of cancer diagnosis and prognosis. Chapters contain protocols and in-depth discussions for commonly used experimental approaches to assess the status and function of components of the pRb pathway, including pRb itself, in cell lines and biological samples.Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, The Retinoblastoma Protein aims to serve as a guide to assist molecular cancer biologists in their search for understanding of the molecular functions of this preeminent tumor suppressor.

Epigenetic Alterations in Oncogenesis
Epigenetic Alterations in Oncogenesis

Author: Adam R. Karpf

Publisher: Springer Science & Business Media

Published: 2012-09-06

Total Pages: 355

ISBN-13: 1441999671

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The purpose of this book is to provide an up to date review of the nature and consequences of epigenetic changes in cancer. Epigenetics literally means “above” genetics, and consists of heritable gene expression or other phenotypic states not accounted for by DNA base sequence. Epigenetic changes are now known to make a large contribution to various aspects of tumorigenesis. These changes include alterations in global and promoter specific DNA methylation, activating and repressive histone modifications, and changes in higher order chromatin structures. Each of these topics will be covered in this book.

Understanding Biophotonics
Understanding Biophotonics

Author: Kevin Tsia

Publisher: CRC Press

Published: 2016-01-05

Total Pages: 757

ISBN-13: 9814411787

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Biophotonics involves understanding how light interacts with biological matter, from molecules and cells, to tissues and even whole organisms. Light can be used to probe biomolecular events, such as gene expression and protein-protein interaction, with impressively high sensitivity and specificity. The spatial and temporal distribution of biochemic

Approximate Analytical Methods for Solving Ordinary Differential Equations
Approximate Analytical Methods for Solving Ordinary Differential Equations

Author: T.S.L Radhika

Publisher: CRC Press

Published: 2014-10-31

Total Pages: 584

ISBN-13: 1466588136

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Approximate Analytical Methods for Solving Ordinary Differential Equations (ODEs) is the first book to present all of the available approximate methods for solving ODEs, eliminating the need to wade through multiple books and articles. It covers both well-established techniques and recently developed procedures, including the classical series solution method, diverse perturbation methods, pioneering asymptotic methods, and the latest homotopy methods. The book is suitable not only for mathematicians and engineers but also for biologists, physicists, and economists. It gives a complete description of the methods without going deep into rigorous mathematical aspects. Detailed examples illustrate the application of the methods to solve real-world problems. The authors introduce the classical power series method for solving differential equations before moving on to asymptotic methods. They next show how perturbation methods are used to understand physical phenomena whose mathematical formulation involves a perturbation parameter and explain how the multiple-scale technique solves problems whose solution cannot be completely described on a single timescale. They then describe the Wentzel, Kramers, and Brillown (WKB) method that helps solve both problems that oscillate rapidly and problems that have a sudden change in the behavior of the solution function at a point in the interval. The book concludes with recent nonperturbation methods that provide solutions to a much wider class of problems and recent analytical methods based on the concept of homotopy of topology.

Laser Manipulation of Cells and Tissues
Laser Manipulation of Cells and Tissues

Author: Michael W. Berns

Publisher: Academic Press

Published: 2007-06-20

Total Pages: 824

ISBN-13:

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The use of laser-based manipulation tools has literally exploded on the cell biology and molecular pathology scene, resulting in as many different laser micromanipulation systems as there are people using them. Laser Manipulation of Cells and Tissues ties all these systems and studies together, describing all of the different kinds of research and practical/analytical applications of laser manipulation. It also provides the reader with the basic information needed to actually build one's own laser micro-manipulation system. The combination of imaging and molecular probe technology with laser micromanipulation greatly extends the use of this technology in molecular, cellular, developmental and patho-biology/medicine. This book should be valuable to scientists, clinicians, and students in the fields of cell and developmental biology, cell physiology, cancer biology, pathology, and stem cell biology. Devotes four chapters to laser catapulting and capture of DNA and other cellular material for biochemical analysis - a major use of this technology that has been adapted for molecular pathology both in clinical medicine and research Discusses the theory of laser tweezers (optical tweezers) and its application to novel problems in biology Covers topics on optoporation (getting things into cells), uncaging of molecules, and the ability to collect and analyze nanomolar amounts of cell material by an array of biochemical/physical tools of particular interest to cell biologists and drug discovery researchers

Laser-Driven Particle Acceleration Towards Radiobiology and Medicine
Laser-Driven Particle Acceleration Towards Radiobiology and Medicine

Author: Antonio Giulietti

Publisher: Springer

Published: 2016-05-04

Total Pages: 326

ISBN-13: 3319315633

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This book deals with the new method of laser-driven acceleration for application to radiation biophysics and medicine. It provides multidisciplinary contributions from world leading scientist in order to assess the state of the art of innovative tools for radiation biology research and medical applications of ionizing radiation. The book contains insightful contributions on highly topical aspects of spatio-temporal radiation biophysics, evolving over several orders of magnitude, typically from femtosecond and sub-micrometer scales. Particular attention is devoted to the emerging technology of laser-driven particle accelerators and their application to spatio-temporal radiation biology and medical physics, customization of non-conventional and selective radiotherapy and optimized radioprotection protocols.