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The near infrared region (700 nm – 2000 nm) of the electromagnetic spectrum provides significant potential for fingermark detection. Many ubiquitous commercial surfaces give luminescent interferences that can present a challenge for latent fingermark enhancement. Background interference from these types of surfaces can be reduced when viewed in the near infrared region. The development of near infrared luminescent techniques for latent fingermarks would improve the possibility of imaging an exploitable fingermark. This research aimed to develop methods for near infrared detection of latent fingermarks across a number of different surface types and assess the effectiveness of the developed techniques by comparing them to conventional detection methods. A mixture of two dyes, styryl 11 and rhodamine 6G (STaR 11), was coated onto a range of metal oxide powders to produce a luminescent fingerprint powder. This was applied as a dry powder for fingermarks on non-porous surfaces as well as a suspension for developing fingermarks on adhesive and wetted surfaces. The dry powder was successful in developing fingermarks and gave comparable results to a commercially available luminescent fingermark powder. The suspension for adhesive surfaces was able to develop fingermarks however when compared to the commercial method, the developed fingermarks were of significantly poor quality. The suspension for wetted surfaces, when used in conjunction with the EcoSpray® device (a pressurised sprayer which delivers the suspension in a fine mist to prevent fingermark damage), had shown significant promise when compared to conventional luminescent SPR. Ultimately, however, the suspension was unable to develop natural fingermarks, which affected its potential for routine use. Styrylisatin was trialled as a potential near infrared luminescent amino acid sensitive reagent for the detection of latent fingermarks on porous surfaces. Styrylisatin was successfully synthesised, however there were s

Fingerprinting is a mainstay of forensic science and has been used in crime investigation for more than one hundred years. However, most fingermarks found at a crime scene are latent; they may become visible through development and enhancement. Among all the fingermark development techniques, conventional luminescence methods are routinely employed, with the advantages of being both sensitive and selective on non-luminescent substrates (i.e., providing high contrast in developed marks). Anti-Stokes luminescence or upconversion is an optical process of converting long-wavelength radiation into a shorter-wavelength emission, which is contrary to conventional Stokes luminescence. Upconversion mainly exits in rare-earth complexes and upconversion materials are referred to as upconverters. Commercially-available upconverters have been widely employed in security inks and biolabels. Upconversion is unusual in both natural surfaces and in consumer products. If the upconverters are applied for fingermark detection and show selective affinity to fingermark materials, theoretically the strong luminescence of the upconverters can be visualised on fingermarks as bright regions on a totally dark background. This means that fingermark detection techniques using upconverters has the potential to eliminate interference from background printing and luminescence. This thesis begins with the review of luminescence-based fingermark detection techniques and previous research on the application of upconverters for fingermark detection. The previous research showed that upconverters have an affinity for fingermark residues and are effective for fingermark detection. Chapter 2 describes issues with respect to imaging the upconversion luminescence. Of the options tested, a 980 nm laser pointer with 700 mW output proved to be the most suitable light source for the excitation of the upconversion luminescence. Long exposure times were needed to record the upconversion luminescence. A Rofin Pol

To derive an optimum design of a MEMS microphone in any application (i.e. photoacoustic instrumentation), it is important to understand the underlying physics that govern the behavior of the device. In addition, one must have a good understanding of the specific requirements imposed on the device in the intended application. In the particular case of photoacoustic detection, signal-to-noise ratio is the overwhelmingly important parameter. Other parameters such as size and required operating voltage may be compromised to achieve the best possible signal-to-noise ratio. An important property, to be shown below, is the low sensitivity to vibration in MEMS microphones due to a much smaller mass of inertia of the sensing diaphragm, when compared to state-of-the-art conventional microphones. In photoacoustic detection, a microphone is used to detect the minute thermal expansion/pressure wave generated in a gas due to molecular absorption, and subsequent release, of energy generated from a light source 1,6. This method is very well suited for molecular fingerprinting, since the absorption versus applied light energy/wavelength is uniquely dependent on the exact molecular structure. The measurement, in which light of various wavelengths is applied, to map the molecular absorption, is referred to as photoacoustic spectroscopy (PAS). Current PAS instrumentation utilizes state-of-the-art conventional microphone technology in combination with high powered light sources to maximize the sensitivity of the system. It is well known from literature (such as 2), that electrostatic, or capacitive, microphones have the highest sensitivity and the lowest self-noise of the known detection principles. while conventional capacitive microphones provide excellent signal-to- noise ratio, there is a significant problem with vibration-borne artifacts.

Denaturing high-performance liquid chromatography (DHPLC) has been used extensively to detect genetic variation. We used this method to detect and identify Yersinia pestis KIM5 ciprofloxacin-resistant isolates by analyzing the quinolone resistance-determining region (QRDR) of the gyrase A gene. Sequencing of the Y. pestis KIM5 strain gyrA QRDR from 55 ciprofloxacin-resistant isolates revealed five mutation types. We analyzed the gyrA QRDR by DHPLC to assess its ability to detect point mutations and to determine whether DHPLC peak profile analysis could be used as a molecular fingerprint. In addition to the five mutation types found in our ciprofloxacin-resistant isolates, several mutations in the QRDR were generated by site-directed mutagenesis and analyzed to further evaluate this method for the ability to detect QRDR mutations. Furthermore, a blind panel of 42 samples was analyzed by screening for two mutant types to evaluate the potential diagnostic value of this method. Our results showed that DHPLC is an efficient method for detecting mutations in genes that confer antibiotic resistance., The original document contains color images. Pub. in Journal of Clinical Microbiology, v71 n7, p3273-3283, Jul 2003.

The importance of DNA abnormalities to tumorigenesis is not in doubt but their clinical utility has not yet been fully elucidated. We performed a pilot study to evaluate detection of occult circulating tumor DNA using tumor-specific microsatellite alterations in subjects previously diagnosed with breast cancer. Using 22 microsatellite markers located at sites of frequent loss of heterozygosity (LOH) in breast cancer, we analyzed DNA from 16 primary tumors (Stage IIA or more advanced) and 30 longitudinally collected plasma specimens. Clinical data at time of plasma collection was obtained. Every tumor (16/16, 100%) was characterized by an individual pattern of LOH, its tOH fingerprint. LOH in plasma DNA was detected in 12/30(40%) plasma DNA samples, from 8/14(57%) subjects. However, the number of LOH in plasma DNA was small (n=15), and the mean proportional Loli was much lower than in tumors (0.05vs0.52). Although they were infrequent, most LORs in plasma DNA (12/15, 80%) were concordant with abnormalities in the paired tumors, suggesting that they were authentic tumor-derived abnormalities. Despite this, we found no association between plasma DNA LOH and original tumor stage or clinical status at time of blood collection (i.e., LOH was seen as often in subjects who had no evident disease as in those with evident disease). In addition, detection of LOH was not consistent between serial samples from half of subjects (5/11, 45%), despite stable clinical condition. No association with clinical outcome was evident, although the sample size may be too small to be conclusive. Microsatellite instability (MI) in plasma DNA was infrequent, non-concordant with paired tumor, and inconsistent in serial samples. The results of this study suggest that detecting microsatellite abnormalities in plasma DNA of breast cancer subjects may nOt be useful either for detecting occult metastases or for monitoring disease. Other techniques may be more promising but it is possible

Optical approaches to breast cancer (BC) detection show promise over conventional methods because they have potential to work in the denser tissue of younger patients and because they do not use ionizing radiation which has been shown to induce cancer in some patients. Three technologies will be combined in this effort. (a) Optical probing, using controlled light at specific wavelengths, is becoming a respected approach to cancer tumor detection. A much-published RADAR-based method known as frequency-domain photon migration (FDPM) gives very high sensitivity. (b) "Spectral fingerprint" analysis of fluorescence emission is the basis for cancer and precancer classification affecting surface cells. This has been shown for cervical and lung cancers. (c) Reading images, such as x-ray films, allows the tumor to stand out relative to the "expected" complex image of the breast. An optical hardware system was envisioned and preliminary testing performed. Hardware was to be based on using technically similar systems used in unrelated industrial applications to make hardware development affordable. Specific systems were identified. Ultimately, collaborators could not be found to modify their systems, and system development proved to be prohibitively costly. Discussions with other researchers has provided a voice for this approach within the community, paving the way for valuable contributions to BC detection., The original document contains color images. All DTIC reproductions will be in black and white.

Caspase-mediated cleavage of PAK2 is a hallmark of the apoptotic response accompanied by strong JNK activation. The purpose of this study is to characterize the PAK-JNK signaling pathway and to identity% signaling components mediating the JNK activation. In this study we isolated and identified a substrate of p21 -activated kinase PAK which might be a downstream regulator in signaling to the Stress-activated kinase module (SAPK) and the morphological response observed in cells undergoing apoptosis. Peptide mass fingerprinting and immunological methods identified this protein as the guanine nucleotide exchange factor GEF-Hl/K1AAO651. GEF- H1 is strongly phosphorylated by PAK. The phosphorylation site was identified using deletion mapping, phosphopeptide/-amino acid analysis and in vitro mutagenesis. The central dbl-homology domain of the exchange factor mediates strong JNK activation, which seems not to be mediated by the known SAPK-regulatory GTPases Rac and Cdc42. Using in vitro exchange factor assays we observe activation of RhoA, but not Raci. Currently, we test the hypothesis of PAK mediating JNK activation via GEF-H1 and RhoGTPases.

Characterization of the cis and trans regulatory factors involved in BRCA1 transcription, was performed. The previously identified positive regulatory region (PRR), was narrowed to 37 bases by deletion mutations. The region consisted of a Polypyrimidine/Polypurine (py/Pu) motif and a CREB like site. The 37 base pair (bp) region was sufficient to direct transcription. Introduction of point mutations impaired BRCA1 transcription. As this region (specifically Py/Pu) was also responsive to estrogen (reported in the previous summary), we biochemically purified the factors which bind it. These factors were identified by peptide mass fingerprinting analysis as subunits of Replication Protein A. The binding of RPA was verified by supershift assays with monoclonal antibodies. Overall the studies indicate that estrogen responsive site within the BRCA1 promoter binds Replication factors. This also suggests that estrogen signaling to BRCA1 promoter maybe mediated by RPA. Aberrant estrogen signaling and/or abnormal functioning of the RPA complex may compromise BRCA1 transcription and contribute to the observed suppression of BRCA1 mRNA in sporadic breast cancers., Original contains color plates: All DTIC reproductions will be in black and white.

The overall goal of this proposal is to test the hypothesis that eosinophils promote DNA oxidative damage in breast carcinoma. DNA oxidative damage is linked to mutation, transformation and cancer development and eosinophil peroxidase (EPO), a hemoprotein secreted from eosinophils, is present in the majority of breast cancer biopsies. Our initial aim was to determine whether EBO promotes oxidative damage of cellular DNA through formation of mutagenic hydroxyl radical (OH) -generated bases. Last year we published a study (Biochemistry, (2O00) 39:5474) that demonstrated activated leukocytes can oxidatively damage DNA, RNA and the nucleotide pool through halide-dependent formation of OH. OH-dependent damage of DNA was quantified by monitoring the content of 8- hydroxyguanine (8OHG), an established OH-specific DNA oxidation product that is mutagenic and implicated in breast cancer development and progression to metastatic disease. To test the hypothesis that EPO promotes DNA oxidative damage in human breast carcinoma, we have identified a family of novel brominated DNA oxidation products. These may serve as molecular fingerprints" for DNA damage by the EPO pathway of eosinophils. The results of our initial studies identifying brominated bases by mass spectrometry were recently published (Biochemistry, (2001) 40:2041-2051) We are presently performing studies aimed at quantifying these EPO-specific brominated bases in a well-characterized repository of breast carcinoma and microscopically normal breast tissue specimens.

In the original fellowship proposal, I planed to profile protease activity using substrate phage display library in the biological samples from mice at different stages of breast cancer. I first used the substrate phage display library I constructed to characterize substrate recognition profiles of two tumor-related proteases, Metalloproteinase-2 (MMP-2) and 9 (MMP-9). Three groups of MMP-2 substrate sequences were novel and found highly selective for MMP-2 over MMP-9. This result supports the hypothesis that substrate phage display library can be used to differentiate diminutive structural difference of proteases. However, I found that phage display library had limited application for in vivo protease profiling because only small quantity of proteases present in the biological samples. Hence, I modified my approach of carrying out the functional study of disease-related proteases and developed a one-pot phage selection system that yield the substrate recognition profile of multiple purified proteases from a single round of selection. This method allows analysis of multiple proteases simultaneously, and prior knowledge of substrate preference is not required. As an illustration, a phage selection with a mixture of thrombin and factor Xa (serine proteases) along with MMP-9 and atrolysin C (metalloproteinases). Peptide substrates were identified that (1) have high kcat/Km ration, (2) are selective for individual proteases, and (3) match the sequences of known physiological substrates. Ultimately, highly selective peptide substrates for disease-related proteases can be obtained, and an array of selective peptide substrates can be assembled and profile protease activity in the biological samples from mice at different stages of breast cancer.

Objects located in highly scattering biomedical media can be imaged with sub-millimeter spatial resolution using the early light selected by time, spatial, and polarization gates. Spectroscopic fingerprints can provide diagnostic potentials for medical screening by utilizing fluorescence, excitation, absorption, and Raman approaches., Proceedings of SPIE, v4082, 26-27 Jul 2000, p8-13. This article is from ADA398019 Optical Sensing, Imaging and Manipulation for Biological and Biomedical Applications Held in Taipei, Taiwan on 26-27 July 2000. Proceedings

We proposed to investigate breast cancer-associated DNA methylation alterations using a technique named Methylation Sensitive Restriction Fragment Length Polymorphism (MS-AFLP) which we developed. We performed NotI-MseI MS-AFLP using clinical specimens of normal and tumor breast DNA. We used both combinations of four NotI and four MseI primers with an additional selective residue at the 3' end (4x4 format) and combinations of four NotI with one additional residue and sixteen MseI with two additional selective residues (4x16 format). We have identified many bands that exhibited alterations in band intensity between normal and tumor breast DNA fingerprints. We have cut out the dried gel slices from those bands, eluted and PCR-amplified DNA fragments, and cloned into a plasmid vector. After transformation of E. coli bacteria, the nucleotide sequences of inserts were determined from several transformant clones. For some of the interesting bands, we have also transferred DNA from the DNA fingerprints by electroblotting onto nylon membranes, hybridized those membranes with fragment probes prepared from individual clones, and determined the identity of the fragments showing alterations. From these studies, we have obtained results indicating the possible associations of homeotic genes and related genes in the pathogenesis of breast cancer.

The overall goal of this proposal was to test the hypothesis that eosinophils promote DNA oxidative damage in breast carcinoma. DNA oxidative damage is linked to mutation, transformation and cancer development and eosinophil peroxidase (EPO), a hemoprotein secreted from eosinophils, is present in the majority of breast cancer biopsies. Our initial aim was to determine whether EPO promotes oxidative damage of cellular DNA through formation of mutagenic hydroxyl radical (OH) -generated bases. In a recent published study (Biochemistry, 39:5474) we show that activated leukocytes oxidatively damage DNA, RNA and the nucleotide pool through halide-dependent formation of OH. *OH-dependent damage of DNA was quantified by monitoring the content of 8-hydroxyguanine (SOHO), an established OH- specific DNA oxidation product that is mutagenic and implicated in breast cancer development and progression to metastatic disease. To test the hypothesis that EPO promotes DNA oxidative damage in human breast carcinoma, we have identified a family of novel brominated DNA oxidation products. These may serve as "molecular fingerprints" for DNA damage by the EPO pathway of eosinophils. We are presently developing GC/Ms methods to quantify these EPO-specific brominated bases in a well-characterized repository of breast carcinoma and microscopically normal breast tissue specimens.

Ductal carcinoma in situ (DCIS) is a pre-invasive stage of breast cancer in which the malignant cells have not penetrated the basement membrane. From our allelotyping study of DCIS, chromosomal regions of 8p, 13q, l6q, 17p and 17q showed loss of heterozygosity (LOR) significantly above background (5%). In our previous report we described the LOR mapping of a region on 8p (30% LOR) containing a putative tumor suppressor gene (TSG) which we refined to a small interval of 1.4 cM between the markers D8S265 to D8SD520. Last year (1998) we completed the construction of a fine structure radiation hybrid map and an integrated YAC/BAC contig spanning the 1.1 -cM deletion region. During the past project year, we mapped eleven expressed sequence tags (ESTs) including 4 previously reported ESTs to this contig. Twenty-three overlapping oligo (overgo) probes were generated using end sequences derived from CITB BAC clones. These overgos were used to screen RPCI-l 1 BAC library. A total of 299 clones including CITB BAC clones were isolated and then fingerprinted. A comprehensive sequence-ready contig that provides a useful resource for sequencing the critical region has been assembled using clones with good fingerprinting data.

Detection of airborne biological particulates is a primary mission of the U.S. Army Edgewood Research, Development and Engineering Center biological defense program. If biological particles could be characterized according to their unique physical and biochemical profiles, detection and perhaps even identification of the particles might be possible. This study focused upon microbial particles, more specifically upon fungal spores, yeast cells, and bacterial cells. Physical characteristics of the particles, it was proposed, could be detected by flow cytometry, while their biochemical profiles could be determined by gas chromatography, and their genetic identity could be obtained by either a suitable genetic probe or by matching its genetic fingerprint. Genetic techniques were not attempted in the work reported here, but the approach was investigated further. Trial results were encouraging.

Before turning to the results of our work in detail, let me briefly provide some background information and discuss the methodology we used for our study. During their deployment associated with the Persian Gulf War, many of the approximately 700,000 veterans of the Gulf War may have been exposed to a variety of potentially hazardous substances. These substances include compounds used to decontaminate equipment and protect it against chemical agents, fuel used as a sand suppressant in and around encampments, fuel oil used to burn human waste, fuel in shower water, leaded vehicle exhaust used to dry sleeping bags, depleted uranium, parasites, pesticides, drugs to protect against chemical warfare agents (such as pyridostigmine bromide), and smoke from oil-well fires. Moreover, DOD acknowledged in June 1996 that some veterans may have been exposed to the nerve agent sarin following the postwar demolition of Iraqi ammunition facilities. Many of these veterans have complained of a wide array of symptoms and disabling conditions since the end of the war in 1991. Some fear that they are suffering from chronic disabling conditions because of exposure to chemicals, pesticides, and other agents used during the war with known or suspected health effects. Accordingly, both DOD and VA established programs through which Gulf War veterans could receive medical examinations and diagnostic services. From 1992 to 1994, VA participants received a regular physical examination with basic laboratory tests. In 1994, VA established a standardized examination to obtain information about exposures and symptoms related to diseases endemic to the Gulf region and to order specific tests to detect the 'biochemical fingerprints' of certain diseases. If a diagnosis was not apparent, veterans could receive up to 22 additional tests and additional specialty consultations., Testimony before the Committee on Government Reform and Oversight, Subcommittee on Human Resources, House of Representatives.

This report describes the work carried out on the specificity of four oncogenic peptide-monoclonal antibody interactions. Monoclonal antibody 172-12A4, which was raised against a peptide from v-erbB was assayed by ELISA with a set of 152 substitution analogs of the antigenic determinant to yield a fingerprint profile of this antigen-antibody interaction. A number of peptide libraries have also been screened and the deconvolution of individual sequences is ongoing. Peptide libraries have been screened against mAb 121-15B10, which was raised against a peptide from v-fes and is homologous with the v-erbB peptide. Peptides with nanomolar affinities have been identified. The antigenic determinants for six antibodies against two different oncogenic peptides were located using omission analogs. Substitution analogs are being synthesized to identify the amino acid specificities of each of these antibodies. Peptide libraries were also screened against these two peptide-antibody interactions, and the deconvolution process is ongoing. The understanding of antibody specificity at the amino acid level will help in the development of these antibodies as early diagnostics for breast cancer.

Major phases of this study included providing the results, critical evaluation, and implications of a literature search in a 'white paper' on advanced analytical methodology for mobility fuels and lubricants applications, assessment of a commercial used engine oil evaluation methodology, and analysis and characterization of new lubricants. In assessing a commercial used oil analysis method, it was concluded that this FTIR-based used oil analysis method should he used only for trend analysis in which the lubricant and the fuel are known and controlled. Efforts were made to characterize and identify lubricants through instrumental analyses, including high-temperature gas chromatography, gel permeation chromatography, elemental analysis, spectroscopic fingerprinting, and establishing FTIR spectral libraries. To increase usefulness of the results of libraries of FTIR spectra data bases have been constructed to include all known specification and inspection data. Calibration curves were established to measure several major lubricating oil additive concentrations in a lubricant base stock, through their FTIR responses. Several increasingly complex experimental protocols are suggested, depending upon the source of lubricant and the needed lubricant property requirements. While a final experimental protocol has not yet been established, a tentative analytical chemical protocol was developed. Present experimental protocol does not address the detection and destruction of antimaterial agents. jg

This report describes concepts and procedures that convert the NATO Reference Mobility Model (NRMM) from a deterministic code to a stochastic one. The motivation for the conversion is the opportunity being presented by advances in computer technology that can allow NRMM to be used in the fast-paced tactical environment of a battlefield. Appropriate components of a stochastic mobility forecast are identified as a speed map, a fingerprint, a mission-rating speed and a range for the mission-rating speed. The speed map and the mission-rating speed are the current NRMM products; the fingerprint and range are new components that describe the performance of NRMM when its data contain errors. Quantification of error performance becomes very important to mobility planning in a tactical setting. The procedures are illustrated by means of a comprehensive and robust example involving four vehicles, four terrain maps and two scenarios. The procedures are somewhat preliminary in the sense that alternative implementations are possible. Minimization of computational bottlenecks motivates the selections; other selections may be more appropriate in the future. Mathematical models; Sensitivity; Mobility; Vehicles, military.