Receptor-DNA interactions: EMSA and footprinting

Defining the precise promoter DNA sequence motifs where nuclear receptors and other transcription factors bind is an essential prerequisite for understanding how these proteins modulate the expression of their specific target genes. The purpose of this chapter is to provide the reader with a detailed guide with respect to the materials and the key methods required to perform this type of DNA-binding analysis. Irrespective of whether starting with purified DNA-binding proteins or somewhat crude cellular extracts, the tried-and-true procedures described here will enable one to accurately access the capacity of specific proteins to bind to DNA as well as to determine the exact sequences and DNA contact nucleotides involved. For illustrative purposes, we primarily have used the interaction of the androgen receptor with the rat probasin proximal promoter as our model system.

Assimilating cell sheets and hybrid scaffolds for dermal tissue engineering

Cell sheets can be used to produce neo-tissue with mature extracellular matrix. However, extensive contraction of cell sheets remains a problem. We devised a technique to overcome this problem and applied it to tissue engineer a dermal construct. Human dermal fibroblasts were cultured with poly(lactic-co-glycolic acid)-collagen meshes and collagen-hyaluronic acid foams. Resulting cell sheets were folded over the scaffolds to form dermal constructs. Human keratinocytes were cultured on these dermal constructs to assess their ability to support bilayered skin regeneration. Dermal constructs produced with collagen-hyaluronic acid foams showed minimal contraction, while those with poly(lactic-co-glycolic acid)-collagen meshes curled up. Cell proliferation and metabolic activity profiles were characterized with PicoGreen and AlamarBlue assays, respectively. Fluorescent labeling showed high cell viability and F-actin expression within the constructs. Collagen deposition was detected by immunocytochemistry and electron microscopy. Transforming Growth Factor-alpha and beta1, Keratinocyte Growth Factor and Vascular Endothelial Growth Factor were produced at various stages of culture, measured by RT-PCR and ELISA. These results indicated that assimilating cell sheets with mechanically stable scaffolds could produce viable dermal-like constructs that do not contract. Repeated enzymatic treatment cycles for cell expansion is unnecessary, while the issue of poor cell seeding efficiency in scaffolds is eliminated.

An alginate-based hybrid system for growth factor delivery in the functional repair of large bone defects

The treatment of challenging fractures and large osseous defects presents a formidable problem for orthopaedic surgeons. Tissue engineering/regenerative medicine approaches seek to solve this problem by delivering osteogenic signals within scaffolding biomaterials. In this study, we introduce a hybrid growth factor delivery system that consists of an electrospun nanofiber mesh tube for guiding bone regeneration combined with peptide-modified alginate hydrogel injected inside the tube for sustained growth factor release. We tested the ability of this system to deliver recombinant bone morphogenetic protein-2 (rhBMP-2) for the repair of critically-sized segmental bone defects in a rat model. Longitudinal [mu]-CT analysis and torsional testing provided quantitative assessment of bone regeneration. Our results indicate that the hybrid delivery system resulted in consistent bony bridging of the challenging bone defects. However, in the absence of rhBMP-2, the use of nanofiber mesh tube and alginate did not result in substantial bone formation. Perforations in the nanofiber mesh accelerated the rhBMP-2 mediated bone repair, and resulted in functional restoration of the regenerated bone. [mu]-CT based angiography indicated that perforations did not significantly affect the revascularization of defects, suggesting that some other interaction with the tissue surrounding the defect such as improved infiltration of osteoprogenitor cells contributed to the observed differences in repair. Overall, our results indicate that the hybrid alginate/nanofiber mesh system is a promising growth factor delivery strategy for the repair of challenging bone injuries.

Evaluation of a hybrid scaffold/cell construct in repair of high-load-bearing osteochondral defects in rabbits

The objective of this study was to evaluate the feasibility and potential of a hybrid scaffold system in large- and high-load-bearing osteochondral defects repair. The implants were made of medical-grade PCL (mPCL) for the bone compartment whereas fibrin glue was used for the cartilage part. Both matrices were seeded with allogenic bone marrow-derived mesenchymal cells (BMSC) and implanted in the defect (4 mm diameter×5 mm depth) on medial femoral condyle of adult New Zealand White rabbits. Empty scaffolds were used at the control side. Cell survival was tracked via fluorescent labeling. The regeneration process was evaluated by several techniques at 3 and 6 months post-implantation. Mature trabecular bone regularly formed in the mPCL scaffold at both 3 and 6 months post-operation. Micro-Computed Tomography showed progression of mineralization from the host–tissue interface towards the inner region of the grafts. At 3 months time point, the specimens showed good cartilage repair. In contrast, the majority of 6 months specimens revealed poor remodeling and fissured integration with host cartilage while other samples could maintain good cartilage appearance. In vivo viability of the transplanted cells was demonstrated for the duration of 5 weeks. The results demonstrated that mPCL scaffold is a potential matrix for osteochondral bone regeneration and that fibrin glue does not inherit the physical properties to allow for cartilage regeneration in a large and high-load-bearing defect site. Keywords: Osteochondral tissue engineering; Scaffold; Bone marrow-derived precursor cells; Fibrin glue

Hybrid robot control and SLAM for persistent navigation and mapping

For a mobile robot to operate autonomously in real-world environments, it must have an effective control system and a navigation system capable of providing robust localization, path planning and path execution. In this paper we describe the work investigating synergies between mapping and control systems. We have integrated development of a control system for navigating mobile robots and a robot SLAM system. The control system is hybrid in nature and tightly coupled with the SLAM system; it uses a combination of high and low level deliberative and reactive control processes to perform obstacle avoidance, exploration, global navigation and recharging, and draws upon the map learning and localization capabilities of the SLAM system. The effectiveness of this hybrid, multi-level approach was evaluated in the context of a delivery robot scenario. Over a period of two weeks the robot performed 1143 delivery tasks to 11 different locations with only one delivery failure (from which it recovered), travelled a total distance of more than 40km, and recharged autonomously a total of 23 times. In this paper we describe the combined control and SLAM system and discuss insights gained from its successful application in a real-world context.

The embodied hybrid space : designing ubiquitous computing towards an amplification of situated real world experiences

The emergence of mobile and ubiquitous computing technology has created what is often referred to as the hybrid space – a virtual layer of digital information and interaction opportunities that sit on top of and augment the physical environment. Embodied media materialise digital information as observable and sometimes interactive parts of the physical environment. The aim of this work is to explore ways to enhance people’s situated real world experience, and to find out what the role and impact of embodied media in achieving this goal can be. The Edge, an initiative of the State Library of Queensland in Brisbane, Australia, and case study of this thesis, envisions to be a physical place for people to meet, explore, experience, learn and teach each other creative practices in various areas related to digital technology and arts. Guided by an Action Research approach, this work applies Lefebvre’s triad of space (1991) to investigate the Edge as a social space from a conceived, perceived and lived point of view. Based on its creators’ vision and goals on the conceived level, different embodied media are iteratively designed, implemented and evaluated towards shaping and amplifying the Edge’s visitor experience on the perceived and lived level.

Operation and control of hybrid microgrid with angle droop controller

This paper compares the performance of two droop control schemes in a hybrid microgrid. With presence of both converter interfaced and inertial sources, the droop controller share power in a decentralized fashion. Both the droop controllers facilitate reactive power sharing based on voltage droop. However in frequency droop control, the real power sharing depends on the frequency, while in angle droop control, it depends on output voltage angle. For converter interfaced sources this reference voltage is tracked while for inertial DG, reference power for the prime mover is calculated from the reference angle with the proposed angle control scheme. This coordinated control scheme shows significant improvement in system performance. The comparison with the conventional frequency droop shows that the angle control scheme shares power with much lower frequency deviation. This is a significant improvement particularly in a frequent load changing scenario.

Differentiation of Rice Tungro Bacilliform Virus Strains by Restriction Analysis and DNA Hybridization

Rice tungro bacilliform virus (RTBV) is one of the two viruses that cause tungro disease. Four RTBV strains maintained in the greenhouse for 4 years, G1, G2, Ic, and L, were differentiated by restriction fragment length polymorphism (RFLP) analysis of the native viral DNA. Although strains G1 and Ic had identical restriction patterns when cleaved with Pst1, BamHI, EcoRI, and EcoRV, they can be differentiated from strains G2 and L by EcoRI and EcoRV digestion. These same endonucleases also differentiate strain G2 from strain L. When total DNA extracts from infected plants were used instead of viral DNA, and digested with EcoRV, identical restriction patterns for each strain (G2 and L) were obtained from roots, leaves, and leaf sheaths of infected plants. The restriction patterns were consistent from plant to plant, in different varieties, and at different times after inoculation. This technique can be used to differentiate RTBV strains and determine the variability of a large number of field samples.

QoS-oriented resource allocation and scheduling of multiple composite Web services in a hybrid cloud using a random-key genetic algorithm

In cloud computing resource allocation and scheduling of multiple composite web services is an important challenge. This is especially so in a hybrid cloud where there may be some free resources available from private clouds but some fee-paying resources from public clouds. Meeting this challenge involves two classical computational problems. One is assigning resources to each of the tasks in the composite web service. The other is scheduling the allocated resources when each resource may be used by more than one task and may be needed at different points of time. In addition, we must consider Quality-of-Service issues, such as execution time and running costs. Existing approaches to resource allocation and scheduling in public clouds and grid computing are not applicable to this new problem. This paper presents a random-key genetic algorithm that solves new resource allocation and scheduling problem. Experimental results demonstrate the effectiveness and scalability of the algorithm.

Performance analysis of hybrid liquid desiccant solar cooling system

This thesis investigates the coefficient of performance (COP) of a hybrid liquid desiccant solar cooling system. This hybrid cooling system includes three sections: 1) conventional air-conditioning section; 2) liquid desiccant dehumidification section and 3) air mixture section. The air handling unit (AHU) with mixture variable air volume design is included in the hybrid cooling system to control humidity. In the combined system, the air is first dehumidified in the dehumidifier and then mixed with ambient air by AHU before entering the evaporator. Experiments using lithium chloride as the liquid desiccant have been carried out for the performance evaluation of the dehumidifier and regenerator. Based on the air mixture (AHU) design, the electrical coefficient of performance (ECOP), thermal coefficient of performance (TCOP) and whole system coefficient of performance (COPsys) models used in the hybrid liquid desiccant solar cooing system were developed to evaluate this system performance. These mathematical models can be used to describe the coefficient of performance trend under different ambient conditions, while also providing a convenient comparison with conventional air conditioning systems. These models provide good explanations about the relationship between the performance predictions of models and ambient air parameters. The simulation results have revealed the coefficient of performance in hybrid liquid desiccant solar cooling systems substantially depends on ambient air and dehumidifier parameters. Also, the liquid desiccant experiments prove that the latent component of the total cooling load requirements can be easily fulfilled by using the liquid desiccant dehumidifier. While cooling requirements can be met, the liquid desiccant system is however still subject to the hysteresis problems.

Recent advances in cancer therapy targeting proteins involved in DNA double-strand break repair

Damage to genetic material represents a persistent and ubiquitous threat to genomic stability. Once DNA damage is detected, a multifaceted signaling network is activated that halts the cell cycle, initiates repair, and in some instances induces apoptotic cell death. In this article, we will review DNA damage surveillance networks, which maintain the stability of our genome, and discuss the efforts underway to identify chemotherapeutic compounds targeting the core components of DNA double-strand breaks (DSB) response pathway. The majority of tumor cells have defects in maintaining genomic stability owing to the loss of an appropriate response to DNA damage. New anticancer agents are exploiting this vulnerability of cancer cells to enhance therapeutic indexes, with limited normal tissue toxicity. Recently inhibitors of the checkpoint kinases Chk1 and Chk2 have been shown to sensitize tumor cells to DNA damaging agents. In addition, the treatment of BRCA1- or BRCA2-deficient tumor cells with poly(ADP-ribose) polymerase (PARP) inhibitors also leads to specific tumor killing. Due to the numerous roles of p53 in genomic stability and its defects in many human cancers, therapeutic agents that restore p53 activity in tumors are the subject of multiple clinical trials. In this article we highlight the proteins mentioned above and catalog several additional players in the DNA damage response pathway, including ATM, DNA-PK, and the MRN complex, which might be amenable to pharmacological interventions and lead to new approaches to sensitize cancer cells to radio- and chemotherapy. The challenge is how to identify those patients most receptive to these treatments.

Spatiotemporal investigations of DNA damage repair using microbeams

Cellular response to radiation damage is made by a complex network of pathways and feedback loops whose spatiotemporal organisation is still unclear despite its decisive role in determining the fate of the damaged cell. Revealing the dynamic sequence of the repair proteins is therefore critical in understanding how the DNA repair mechanisms work. There are also still open questions regarding the possible movement of damaged chromatin domains and its role as trigger for lesion recognition and signalling in the DNA repair context. The single-cell approach and the high spatial resolution offered by microbeams provide the perfect tool to study and quantify the dynamic processes associated with the induction and repair of DNA damage. We have followed the development of radiation-induced foci for three DNA damage markers (i.e. γ-H2AX, 53BP1 and hSSB1) using normal fibroblasts (AG01522), human breast adenocarcinoma cells (MCF7) and human fibrosarcoma cells (HT1080) stably transfected with yellow fluorescent protein fusion proteins following irradiation with the QUB X-ray microbeam (carbon X-rays <2 µm spot). The size and intensity of the foci has been analysed as a function of dose and time post-irradiation to investigate the dynamics of the above-mentioned DNA repair processes and monitor the remodelling of chromatin structure that the cell undergoes to deal with DNA damage.