Role of Human and Mouse Rad54 in DNA Recombination and Repair

DNA double-strand breaks (DSBs) which can be induced by endogenously produced radicals or by ionizing radiation are among the most genotoxic DNA lesions. Repair of DSBs is of cardinal importance for the prevention of chromosomal fragmentation, translocations, and deletions. The genetic instability resulting from persistent or incorrectly repaired DSBs can eventually result in cancer. Therefore, to understand the biological consequences of exposure to ionizing radiation, insight into the mechanisms of DSB repair in mammalian cells is essential. The pace of identification of mammalian DSB repair genes has rapidly increased over the last few years. However, the functional analysis of the encoded proteins and the analysis of the role of the different DSB repair mechanisms in mammals are far from complete. This thesis describes the generation and phenotypic characterization of cells and mice, with a defect in one of the DSB repair genes, the RAD54 recombinational DNA repair gene. Furthermore, the initial characterization and cellular behavior of the mammalian Rad54 protein is described. Chapter 1 outlines the current knowledge on the role and molecular mechanisms of the multiple pathways that have evolved for the repair of DSBs. Our main findings concerning mammalian Rad54 at the protein and cellular level are discussed and integrated in the emerging picture of the DSB repair mechanisms in mammals. Chapters 2 and 3 describe the isolation of mammalian RAD54 genes and genomic characterization of the mouse RAD54 gene. Chapters 4 and 5 describe the generation and phenotypic characterization of RAD54 knockout cells and mice. Chapters 6 and 7 describe the characterization of the in vitro activities of the purified human Rad54 protein and the cellular behavior of the mouse Rad54 protein upon induction of DNA damage

Molecular Imaging of Inflammation in Aortic Aneurysmal Disease

The high mortality rate of diseases of the aorta has its foundation in imaging methods that define anatomy and disease burden but less so upon the diagnosis of asymptomatic conditions, rate of aneurysm expansion, or prediction of rupture. However, anatomical features can now be co-localized with molecular and physiological activity. The advancement of nanoparticles based upon iron oxide will also serve to bring a trio of magnetic, radionuclide, and optical imaging modalities together. The combinations of these technologies are still at the preclinical refinement stage but already enzyme-activatable probes have b

Characteristics of DNA-binding proteins determine the biological sensitivity to high-linear energy transfer radiation

Non-homologous end-joining (NHEJ) and homologous recombination repair (HRR), contribute to repair ionizing radiation (IR)-induced DNA double-strand breaks (DSBs). Mre11 binding to DNA is the first step for activating HRR and Ku binding to DNA is the first step for initiating NHEJ. High-linear energy transfer (LET) IR (such as high energy charged particles) killing more cells at the same dose as compared with low-LET IR (such as X or γ rays) is due to inefficient NHEJ. However, these phenomena have not been demonstrated at the animal level and the mechanism by which high-LET IR does not affect the efficiency of HRR remains unclear. In this study, we showed that although wild-type and HRR-deficient mice or DT40 cells are more sensitive to high-LET IR than to low-LET IR, NHEJ deficient mice or DT40 cells are equally sensitive to high- and low-LET IR. We also showed that Mre11 and Ku respond differently to shorter DNA fragments in vitro and to the DNA from high-LET irradiated cells in vivo. These findings provide strong evidence that the different DNA DSB binding properties of Mre11 and Ku determine the different efficiencies of HRR and NHEJ to repair high-LET radiation induced DSBs

The human RAD54 recombinational DNA repair protein is a double-stranded DNA-dependent ATPase

DNA double-strand break repair through the RAD52 homologous recombination pathway in the yeast Saccharomyces cerevisiae requires, among others, the RAD51, RAD52, and RAD54 genes. The biological importance of homologous recombination is underscored by the conservation of the RAD52 pathway from fungi to humans. The critical roles of the RAD52 group proteins in the early steps of recombination, the search for DNA homology and strand exchange, are now becoming apparent. Here, we report the purification of the human Rad54 protein. We showed that human Rad54 has ATPase activity that is absolutely dependent on double-stranded DNA. Unexpectedly, the ATPase activity appeared not absolutely required for the DNA repair function of human Rad54 in vivo. Despite the presence of amino acid sequence motifs that are conserved in a large family of DNA helicases, no helicase activity of human Rad54 was observed on a variety of different DNA substrates. Possible functions of human Rad54 in homologous recombination that couple the energy gained from ATP hydrolysis to translocation along DNA, rather than disruption of base pairing, are discussed

VE-cadherin and claudin-5: it takes two to tango

Endothelial barrier function requires the adhesive activity of VE-cadherin and claudin-5, which are key components of adherens and tight endothelial junctions, respectively. Emerging evidence suggests that VE-cadherin controls claudin-5 expression by preventing the nuclear accumulation of FoxO1 and -catenin, which repress the claudin-5 promoter. This indicates that a crosstalk mechanism operates between these junctional structures

The hematopoietic bone marrow niche ecosystem

The bone marrow (BM) microenvironment, also called the BM niche, is essential for the maintenance of fully functional blood cell formation (hematopoiesis) throughout life. Under physiologic conditions the niche protects hematopoietic stem cells (HSCs) from sustained or overstimulation. Acute or chronic stress deregulates hematopoiesis and some of these alterations occur indirectly via the niche. Effects on niche cells include skewing of its cellular composition, specific localization and molecular signals that differentially regulate the function of HSCs and their progeny. Importantly, while acute insults display only transient effects, repeated or chronic insults lead to sustained alterations of the niche, resulting in HSC deregulation. We here describe how changes in BM niche composition (ecosystem) and structure (remodeling) modulate activation of HSCs in situ . Current knowledge has revealed that upon chronic stimulation, BM remodeling is more extensive and otherwise quiescent HSCs may be lost due to diminished cellular maintenance processes, such as autophagy, ER stress response, and DNA repair. Features of aging in the BM ecology may be the consequence of intermittent stress responses, ultimately resulting in the degeneration of the supportive stem cell microenvironment. Both chronic stress and aging impair the functionality of HSCs and increase the overall susceptibility to development of diseases, including malignant transformation. To understand functional degeneration, an important prerequisite is to define distinguishing features of unperturbed niche homeostasis in different settings. A unique setting in this respect is xenotransplantation, in which human cells depend on niche factors produced by other species, some of which we will review. These insights should help to assess deviations from the steady state to actively protect and improve recovery of the niche ecosystem in situ to optimally sustain healthy hematopoiesis in experimental and clinical settings

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD)

Monitoring daily physical activity of upper extremity in young and adolescent boys with Duchenne muscular dystrophy:A pilot study

Introduction Accelerometry of the upper extremity (UE) potentially provides information on the extent of activities in daily life in patients with Duchenne muscular dystrophy (DMD). The objective of this study is to evaluate the validity of home measurements of UE accelerometry. Methods This was a cross-sectional study in 16 patients with DMD (aged 7-17 years). Patients were monitored for 1 to 3 days with two accelerometers on the UE and one accelerometer on the wheelchair. Results The mean intensity of activity and the mean frequency of transfers of arm elevation from low to middle were approximately twofold higher in patients with a Brooke scale score of 1 or 2 than in patients with a Brooke scale score of 3 or 4. Correlations with the Performance of Upper Limb scale score were high for intensity and for the total frequency of arm elevations per hour. Discussion Intensity, percentage of time in middle orientation, and frequency of transfers of the upper arm correlated well with functional measurements