Tissue engineering active biological machines : bio-inspired design, directed self-assembly, and characterization of muscular pumps simulating the embryonic heart

Biological machines are active devices that are comprised of cells and other biological components. These functional devices are best suited for physiological environments that support cellular function and survival. Biological machines have the potential to revolutionize the engineering of biomedical devices intended for implantation, where the human body can provide the required physiological environment. For engineering such cell-based machines, bio-inspired design can serve as a guiding platform as it provides functionally proven designs that are attainable by living cells. In the present work, a systematic approach was used to tissue engineer one such machine by exclusively using biological building blocks and by employing a bio-inspired design. Valveless impedance pumps were constructed based on the working principles of the embryonic vertebrate heart and by using cells and tissue derived from rats. The function of these tissue-engineered muscular pumps was characterized by exploring their spatiotemporal and flow behavior in order to better understand the capabilities and limitations of cells when used as the engines of biological machines.

Revealing, illuminating, and modifying proteins in human diseases using noncanonical amino acids

To better understand human diseases, much recent work has focused on proteins to either identify disease targets through proteomics or produce therapeutics via protein engineering. Noncanonical amino acids (ncAAs) are tools for altering the chemical and physical properties of proteins, providing a facile strategy not only to label proteins but also to engineer proteins with novel properties. My thesis research has focused on the development and applications of noncanonical amino acids in identifying, imaging, and engineering proteins for studying human diseases. Chapter 1 introduces the concept of ncAAs and reveals insights to how I chose my thesis projects.

ncAAs have been incorporated to tag and enrich newly synthesized proteins for mass spectrometry through a method termed BONCAT, or bioorthogonal noncanonical amino acid tagging. Chapter 2 describes the investigation of the proteomic response of human breast cancer cells to induced expression of tumor suppressor microRNA miR-126 by combining BONCAT with another proteomic method, SILAC or stable isotope labeling by amino acids in cell culture. This proteomic analysis led to the discovery of a direct target of miR-126, shedding new light on its role in suppressing cancer metastasis.

In addition to mass spectrometry, ncAAs can also be utilized to fluorescently label proteins. Chapter 3 details the synthesis of a set of cell-permeant cyclooctyne probes and demonstration of selective labeling of newly synthesized proteins in live mammalian cells using azidohomoalanine. Similar to live cell imaging, the ability to selectively label a particular cell type within a mixed cell population is important to interrogating many biological systems, such as tumor microenvironments. By taking advantage of the metabolic differences between cancer and normal cells, Chapter 5 discusses efforts to develop selective labeling of cancer cells using a glutamine analogue.

Furthermore, Chapter 4 describes the first demonstration of global replacement at polar amino acid positions and its application in developing an alternative PEGylation strategy for therapeutic proteins. Polar amino acids typically occupy solvent-exposed positions on the protein surface, and incorporation of noncanonical amino acids at these positions should allow easier modification and cause less perturbation compared to replacements at the interior positions of proteins.

Designing conformational control of human tissue transglutaminase

Understanding the mechanisms of enzymes is crucial for our understanding of their role in biology and for designing methods to perturb or harness their activities for medical treatments, industrial processes, or biological engineering. One aspect of enzymes that makes them difficult to fully understand is that they are in constant motion, and these motions and the conformations adopted throughout these transitions often play a role in their function.

Traditionally, it has been difficult to isolate a protein in a particular conformation to determine what role each form plays in the reaction or biology of that enzyme. A new technology, computational protein design, makes the isolation of various conformations possible, and therefore is an extremely powerful tool in enabling a fuller understanding of the role a protein conformation plays in various biological processes.

One such protein that undergoes large structural shifts during different activities is human type II transglutaminase (TG2). TG2 is an enzyme that exists in two dramatically different conformational states: (1) an open, extended form, which is adopted upon the binding of calcium, and (2) a closed, compact form, which is adopted upon the binding of GTP or GDP. TG2 possess two separate active sites, each with a radically different activity. This open, calcium-bound form of TG2 is believed to act as a transglutaminse, where it catalyzes the formation of an isopeptide bond between the sidechain of a peptide-bound glutamine and a primary amine. The closed, GTP-bound conformation is believed to act as a GTPase. TG2 is also implicated in a variety of biological and pathological processes.

To better understand the effects of TG2’s conformations on its activities and pathological processes, we set out to design variants of TG2 isolated in either the closed or open conformations. We were able to design open-locked and closed-biased TG2 variants, and use these designs to unseat the current understanding of the activities and their concurrent conformations of TG2 and explore each conformation’s role in celiac disease models. This work also enabled us to help explain older confusing results in regards to this enzyme and its activities. The new model for TG2 activity has immense implications for our understanding of its functional capabilities in various environments, and for our ability to understand which conformations need to be inhibited in the design of new drugs for diseases in which TG2’s activities are believed to elicit pathological effects.

A workbook-based methodology for implementing concurrent engineering

Concurrent Engineering demands a new way of working and many organisations experience difficulty during implementation. The research described in this paper has the aim to develop a paper-based workbook style methodology that companies can use to increase the benefits generated by Concurrent Engineering, while reducing implementation costs, risk and time. The three-stage methodology provides guidance based on knowledge accumulated from implementation experience and best practitioners. It encourages companies to learn to manage their Concurrent Engineering implementation by taking actions which expose them to new and valuable experiences. This helps to continuously improve understanding of how to maximise the benefits from Concurrent Engineering. The methodology is particularly designed to cater for organisational and contextual uniqueness, as Concurrent Engineering implementations will vary from company to company. Using key actions which improve the Concurrent Engineering implementation process, individual companies can develop their own 'best practice' for product development. The methodology ensures that key implementation issues, which are primarily human and organisational, are addressed using simple but proven techniques. This paper describes the key issues that the majority of companies face when implementing Concurrent Engineering. The structure of the methodology is described to show how the issues are addressed and resolved. The key actions used to improve the Concurrent Engineering implementation process are explained and their inclusion in the implementation methodology described. Relevance to industry. Implementation of Concurrent Engineering concepts in manufacturing industry has not been a straightforward process. This paper describes a workbook-style tool that manufacturing companies can use to accelerate and improve their Concurrent Engineering implementation. © 1995.

Interfacial recognition of human prostate-specific antigen by immobilized monoclonal antibody: effects of solution conditions and surface chemistry.

The specific recognition between monoclonal antibody (anti-human prostate-specific antigen, anti-hPSA) and its antigen (human prostate-specific antigen, hPSA) has promising applications in prostate cancer diagnostics and other biosensor applications. However, because of steric constraints associated with interfacial packing and molecular orientations, the binding efficiency is often very low. In this study, spectroscopic ellipsometry and neutron reflection have been used to investigate how solution pH, salt concentration and surface chemistry affect antibody adsorption and subsequent antigen binding. The adsorbed amount of antibody was found to vary with pH and the maximum adsorption occurred between pH 5 and 6, close to the isoelectric point of the antibody. By contrast, the highest antigen binding efficiency occurred close to the neutral pH. Increasing the ionic strength reduced antibody adsorbed amount at the silica-water interface but had little effect on antigen binding. Further studies of antibody adsorption on hydrophobic C8 (octyltrimethoxysilane) surface and chemical attachment of antibody on (3-mercaptopropyl)trimethoxysilane/4-maleimidobutyric acid N-hydroxysuccinimide ester-modified surface have also been undertaken. It was found that on all surfaces studied, the antibody predominantly adopted the 'flat on' orientation, and antigen-binding capabilities were comparable. The results indicate that antibody immobilization via appropriate physical adsorption can replace elaborate interfacial molecular engineering involving complex covalent attachments.

Characterising the information requests of aerospace engineering designers

During product development, engineering designers raise several information requests that make them search through human and documentary sources. This paper reports research to characterise, in detail, these requests for designers working in a major aerospace engineering company. The research found that at a high level, a distinction can be made between requests to acquire information and to process information. The former are raised to access design and domain information. The latter, instead, are formed to define designs. For researchers, this study extends existing knowledge of information requests by characterising key differences in their nature and explaining how they are used in the design process. For practitioners, these findings can be used as a basis to understand the diverseness of information requests and how to channel efforts to support designers in information seeking. In particular, the research indicates that a strategy to support designers should enable the development of engineering communities that share information effectively and the introduction of techniques that facilitate the documentation of information. © 2012 Springer-Verlag London Limited.

Parallel coordinates in computational engineering design

Modern Engineering Design involves the deployment of many computational tools. Re- search on challenging real-world design problems is focused on developing improvements for the engineering design process through the integration and application of advanced com- putational search/optimization and analysis tools. Successful application of these methods generates vast quantities of data on potential optimum designs. To gain maximum value from the optimization process, designers need to visualise and interpret this information leading to better understanding of the complex and multimodal relations between param- eters, objectives and decision-making of multiple and strongly conflicting criteria. Initial work by the authors has identified that the Parallel Coordinates interactive visualisation method has considerable potential in this regard. This methodology involves significant levels of user-interaction, making the engineering designer central to the process, rather than the passive recipient of a deluge of pre-formatted information. In the present work we have applied and demonstrated this methodology in two differ- ent aerodynamic turbomachinery design cases; a detailed 3D shape design for compressor blades, and a preliminary mean-line design for the whole compressor core. The first case comprises 26 design parameters for the parameterisation of the blade geometry, and we analysed the data produced from a three-objective optimization study, thus describing a design space with 29 dimensions. The latter case comprises 45 design parameters and two objective functions, hence developing a design space with 47 dimensions. In both cases the dimensionality can be managed quite easily in Parallel Coordinates space, and most importantly, we are able to identify interesting and crucial aspects of the relationships between the design parameters and optimum level of the objective functions under con- sideration. These findings guide the human designer to find answers to questions that could not even be addressed before. In this way, understanding the design leads to more intelligent decision-making and design space exploration. © 2012 AIAA.

basic research in computer science and software engineering at sklcs

The State Key Laboratory of Computer Science (SKLCS) is committed to basic research in computer science and software engineering. The research topics of the laboratory include: concurrency theory, theory and algorithms for real-time systems, formal specifications based on context-free grammars, semantics of programming languages, model checking, automated reasoning, logic programming, software testing, software process improvement, middleware technology, parallel algorithms and parallel software, computer graphics and human-computer interaction. This paper describes these topics in some detail and summarizes some results obtained in recent years.

Purification of human tissue prokallikrein excreted from insect cells by liquid chromatography

Tissue kallikrein, generally existing in living bodies as prokallikrein, is a serine proteinase that has proven of great significance to treat hypertension, cardiopathy and nephropathy. Although the extraction of tissue kallikrein from human urine is the most commonly used method to obtain such a protein, not only the yield is very little, but also the procedure is rather complex. Furthermore, the biological safety is uncertain. Therefore, the preparation of such a protein by genetic engineering method, including gene expression, cell culture, separation and purification, is very important. In this paper, a new method to obtain purified tissue prokallikrein excreted from insect cells by liquid chromatography has been proposed. In contrast to the previously published papers, the purification procedure is simplified to only three steps with the final yield of 57% and the purity of 95%, which is not only convenient, but also low-cost and suitable for the large-scale preparation of such a protein. The purified protein is further validated as prokallikrein by high performance liquid chromatography-mass spectrometry and amino acid sequencing. (c) 2005 Elsevier B.V. All rights reserved.