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GRADUATE STUDENTS

Maria Angelica Mendoza Baez "Gela", Ph. D.

Cannabis sativa L., marijuana, plants can be easily identified through morphological examination and chemical analysis; however, there is a need for a DNA method with the potential to differentiate between individual plants. In so doing, this method can be used as a means of associating criminal cases and tracking cannabis distribution networks. DNA typing of cannabis will also help generate a frequency database which can be used to identify plant origin and or source. This research project aims to develop and validate a ten STR multiplex reaction for Cannabis sativa. Several cannabis cultivars (with high THC content from different origins) will be typed using the multiplex reaction in hopes of generating a frequency database which will be used to assess locus polymorphism levels.

Jill Fetscher

Mixtures of cells are often found in a variety of matrices, making it difficult to identify an individual contributor from the mixture. For example, sperm and epithelial cells from sexual assault evidence are common mixtures that prove difficult to analyze. The samples are most often obtained via a vaginal swab from the victim and contain both victim and attacker cell fractions often making it extremely difficult to individualize the suspected rapist. In order to acquire a single source of cells from a mixed sample, cells should be separated prior to DNA extraction and amplification. The objective of this research is to use magnetic nanoparticles/beads modified with hyperbranched dendrimers having multiple cell-surface specific monoclonal antibodies bound to the reactive groups of the outermost dendrimer generation. This modification should significantly increase the strength of target cell binding by increasing the surface area on the magnetic particles thus providing excess binding sites for cell attachment. The target cells will be bound to antibodies and non-target cells will be washed away. Subsequent lysis of the target cells will release the target cell’s DNA for amplification without further purification steps.

Beatrice Kallifatidis

The ability to rapidly and reliably determine the sex of birds is very important for successful captive bird breeding programs, as well as for field research. In nestlings and monomorphic species it is difficult, if not impossible, to determine the sex by sight only. It is estimated that adult females appear identical to males in over 50% of the world’s bird species. The purpose of this research project is avian sex determination with DNA extracted from feathers using real-time PCR. In birds the female is the heterogametic sex (ZW) and the male is the homogametic sex (ZZ). The gene encoding chromo-helicase-DNA binding protein 1 (CHD-1) has been found on both the Z and W chromosomes, however, the alleles are not identical. The CHD-1 gene has at least one intron of different sizes in the W and Z alleles, which provides an opportunity to differentiate between males and females using PCR. Thus, by amplifying homologous sections of both genes (CHD-W gene and CHD-Z gene) and by incorporating introns whose lengths differ in the W and Z chromosome the sex of birds can be determined. After running an agarose gel we expect to see a single band in males (derived from the Z-chromosome) and two bands in females (derived from the Z and W chromosome).

Ashley Lekas-Diaz

Soil microbial-forensics a new area of research, initializing a specialized interest in the microbial communities within the soil composition. The profiling of the microorganisms present in soil has the ability to introduce and validate a new discipline for the forensic science community. This
project focuses on universal primer sets that amplify hypervariable molecular markers from the microorganisms in the soil. Among the microorganisms that were queried are bacteria, fungi, archaea, plant and
nematodes. Amplified Length Heterogeneity-PCR technique will be used to amplify hypervariable domains that can identify the presence of the aforementioned microorganisms. The expected outcome is to develop a forensic application or kit that will select for multiple taxa, decreasing the amount of work, increasing the turn around time for results and most importantly increasing the power of discrimination for any evidence matches. In
addition to a forensic application, this multiplex approach has great potential for the use in microbial ecology as well.

Natalie Leyva The purpose of my thesis project is to assess the genetic health and degree of inbreeding within the wild horses inhabiting the Ochoco National Forest in Oregon. This will enable the US Forest Service to better manage the herd size and the genetic wellbeing of the herd. The project can be divided into three objectives: 1) assessing genetic relatedness within the herd using microsatellite analyses and non-invasive sampling techniques (i.e., hair samples); 2) based on the molecular data, aid in the census of horses occupying the rangeland; and 3) forensically, be able to discriminate between the Ochoco wild horses and those known as “trespass” horses, those that do not belong on the managed lands. Presently, I am working on a DNA pooling technique to further increase the speed of the research and make the analysis more efficient. The full identification of the individuals will be based on the height of the frequency peaks, it will not profile a single horse; but will give an estimate to the number of individuals making up the herd.

Molecular and Chemical Characterization of Three Miami-Dade Soil Types for Forensic Comparison Lilliana I. Moreno
The usefulness of any evidence relies on the number of significant variations that may be encountered in the material, which can be easily measured, observed and matched. Soil is a ubiquitous material that is easily transferred from one place to another. Traditional soil characterizations tend to be too broad for specific location identification, thus providing limited use in forensics. However, soil forensics has been gaining importance as newer techniques prove to be useful in its characterization and comparison. Locally, Miami-Dade County soil types are broadly divided into six categories. This study proposes to use both chemical and molecular methods to supplement traditional methods to further characterize three of the Miami-Dade soil types. Pristine soil samples will be collected bi-annually and, in addition, an arsenic contaminated site will be used as an internal control to determine how contamination influences the basic soil characteristics. All soil samples will be subjected to standard physical analysis such as nutrient content, color, pH, and moisture. Inductively coupled plasma mass spectrometry (ICP-MS) will be used to obtain a refinied chemical profile. Molecular characterization will involve amplification of bacterial16S rRNA genes heterogeneous regions that will provide a profile that is unique to each soil sample. The length heterogeneitypolymerase chain reaction (LH-PCR) technique, which is not commonly known by forensic investigators, will be used. The chemical profile will be compared with the physical and amplicon/microbial data to determine the best sensor for soil discrimination.
Melissa S. Doud	Since the anthrax letters of 2001, the threat of biowarfare has become a reality. Recently the United States government has asked researchers to join forces with them to fight against bioterrorism. Microbial forensics is a scientific discipline that is designed to analyze evidence from a bioterrorism act or a biocrime. This area of science seeks to find quick ways to detect and identify agents that are used in biowarfare, establish the source of the agent and thus track down potential suspects to the crime.
Amplicon length heterogeneity-polymerase chain reaction (LH-PCR) is one method that can be used to study bacterial communities that contain potential pathogens. LH-PCR detects differences between eubactaria by analyzing the hypervariable regions of the 16S rRNA gene. Using sputum from cystic fibrosis (CF) patients as my model system, I propose to optimize the LH-PCR technique so that it may be used in eubacterial samples that are contaminated with human DNA. Samples of CF sputum from different individuals will be profiled in triplicate to establish the reproducibility of the technique and the uniqueness of each profile. Also, a reference ladder containing the most common eubacteria in CF lungs will be developed to increase the efficiency of identifying organisms present in the sample. LH-PCR has the potential to be used the fight against bioterrorism and even in routine casework seen in crime laboratories. This is the research being performed by Melissa S. Doud, B.A. graduated from Elmira College 2003. Currently working under Dr. Kalai Mathee

Microsatellite markers are a powerful tool for DNA testing in animals, with applications that span from conservation biology to forensic science. The USDA Forest Service is interested in using fecal samples from equines to estimate the population size of a herd and to monitor the genetic health of the population for signs of inbreeding. This will involve the development of the best preservation method and DNA extraction method for use with fecal samples. The real time-polymerase chain reaction (RT-PCR) will be used to quantify equine DNA from fecal samples and this procedure will be optimized. An existing commercial equine short tandem repeat (STR) multiplex kit will be used to profile wild mustang fecal samples provided by the USDA Forest Service, and a database will be created. Furthermore, an in-house equine multiplex STR kit will be developed using published primers in order to be more independent of Applied Biosystems and to reduce costs of equine profiling services. Subsequently, a database will be developed using this new kit.	Margaret Shekarkhar
UNDERGRADUATEs Alejandro Castaneda I used soil slurries that were subjected to different agricultural chemical treatments to inoculate BIOLOG Ecoplates™ and analyzed the microbial communities therein. BIOLOG EcoPlates™ consist of 96 well plates containing 31 different carbon sources and a blank in triplicate and were developed specifically for ecological studies to measure the community level physiological response. Formation of purple color occurswhen the microbes utilize the carbon source and begin to respire. The respiration of the cells reduces a tetrazolium dye that is included with each carbon source and the color change is read with the spectrophotometer. The treatments included two antibiotics, two fungicides, elemental sulfur to lower soil pH and a control treatment. Based on the results I compared the effect of the treatments on the metabolic diversity of the soil community compared to the control soil (no treatment). The results showed the fungicides increased community metabolic diversity while the antibiotics decreased it and the sulfur treatment was no different from the control. The experiments will be repeated with pulsed addition of the chemicals to see if chronic application of these agricultural chemicals impact the communities to the point they cannot fully recover or drives permanent changes in the community structure and function over time. Christopher Abin In close collaboration with two of my colleagues, our goal is to elucidate the link between microbial community structure and function and the environmental factors that drive them. I approached Dr. Mills about conducting research in her lab after I was accepted to the RISE program during the spring of 2009. As an aspiring microbial ecologist, I found Dr. Mills’ research on soil microbiology to be quite interesting and potentially fulfilling. I hope to attend graduate school with aspirations of becoming a microbial ecologist. After earning my Ph.D. and completing a post-doctoral fellowship, I will strive to secure a faculty position at a university, where I will continue my research and teach future generations of students. Merly Suarez The goal is to assess the genetic health of the wild mustangs of the Ochoco National Forest in Oregon. This will be accomplished by using non-invasive sampling techniques for performing genetic, population estimates and inbreeding analysis. Hair samples were obtained (some from captured horses and others from pine trees which horses rub against and leave behind hair) and are going to be used to determine if this herd of mustangs may have specific inherited genetic diseases (since the herd is believed to be inbred). The diseases that will be assessed are: Hyperkalemic periodic paralysis (HYPP, which results in the horse having muscle paralysis), Severe combined immunodeficiency (SCID, foal dies by 5 months of age), Glycogen branching enzyme deficiency, and Polysaccharide storage myopathy. Guillermo Chavez Testing the efficacy of cleaners, and measuring their effects on Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Several different tests were done using these three bacteria as model systems, such as disc diffusion assays and exposing bacterial samples to disinfectants according to their recommended exposure time and subsequent plating on growth media in order to determine the efficacy of the cleaner being tested. The study’s aim was to see whether all the cleaners tested would be equally effective irregardless of there intended usage (commercial or household).

	Maria Angelica Mendoza Baez "Gela", Ph. D. Cannabis sativa L., marijuana, plants can be easily identified through morphological examination and chemical analysis; however, there is a need for a DNA method with the potential to differentiate between individual plants. In so doing, this method can be used as a means of associating criminal cases and tracking cannabis distribution networks. DNA typing of cannabis will also help generate a frequency database which can be used to identify plant origin and or source. This research project aims to develop and validate a ten STR multiplex reaction for Cannabis sativa. Several cannabis cultivars (with high THC content from different origins) will be typed using the multiplex reaction in hopes of generating a frequency database which will be used to assess locus polymorphism levels.

	Jill Fetscher Mixtures of cells are often found in a variety of matrices, making it difficult to identify an individual contributor from the mixture. For example, sperm and epithelial cells from sexual assault evidence are common mixtures that prove difficult to analyze. The samples are most often obtained via a vaginal swab from the victim and contain both victim and attacker cell fractions often making it extremely difficult to individualize the suspected rapist. In order to acquire a single source of cells from a mixed sample, cells should be separated prior to DNA extraction and amplification. The objective of this research is to use magnetic nanoparticles/beads modified with hyperbranched dendrimers having multiple cell-surface specific monoclonal antibodies bound to the reactive groups of the outermost dendrimer generation. This modification should significantly increase the strength of target cell binding by increasing the surface area on the magnetic particles thus providing excess binding sites for cell attachment. The target cells will be bound to antibodies and non-target cells will be washed away. Subsequent lysis of the target cells will release the target cell’s DNA for amplification without further purification steps.
	Beatrice Kallifatidis The ability to rapidly and reliably determine the sex of birds is very important for successful captive bird breeding programs, as well as for field research. In nestlings and monomorphic species it is difficult, if not impossible, to determine the sex by sight only. It is estimated that adult females appear identical to males in over 50% of the world’s bird species. The purpose of this research project is avian sex determination with DNA extracted from feathers using real-time PCR. In birds the female is the heterogametic sex (ZW) and the male is the homogametic sex (ZZ). The gene encoding chromo-helicase-DNA binding protein 1 (CHD-1) has been found on both the Z and W chromosomes, however, the alleles are not identical. The CHD-1 gene has at least one intron of different sizes in the W and Z alleles, which provides an opportunity to differentiate between males and females using PCR. Thus, by amplifying homologous sections of both genes (CHD-W gene and CHD-Z gene) and by incorporating introns whose lengths differ in the W and Z chromosome the sex of birds can be determined. After running an agarose gel we expect to see a single band in males (derived from the Z-chromosome) and two bands in females (derived from the Z and W chromosome).