STudent Projects

 
GRADUATE STUDENTS
 

 

 

Natalie Damaso -combined MSFS/PhD in Biology student

Soils are very important ecosystem components that are usually under appreciated. They contain a vast array of information, both abiotic and biotic, and are one of the most challenging natural environments to study, especially for microbiologists. The current ecological hypothesis states that the soil type (e.g., chemical and physical properties) determines which microbes occupy a particular soil and provides the foundation for soil provenance studies. Therefore, soil metagenomics profiling should produce a unique biotic profile at the community level and provide rapid and efficient methods to see a snapshot of the patterned diversity within the communities. Microbial profiling effectiveness is dependent on the uniqueness among different habitat types, level of heterogeneity within a habitat, and stochastic processes in the community over time. Temporal variability (over several year’s span) also needs to be considered, as limited temporal variability should be observed as the soil microbial community should not change substantially over time in order to use pattern modeling of community analysis for soil classification and provenance. Elucidating soil microbial communities’ spatial and temporal variability is critical to improving our understanding of the factors regulating their structure and function. The overall goal of this project is to look at abiotic parameters and the biotic soil content at the structural and functional gene levels to better understand microbial distributions and interactions within and across different soil types. The correlation between microbial structural diversity and functional diversity in soil is understudied, but biodiversity is assumed to influence ecosystem stability, productivity, and resilience toward stress and disturbance. In this study, modeling approaches will be used to study the microbial patterns and drivers of the variability by observing the spatial and temporal distribution of microbes using abiotic and biotic information. Correlating the abiotic factors of soil with the biotic information can help understand ecological factors that regulate the soil biota and their functional guilds.

 


 

   

Julian Mendel - combined MSFS/PhD in Biology student

 

PhD project: Disease, Drones and Dogs: Early detection of laurel wilt disease in the avocado groves of South Florida

The invasive “Redbay” ambrosia beetle, first detected in Savannah Georgia ten years ago, is a vector of the fungal pathogen Raffaelea lauricola. Since then the laurel wilt disease has become a major threat to the thriving avocado industry in Florida causing a shutdown of the water transport system and death of the trees in a matter of weeks. This project aims to explore and understand the symbiotic relationship between the fungus and the beetle as well as the transformation the fungus goes through during its life stages both morphologically and at the DNA expression level.  In addition, early detection is critical to the containment of the disease as it rapidly spreads through the root system to neighboring trees. Currently PCR methods are used for identification but this study aims to utilize spectral imaging to detect stressed trees, coupled with confirmation with canine units (scent dogs) that will be trained to detect the volatile compounds the fungus produces.

 

 


 

            

Beatrice Kallifatidis

The differentiation of hallucinogenic from non-hallucinogenic fungi is of interest from a forensic standpoint, in order to prove possession and trafficking of illegal mushrooms. The identification of toxic mushroom species is of importance from a public health point of view, as mushroom poisoning is a quite common phenomenon caused due to morphologically misidentification of mushrooms that are mistaken for hallucinogenic or edible species.

The goal of this study is to develop a reliable/reproducible, sensitive and rapid DNA based approach for the identification of hallucinogenic and toxic fungi species, which can be easily integrated into forensic labs. The proposed method is random amplified microsatellites (RAMS).

In microsatellite analysis prior sequence information is usually needed and the method is most often developed for species specific identification. Since there is limited information regarding the DNA sequences of many fungi, RAMS-PCR will be an advantageous method, as no prior genomic information is needed to develop the microsatellites. Thus, by developing several ‘universal microsatellites’ we might be able to profile any fungi species and establish a database of hallucinogenic and toxic fungal DNA profiles. The accuracy and reproducibility of species identification by RAMS will be confirmed with DNA sequencing.

 

 


 

                                                        

Ashley Lekas-Diaz

My M.F.S. project focused on universal primer sets that amplify hypervariable molecular markers from the microorganisms in the soil.  Among the microorganisms that were queried were bacteria, fungi, plants and nematodes.  Amplified Length Heterogeneity-PCR technique was used to amplify hypervariable domains that can identify the presence of the aforementioned microorganisms. Future applications of this work would include developing a kit which can be utilized in the forensic community; selecting for multiple taxa, decreasing the amount of work, increasing the turn-around time and most importantly increasing the power of discrimination for any linkage/dissociation of evidence.  In addition to a forensic application, this multiplex approach has great potential for the use in microbial ecology as well.

Currently, I am working in the Sequencing Core at the John P. Hussman Institute for Human Genomics in the University of Miami’s Miller School of Medicine as a Research Associate II. We run high-throughput protocols for various next-generation sequencing projects including studies of Autism, Parkinson’s disease, Charcot Marie Tooth Disease, Alzheimer’s disease and many others.


 

 
                 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).