Presumptive Identification of the Fungal Communities in Cystic Fibrosis Patients' Sputum Using Amplicon Length Heterogeneity Polymerase Chain Reaction

This thesis would not have been possible without the aid of my family, friends, laboratory members, and professors. First and foremost, I would like to thank Dr. Kalai Mathee for allowing me to enter her lab in August 2007 and enabling to embark on this journey. This experience has transformed me into more mature scientist, teaching me how to ask the right questions and the process needed to solve them. I would also like to acknowledge Dr. Lisa Schneper. She has helped me throughout the whole process, by graciously giving me input at every step of the way. I would like to express gratitude to Dr. Jennifer Richards for all her input in writing the thesis. She has been a great teacher and being in her class has been a pleasure. Moreover, I would like to thank all the committee members for their constructive criticism throughout the process. When I entered the lab in August, there was one person who literally was by my side, Melissa Doud. Without your input and guidance I would not have even been able to do these experiments. I would also like to thank you and Dr. Light for allowing me to meet some cystic fibrosis patients. It has allowed me to put a face on the disease, and help the patients' fight. For a period before I had entered the lab, Ms. Doud had an apprentice, who started the fungal aspect of the project, Caroline Veronese. Her initial work has enabled me to prefect the protocols and complete the ITS 1 region.One very unique aspect about Dr. Mathee's lab is the camaraderie. I would like to thank all the lab members for the good times in and out of the lab. These individuals have been able to make smile and laugh in parties and lab meetings. I would like to individually thank Balachandar Dananjeyan, Deepak Balasubramanian, and V arinderpal Singh Pannu for all the PCR help and Natalie Maricic for the laughs and being a great classmate. Last, but not least, I would like to acknowledge my family and friends for their support and keeping me sane: Cecilia, my mother, Mohammad, my father, Amir, my older brother, Billal, my younger brother, Ouday Akkari and Stephanie De Bedout, my best friends.

The use of quantitative polymerase chain reaction amplification method to determine sex ratio of human spermatozoa in semen and to determine maternal contamination of amniotic fluid samples

We have modified a technique which uses a single pair of primer sets directed against homologous but distinct genes on the X and Y chromosomes, all of which are coamplified in the same reaction tube with trace amounts of radioactivity. The resulting bands are equal in length, yet distinguishable by restriction enzyme sites generating two independent bands, a 364 bp X-specific band and a 280 bp Y-specific band. A standard curve was generated to show the linear relationship between X/Y ratio average vs. %Y or %X chromosomal content. Of the 51 purified amniocyte DNA samples analyzed, 16 samples showed evidence of high % X contamination while 2 samples demonstrated higher % Y than the expected 50% X and 50% Y chromosomal content. With regards to the 25 processed sperm samples analyzed, X-sperm enrichment was evident when compared to the primary sex ratio whereas Y-sperm was enriched when we compared before and after selection samples.

Cytokine analysis in Atlantic bottlenose dolphins: Molecular characterization of IL-4, IL-6, and IL-10

The health status of wild and captive Atlantic Bottlenose dolphins ( Tersiops truncatis) is difficult to ascertain. Mass strandings of these animals have been attributed to pollutants, as well as bacterial infections. Using human Enzyme Linked Immuno-Assays (ELISA) for immunological cytokines, I measured soluble cytokine levels with respect to their health status. In a retrospective analysis of dolphin sera, there was a trend of higher cytokine levels in “sick” animals. I cultured dolphin lymphocytes in the presence of a mitogen (PHA), a super antigen (Staph-A), Lipopolysaccharide (LPS), and a calcium flux inducer (PMA). Levels of messenger RNA, from these cultured cells, were assayed with Polymerase Chain Reaction (PCR) using primers for the human cytokines IL-2, IL-4, IL-6, IL-10, Tumor Necrosis Factor, and Interferon gamma. Only IL-4, IL-6, and IL-10 messages were obtained, inferring similar nucleotide homology to the human primer sequences. The PCR products were sequenced. Sixteen IL-4 sequences, twelve IL-6 sequences and seven IL-10 sequences were obtained and analyzed. Each cytokine exhibited the same nucleotide sequence in all dolphins examined. There was no difference in the cytokine profile in response to the various stimuli. The derived amino acid composition for each of the dolphin cytokines was used for molecular modeling, which showed that dolphin IL-4, IL-6, and IL-10 were structurally similar to the corresponding proteins of Perissodactyla. ^

Expression of Bf/C2 Gene(S) in the Japanese Medaka Fish

Complement factor B and C2 are two central serine proteases of the alternative and classical complement pathways, respectively, that serve as the catalytic subunits of the C3 convertase. Research has been completed using a female Japanese medaka fish, (Oryzias latipes), and other teleost and elasmobrach species in order to isolate eDNA clones and perform linkage analysis of the Bf/C2 gene(s). To further analyze the evolution of the complement system in teleosts, different tissues than the ones from previous studies of medaka fish were analyzed for the constitutive gene expression of factor B and C2. Bf/C2 sequences were amplified by reverse transcription-polymerase chain reaction with primers corresponding to the common amino acid sequences shared by mammalian Bf and C2. Agarose gel electrophoresis was used to visualize sample bands and to calculate the concentration of gene expression of the Bf/C2 gene(s) in each tissue. All five tissue types, kidney, liver, muscle, testis, and spleen from a male medaka fish demonstrated Bf/C2 gene(s) expression, confirming that the messages of Bf/C2 gene(s) are distributed throughout the medaka fish. Tissues of the spleen, liver, and kidney contained the highest concentrations of expression of Bf/C2 gene( s ), while tissues of the muscle and testis contained the lowest concentrations. This research also determined that RT-PCR allowed for more sensitive analysis of gene expression than other molecular biology techniques such as Northern blotting analysis.