Chih-ming Ho, Ph.D. - US grants

This award provides funding to the University of California at Los Angeles, Dr. James C. Liao, Principal Investigator, for a three year Combined Research-Curriculum Development award entitled, "A Laboratory Curriculum for Gene Chip Technology." A novel laboratory course for gene chip technology will be developed. The purpose of this course is to educate students in this cutting-edge area of biotechnology. Gene chip technology combines microfabrication processes with biomolecular recognition, and has begun to revolutionize research and application of life sciences and the biomedical industry. This emerging field draws on engineering talents as well as biological knowledge. Engineers have an unprecedented, significant role to play in life science research and related industry. The objective of this course is to impart the basic principles of gene chip fabrication and the processing of gene chip data. Students will apply the technology to investigate the gene expression profiles of E coli, a microorganism of importance to both biotechnology and biomedicine. Results will be published as a database via the Internet. Multi-media and web-based instruction will be used in the course. Expertise in instructional development will be used for course development and evaluation.

Under the influence of light, electricity, or chemical reagents, certain interlocked molecules, known as catenanes and rotaxanes-which comprise appropriately matched ring and dumbbell-shaped components-will perform motions (e.g., rotary and linear) at a molecular level reminiscent of the moving parts of macroscopic machines. Such molecular motors hold promise as the intelligent" building blocks for the construction of devices and machines. A team of chemists and engineers from two different institutions (UCLA and nearby CALTECH) will address the fundamental scientific issues surrounding the relationships between controllable molecular machines, nanoscate devices, and the predictable movements of machine components at a macroscopic level.

The aims of this collaborative project-which focuses on the NSE RESEARCH THEME of Nanoscale Devices and System Architecture-are to (I) develop the template-directed synthesis (self-assembly) of interlocked molecules (switchable catenanes and rotaxanes) and interpenetrating supermolecules (addressable pseudorotaxanes) as a forerunner to (2) attaching them covalently to frameworks (e.g., silica, alumina) whose (3) synthesis (self-organization) must be established prior to (4) demonstrating the abilities of these machine-like (super)molecules to express different kinds of coherent movements (mainly linear but also possibly rotary ones) characteristic of macroscopic machines when (5) they are activated by chemicals (acids/bases or oxidizing/reducing agents) or electrons or light (redox and electron transfer processes) as a prelude to (6) transducing and amplifying the coherent molecular level movements into macroscopic motions.

The specific objectives of the team are to demonstrate transduction of force and motion from the relative mechanical movements of the components present in catenanes, rotaxanes and pseudorotaxanes through the development-on the nanoscale level-of actuating materials and devices reminiscent of (1) engines, (2) levers, (3) muscles, and (4) valves.

In thc first instance, we envisage constructing supramolecular two-stroke engines based on two-station pseudorotaxanes with the ring component lodged covalently in appropriately-sized silica pores, leaving the semi-dumbbell-shaped component to act as the piston. In the second example, we propose to design mechanical levers to amplify nanometer motions generated by suitable molecular or supramolecular machines. In the third instance, we propose to graft the ring and thread components of pseudorotaxanes onto separate carbon nanotubes using an aromatic polymer which we have demonstrated wraps itself helically around carbon nanotubes in order to realize artificial muscles and actuators. And, in the final example, we intend to develop molecular valves at the necks of suitably-sized silica pores, lined with pseudorotaxanes that can be induced to associate and dissociate (rings from threads) such that guest molecules located within the pores are, respectively, trapped or free to escape.

The anticipated outcome of the proposed program of research includes (I) the synthesis of new molecular motors capable of operating as machines, (2) the synthesis of integrated power supplies for the machines, (3) a bottom-up and top-down integration of frameworks for the machines, (4) new fundamental understanding of forces, friction, etc., on the nanoscale, and (5) a group of students with both broad perspectives and individual expertise in nanoscicnce.

With chemists and engineers working side-by-side, this highly integrated project seeks to transform molecular machines from being scientific curiosities into functioning nanosystems with technological potential, to enrich the education of both graduate and undergraduate students, and to promote the public awareness of nano-science and technology through community outreach.

The goal of this interdisciplinary research is to analyze the vast amounts of MEMS sensor data using datamining techniques to discover relationships among actions at MEMS actuators and their impact on the system state. These relationships (captured in the form of rules) are then used to build a feedback loop for aircraft control. The input-output relationships for most systems (e.g., the delta wing aircraft) are highly non-linear. Traditional datamining approaches discard much important information from the datasets and cannot provide sufficient transfer function information, which makes them unsuitable for system control. This project develops a scalable multivariate datamining technique that discovers full sensor-actuator relationships and predictive models under a wide range of conditions (dynamic, temporal, spatial, etc.). The research includes collecting data for dynamic system behavior, extending the datamining algorithms for summarizing temporal rules, developing the rule selection strategy for actuation schema, and developing wind tunnel experiments to validate the approach. This work has the potential to advance the state-of-the-art in data mining substantially, as this problem has many features (real time feedback, spatio-temporal nature) that are not commonly found in other applications. The success of data mining techniques is expected to advance the MEMS sensor and actuation technology in system monitoring and control and in other engineering problems.

This Nanoscale Interdisciplinary Research Teams (NIRT) project will address the common problem of large pressure drops in microfluidics by nano-engineering novel channel surfaces and controlling their surface properties. The consequences are expected to be both dramatic and far-reaching. The research project is to develop a nano-engineered surface to drastically reduce viscous drag. Despite the explosive growth in microfluidics, as represented by such high-profile applications as biochips and lab-on-a-chip, this fundamental problem associated with miniaturization remains unsolved: the disproportional increase in the relative pressure drop and the power consumption as devices are reduced in size. Due to the severe retardation of velocity at the surface, transport of liquids through long, nano/microscale channels encounter to high losses to be practical. Fabrication of these surfaces will be developed by integrating the rich arsenal of MEMS and Nano-technologies with the extensive knowledge of surface and biomaterial sciences, based upon the specialized expertise of the four principal investigators. Following development and characterization of the novel surfaces, an electrically re-configurable bioreactor chip will be developed as a capstone device, which further promotes synergistic integration among the team members as well as public awareness.

Fusion of the traditionally disjoint areas in this research - mechanical engineering and chemistry - start from students, who will take a set of formal courses developed and cross-offered between two schools for nanoscale science and engineering. The students continue to develop their interdisciplinary mind from monthly team meetings and weekly task meetings for research.

This Major Research Instrumentation (MRI) award provides funds for the purchase of a scanning electron microscope and a Fourier transformation infrared spectrometer. This equipment will be used to support research on development of three-dimensional nanomanufacturing technologies and on those technologies' applications in nanophotonic structures and devices. Most nanophotonic structures with designed functionalities are complicated and three-dimensional, therefore three-dimensional nanomanufacturing technologies are essential. The scanning electron microscope can provide images with high resolution, and the Fourier transformation infrared spectrometer can measure the essential optical properties of photonic structures and devices. These structural and optical properties will provide important information and guidance for the development and optimization of nanomanufacturing processes.
The research conducted using this equipment is interdisciplinary and involves the collaborative efforts of researchers from various departments. This will likely lead to advances in the sciences and technologies of nanomanufacturing, photonic material, and devices. By creating a tight-nit environment of mechanical engineers, electrical engineers, and chemists, a new generation of engineers with interdisciplinary knowledge and research experience will be trained through participating in these research activities. The advances in nanomanufacturing and education will benefit society by helping the United States maintain a competitive edge in this high technology industry and prepare a high quality workforce for the nation's economy and security.

Control over reversible biological states, such as the stepwise progression of cells toward apoptosis before the point of no return, holds promise for eradicating mankind's greatest killers, including cancer, diabetes and heart disease. A key mutable biological process that regulates cell survival and function is the formation of the dynamic cytoskeleton, in which microtubules play a leading role. Microtubules regulate intracellular transport, motility, shape, membrane trafficking, subcellular organization, cell division and responses to the environment. Not surprisingly, microtubules have become a main target for disease therapeutics, especially in cancer. Molecular technologies that increase our local and cell-wide understanding of dynamic microtubule processes are essential for gaining control over processes that depend on microtubules, such as reversible stages in cell death. A set of unique nanotech tools has already developed in our group and enables us to precisely address the aims outlined in the NIH nanomedicine initiative, which is "... to characterize quantitatively the molecular scale components ...in living cells to improve human health". We will integrate novel nanotech-based technologies into a generic platform for interrogating and controlling a wide class of cellular functions. These technologies include 1) optoelectronic tweezer technology that can precisely move cells in a programmable bio-reactor system. 2) AFM modified with multiwalled carbon nanotubes that can measure nanoscale molecular interactions occurring at the cell surface. 3) Metallo-dielectric multilayer superlens that can reach an ultimate spatial resolution of 20-30nm. 4) An "intelligent" drug which selectively targets diseased cells such as cancer cells. Coupling these nanotechnologies possessing molecular level resolution, we will be able to directly visualize and control key molecules towards enhanced intervention over processes such as apoptosis, and elucidate new targets or mechanisms for therapy.

PROPOSAL NO.: CTS-0502767
PRINCIPAL INVESTIGATORS: C-M HO
INSTITUTION: UCLA

INTERNATIONAL BIO-NANO-INFORMATION (BNI) FUSION CONFERENCE

This grant is for partial funding for the Bio-Nano-Information (BNI) Fusion Conference to be held in Marina del Rey, CA on July 20-22, 2005. NSF support will pay travel expenses and meeting costs for graduate students, and enable approximately 40 students to attend the conference. The intellectual merit of the proposed conference involves primarily the exchange of technical information in an emerging area. Observation of processes in nature (e.g. cellular function at the nano scale resulting in macroscopic structures and higher-order functionalities) leads towards a compelling approach consisting of fusing biotechnology, nanotechnology, and information science, which will enrich the development of revolutionary application-specific technologies. Fusion of bio-nano-informatics will culminate in systemic architectures that will rival those occurring in nature. Fundamental comprehension of how the interplay of these three areas can be manipulated on the molecular level to produce enhanced, emergent properties and will address the question of how much further it is possible to "push the envelope". The broader impacts of the proposal are as follows. Making advancements in emergent functionality through technology fusion is an ambitious and challenging task, which needs the participation of researchers from around the world. This conference will bring leading researchers together to share their accomplishments in bio, nano and informatics technologies, as well as to explore the potential of BNI fusion. The BNI Fusion Conference will provide students a unique opportunity to experience lectures given by preeminent specialists covering wide varieties of interdisciplinary fields. A poster session will be arranged for young researchers to present their findings. In addition to the proceedings (published on CD-ROM to facilitate multimedia) provided to each participant at the time of the symposium, selected papers are expected to be published in a special issue of an archival journal.

In human disease, various cellular signaling and molecular assemblies may behave or interact aberrantly compared to their healthy state counterparts. Often, it is unclear which molecular nano-complex in the complex network is the most important to control for treating a human disease. Recent work in myeloma provides evidence that multiple myeloma can become addicted to a normal, unmutated cellular components, in contrast to the usual idea that it is only a singular broken cellular component that is a candidate for engineering molecular interventions. The most effective way to treat a problem of this complexity is to attack on many fronts at once, with optimized drug combination treatments exemplifying this approach. CCC developed the feedback system control (FSC) scheme to rapidly and iteratively arrive at an optimized set of drugs and dosages that achieves a desired therapeutic outcome. In addition, PhosphoFlow is a multi- parameter, single-cell phosphorylation analysis methodology that CCC use to identify key protein complexes that control outcomes. Importantly, PhosphoFlow also is used to exclude candidate molecular assemblies that are minor drivers or not involved in specific system outcomes. With this methodology, key protein complexes were rapidly identified, such as the S6-regulated ribosome translation complex that behaves aberrantly in HSV-1 infection. We have also applied this powerful yet broadly applicable FSC and PhosphoFlow two-step approach to investigate another major disease, cancer. In non-small cell lung cancer (NSCLC) and WEHl-231 leukemia cell line tests, high levels of S6 ribosomal complex activity were also observed. The goals of CCC is to i) identify and manipulate/engineer interactions between the key molecular assemblies such as S6-regulated ribosome assembly to improve the treatment of representatives of two major disease classes, cancer and infection, ii) apply our newly gained molecular and cell-based knowledge on manipulating the ribosome to small animal model preclinical tests, and iii) further advance our lung cancer clinical tests by applying this new knowledge in manipulating the ribosome and interacting/regulating pathways. RELEVANCE (See instructions): Lung cancer is the deadliest form of cancer in the U.S. and is responsible for more deaths each year than breast, prostate, colon, hepatic, renal, and skin cancers combined. HSV-1 is one ofthe most pervasive infections with as much as 90% of adults having been exposed to HSV in their lifetime. In the first 4-year NDC period, we were able to showed the advantage of a two-drug therapy in mouse model and clinical tests and identify kev mqlectjlar components in inhibiting HSV infection.

This project studies the mechanisms by which vascular-derived stem cells spontaneously organize into patterns, and then uses these mechanisms to devise some simple controls on pattern formation. Building on our previous work using Partial Differential Equation models of the developing stem cell culture system, we will develop a series of predictions addressing how altered boundary conditions change the occurrence, size, shape and other parameters of the pattern. We will extend our previous modeling efforts by incorporating cells and cell movement in the model, which had previously been purely chemical. We will also study how the mechanical and chemical properties of the substrate alter the evolving pattern, and how the features of cell proliferation and movement affect the pattern. Predictions will be tested in an experimental culture system of vascular-derived adult stem cells.

The process by which cells self-organize into structured patterns ("pattern formation") is essential to many biological processes. The development of the embryo, for example, can be viewed as a sequence of these processes. It is clearly important to understand the mechanisms of these processes in order to be able to do successful ?tissue engineering?. In the past, a common approach to tissue engineering has been to decide what patterns are desired, and then to attempt to use direct external controls to produce that pattern, without an understanding of the underlying biological organizational principles. However, biological systems are characterized by their capacity for self-organization, which can defeat and frustrate direct attempts at pattern control. Here, we propose to take advantage of the self-organizational behavior of stem cells to derive engineering approaches to the inverse problem of coaxing complex biological systems into a desired form. We believe that the shape of the boundary, the elastic and chemical properties of the surface on which they are grown, and the migratory tendencies of the cells are important contributors to pattern formation. We expect that insights gained from this study will have a positive impact on attempts to achieve tissue engineering of biological complex structures.

PROJECT SUMMARY The authors have developed a computational platform to rapidly identify optimal drug and dose combinations from the innumerable possibilities. By testing this technique termed Phenotypic Personalized Medicine (PPM) in a diverse number of experimental systems representing different diseases, they have found that the response of biological systems to drugs can be described by a low order, smooth multidimensional surface. The main consequence of this is that optimal drug combinations can be found in a small number of tests and that translation from in vitro to in vivo and ultimately to clinical application is enabled through a re- optimization process. This input?output relationship that is always based on experimental data in lieu of predicted responses may also lead to a straightforward solution for handling human diversity in cancer therapeutics, among other diseases. In these series of studies they will test the hypothesis that PPM can be developed and validated for clinical use by using it to find novel drug combinations of repurposed/repositioned drugs to treat hepatocellular carcinoma. The goal is that by the end of year 3 of this project, they will be able to initiate a clinical trial using these novel combination or combinations. This group has previously used PPM-based optimization to find novel drug combinations in in vitro and in vivo models of cancer and infection. They have shown that this approach was able to markedly improve the efficacy of colorectal cancer therapy in vivo in mouse models. Translationally in a first-in-human clinical trial, they recently completed a prospective clinical study involving 4 PPM-dosed patients and 4 control (standard of care dosed) patients. They calculated the tacrolimus dosing regimen using the PPM process. Because PPM does not require a priori knowledge of disease mechanism and because it is a dynamic process that can accommodate a changing system, it can efficiently find personalized drug dosing over a varying range of time, having a profound stabilizing effect on the tacrolimus trough levels. For this application, they have selected hepatocellular carcinoma (HCC, liver cancer) to be the human disease for PPM application. The key rationale for this clinical selection is that they have a wealth of in vitro data on HCC, an active HCC tumor biorepository, and a large clinical volume of patients with HCC. These existing resources, both in vitro and clinical, allow for the immediate exploration of combination discovery followed by a clinical validation of discovered combination candidates in patients with unresectable HCC.