The ethnopharmacology of plants used as both food and medicine in a Totonac community of Mexico

Most ethnopharmacological studies overlook food plants, yet many edible plants, also have medicinal value. I documented plants that are used as both food and medicine by the Totonac of Zapotitlan de Mendez, Mexico and recorded the presence of selected secondary compounds, and physical characteristics in these plants. Photoactivity, antimicrobial, and antifungal assays also were performed. The presence of these properties were compared among food/medicine plants, food, medicinal, and randomly selected plants. I predicted that a higher percentage of medicinal plants would contain the secondary compounds, physical characteristics, and bioactivity compared to the other groups. Phenolics and cyanogenic glycosides in the medicinal group were significantly greater than in the food/medicine group. The food plants did not differ greatly from the medicinal plants. This research indicates that including food plants in ethnomedical studies could provide a more complete knowledge of peoples therapeutic resources and practices. ^

Enzyme Differences between Adjacent Hybrid and Parent Populations of Typha

Enzyme variation among three adjacent Typha populations was analyzed by means of disc electrophoresis, isoelectric focusing, and enzyme assays. Esterase isozyme analysis of seedlings of Typha latifolia L.,T . x glauca Godr., and T. angustifolia L. indicated that there was little or no variation within each population. Additional analyses of esterase, malate dehydrogenase, glutamate dehydrogenase, and alcohol dehydrogenase of pooled samples of pollen and seedlings indicated that the Typha x glauca population was the product of hybridization between the adjacent parent populations. The hybrid population had more isozymes, but lower specific activities, than the parents.

Emergence and Fate of Siloxanes in Waste Streams: Release Mechanisms, Partitioning and Persistence in Three Environmental Compartments

Siloxanes are widely used in personal care and industrial products due to their low surface tension, thermal stability, antimicrobial and hydrophobic properties, among other characteristics. Volatile methyl siloxanes (VMS) have been detected both in landfill gas and biogas from anaerobic digesters at wastewater treatment plants. As a result, they are released to gas phase during waste decomposition and wastewater treatment. During transformation processes of digester or landfill gas to energy, siloxanes are converted to silicon oxides, leaving abrasive deposits on engine components. These deposits cause increased maintenance costs and in some cases complete engine overhauls become necessary. The objectives of this study were to compare the VMS types and levels present in biogas generated in the anaerobic digesters and landfills and evaluate the energetics of siloxane transformations under anaerobic conditions. Siloxane emissions, resulting from disposal of silicone-based materials, are expected to increase by 29% within the next 10 years. Estimated concentrations and the risk factors of exposure to siloxanes were evaluated based on the initial concentrations, partitioning characteristics and persistence. It was determined that D4 has the highest risk factor associated to bioaccumulation in liquid and solid phase, whereas D5 was highest in gas phase. Additionally, as siloxanes are combusted, the particle size range causes them to be potentially hazardous to human health. When inhaled, they may affix onto the alveoli of the lungs and may lead to development of silicosis. Siloxane-based COD-loading was evaluated and determined to be an insignificant factor concerning COD limits in wastewater. Removal of siloxane compounds is recommended prior to land application of biosolids or combustion of biogas. A comparison of estimated costs was made between maintenance practices for removal of siloxane deposits and installation/operation of fixed-bed carbon absorption systems. In the majority of cases, the installation of fixed-bed adsorption systems would not be a feasible option for the sole purpose of siloxane removal. However they may be utilized to remove additional compounds simultaneously.

Emergence and fate of siloxanes in waste streams: Release mechanisms, partitioning and persistence in three environmental compartments

Siloxanes are widely used in personal care and industrial products due to their low surface tension, thermal stability, antimicrobial and hydrophobic properties, among other characteristics. Volatile methyl siloxanes (VMS) have been detected both in landfill gas and biogas from anaerobic digesters at wastewater treatment plants. As a result, they are released to gas phase during waste decomposition and wastewater treatment. During transformation processes of digester or landfill gas to energy, siloxanes are converted to silicon oxides, leaving abrasive deposits on engine components. These deposits cause increased maintenance costs and in some cases complete engine overhauls become necessary. ^ The objectives of this study were to compare the VMS types and levels present in biogas generated in the anaerobic digesters and landfills and evaluate the energetics of siloxane transformations under anaerobic conditions. Siloxane emissions, resulting from disposal of silicone-based materials, are expected to increase by 29% within the next 10 years. Estimated concentrations and the risk factors of exposure to siloxanes were evaluated based on the initial concentrations, partitioning characteristics and persistence. It was determined that D4 has the highest risk factor associated to bioaccumulation in liquid and solid phase, whereas D5 was highest in gas phase. Additionally, as siloxanes are combusted, the particle size range causes them to be potentially hazardous to human health. When inhaled, they may affix onto the alveoli of the lungs and may lead to development of silicosis. Siloxane-based COD-loading was evaluated and determined to be an insignificant factor concerning COD limits in wastewater. ^ Removal of siloxane compounds is recommended prior to land application of biosolids or combustion of biogas. A comparison of estimated costs was made between maintenance practices for removal of siloxane deposits and installation/operation of fixed-bed carbon absorption systems. In the majority of cases, the installation of fixed-bed adsorption systems would not be a feasible option for the sole purpose of siloxane removal. However they may be utilized to remove additional compounds simultaneously.^

A multi-faceted diagnostic approach to lung infections in patients with cystic fibrosis

One in 3,000 people in the US are born with cystic fibrosis (CF), a genetic disorder affecting the reproductive system, pancreas, and lungs. Lung disease caused by chronic bacterial and fungal infections is the leading cause of morbidity and mortality in CF. Identities of the microbes are traditionally determined by culturing followed by phenotypic and biochemical assays. It was first thought that the bacterial infections were caused by a select handful of bacteria such as S. aureus, H. influenzae, B. cenocepacia, and P. aeruginosa. With the advent of PCR and molecular techniques, the polymicrobial nature of the CF lung became evident. The CF lung contains numerous bacteria and the communities are diverse and unique to each patient. The total complexity of the bacterial infections is still being determined. In addition, only a few members of the fungal communities have been identified. Much of the fungal community composition is still a mystery. This dissertation addresses this gap in knowledge. A snap shot of CF sputa bacterial community was obtained using the length heterogeneity-PCR community profiling technique. The profiles show that south Florida CF patients have a unique, diverse, and dynamic bacterial community which changes over time. The identities of the bacteria and fungi present were determined using the state-of-the-art 454 sequencing. Sequencing results show that the CF lung microbiome contains commonly cultured pathogenic bacteria, organisms considered a part of the healthy core biome, and novel organisms. Understanding the dynamic changes of these identified microbes will ultimately lead to better therapeutical interventions. Early detection is key in reducing the lung damage caused by chronic infections. Thus, there is a need for accurate and sensitive diagnostic tests. This issue was addressed by designing a bacterial diagnostic tool targeted towards CF pathogens using SPR. By identifying the organisms associated with the CF lung and understanding their community interactions, patients can receive better treatment and live longer.

Anticancer activity of 4-N gemcitabine analogues

Gemcitabine (2', 2'-difluoro-2'-deoxycytidine or dFdC) has become a standard chemotherapeutic agent in the treatment of several cellular and solid tumor- related malignancies. Gemcitabine's anti-cancer activity has been attributed to its inhibitory effects on the cell's DNA synthetic machinery resulting in the induction of cell arrest and apoptosis. Despite its broad application, treatment capacity with this drug is limited due to complicated administration schedules stemming from low bioavailability and tumor resistance associated with its rampant intracellular enzymatic inactivation. The aim of this study is to characterize the anti-cancer activity of novel designed and synthesized gemcitabine analogues, that were modified with long alkyl chains at the 4-amino group of the cytosine ring. This study proposes the use of these alternative derivatives of gemcitabine that not only uphold current drug standards for potency, but additionally confer chemical stability against enzymatic inactivation. During screening conducted to identifY prospective gem-analogue candidates, I observed the potent anticancer properties ofthree 4-N modified compounds on MCF-7 breast adenocarcinoma cells. Experiments described here with these compounds referred to as LCO, LCAO, and Gvaldo, evaluate their cytotoxicity on MCF-7 cells at the concentrations of 25flM and 2.5flM, and assess their inhibitory effects on DNA synthesis and cell cycle progression using sulphorhodamine B and bromodeoxyuridine assays as well as flow cytometric analyses, respectively. Among the compounds tested, LCO was shown to be most active inhibitor of DNA synthesis (a=.05; p<.OOl) as reflected as a distinct GO/Gl versus S-phase arrest in the 25flM and 2.5flM treatments, respectively. Together, these experiments provide preliminary evidence for the clinical application of LCO-like gemcitabine derivatives as a novel treatment for breast cancer.

Pseudomonas aeruginosa AMPR transcriptional regulatory network

In Enterobacteriaceae, the transcriptional regulator AmpR, a member of the LysR family, regulates the expression of a chromosomal β-lactamase AmpC. The regulatory repertoire of AmpR is broader in Pseudomonas aeruginosa, an opportunistic pathogen responsible for numerous acute and chronic infections including cystic fibrosis. Previous studies showed that in addition to regulating ampC, P. aeruginosa AmpR regulates the sigma factor AlgT/U and production of some quorum sensing (QS)-regulated virulence factors. In order to better understand the ampR regulon, the transcriptional profiles generated using DNA microarrays and RNA-Seq of the prototypic P. aeruginosa PAO1 strain with its isogenic ampR deletion mutant, PAOΔampR were analyzed. Transcriptome analysis demonstrates that the AmpR regulon is much more extensive than previously thought influencing the differential expression of over 500 genes. In addition to regulating resistance to β-lactam antibiotics via AmpC, AmpR also regulates non-β-lactam antibiotic resistance by modulating the MexEF-OprN efflux pump. Virulence mechanisms including biofilm formation, QS-regulated acute virulence, and diverse physiological processes such as oxidative stress response, heat-shock response and iron uptake are AmpR-regulated. Real-time PCR and phenotypic assays confirmed the transcriptome data. Further, Caenorhabditis elegans model demonstrates that a functional AmpR is required for full pathogenicity of P. aeruginosa. AmpR, a member of the core genome, also regulates genes in the regions of genome plasticity that are acquired by horizontal gene transfer. The extensive AmpR regulon included other transcriptional regulators and sigma factors, accounting for the extensive AmpR regulon. Gene expression studies demonstrate AmpR-dependent expression of the QS master regulator LasR that controls expression of many virulence factors. Using a chromosomally tagged AmpR, ChIP-Seq studies show direct AmpR binding to the lasR promoter. The data demonstrates that AmpR functions as a global regulator in P. aeruginosa and is a positive regulator of acute virulence while negatively regulating chronic infection phenotypes. In summary, my dissertation sheds light on the complex regulatory circuit in P. aeruginosa to provide a better understanding of the bacterial response to antibiotics and how the organism coordinately regulates a myriad of virulence factors.

Cocaine Enhances HIV-1 Infectivity in Monocyte Derived Dendritic Cells by Suppressing microRNA-155

Cocaine and other drugs of abuse increase HIV-induced immunopathogenesis; and neurobiological mechanisms of cocaine addiction implicate a key role for microRNAs (miRNAs), single-stranded non-coding RNAs that regulate gene expression and defend against viruses. In fact, HIV defends against miRNAs by actively suppressing the expression of polycistronic miRNA cluster miRNA-17/92, which encodes miRNAs including miR-20a. IFN-g production by natural killer cells is regulated by miR-155 and this miRNA is also critical to dendritic cell (DC) maturation. However, the impact of cocaine on miR-155 expression and subsequent HIV replication is unknown. We examined the impact of cocaine on two miRNAs, miR-20a and miR-155, which are integral to HIV replication, and immune activation. Using miRNA isolation and analysis, RNA interference, quantitative real time PCR, and reporter assays we explored the effects of cocaine on miR-155 and miR-20 in the context of HIV infection. Here we demonstrate using monocyte-derived dendritic cells (MDCCs) that cocaine significantly inhibited miR-155 and miR-20a expression in a dose dependent manner. Cocaine and HIV synergized to lower miR-155 and miR-20a in MDDCs by 90%. Cocaine treatment elevated LTR-mediated transcription and PU.1 levels in MDCCs. But in context of HIV infection, PU.1 was reduced in MDDCs regardless of cocaine presence. Cocaine increased DC-SIGN and and decreased CD83 expression in MDDC, respectively. Overall, we show that cocaine inhibited miR-155 and prevented maturation of MDDCs; potentially, resulting in increased susceptibility to HIV-1. Our findings could lead to the development of novel miRNA-based therapeutic strategies targeting HIV infected cocaine abusers.

Novel insights into the mechanisms of regulation of tyrosine kinase receptors by Ras Interference 1

Receptor-tyrosine kinases (RTKs) are membrane bound receptors characterized by their intrinsic kinase activity. RTK activities play an essential role in several human diseases, including cancer, diabetes and neurodegenerative diseases. RTK activities have been regulated by the expression or silencing of several genes as well as by the utilization of small molecules. Ras Interference 1 (Rin1) is a multifunctional protein that becomes associated with activated RTKs upon ligand stimulation. Rin1 plays a key role in receptor internalization and in signal transduction via activation of Rab5 and association with active form of Ras. This study has two main objectives: (1) It determines the role of Rin1 in the regulation of several RTKs focusing on insulin receptor. This was accomplished by studying the Rin1-insulin receptor interaction using a variety of biochemical and morphological assays. This study shows a novel interaction between the insulin receptor and Rin1 through the Vps9 domain. Two more RTKs (epidermal growth factor receptor and nerve growth factor receptor) also interacted with the SH2 domain of Rin1. The effect of the Rin1-RTK interaction on the activation of both Rab5 and Ras was also studied during receptor internalization and intracellular signaling. Finally, the role of Rin1 was examined in two differentiation processes (adipogenesis and neurogenesis). Rin1 showed a strong inhibitory effect on 3T3-L1 preadipocyte differentiation but it seems to show a modest effect in PC12 neurite outgrowth. These data indicate a selective function and specific interaction of Rin1 toward RTKs. (2) It examines the role of the small molecule Dehydroleucodine (DhL) on several key signaling molecules during adipogenesis. This was accomplished by studying the differentiation of 3T3-L1 preadipocytes exposed to different concentrations of DhL in different days of the adipocyte formation process. The results indicate that DhL selectively blocked adipocyte formation, as well as the expression of PPARγ, and C/EBP&agr;. However, DhL treatment did not affect Rin1 or Rab5 expression and their activities. Taken together, the data indicate a potential molecular mechanism by which proteins or small molecules regulate selective and specific RTK intracellular membrane trafficking and signaling during cell growth and differentiation in normal and pathological conditions.

A Multi-Faceted Diagnostic Approach to Lung Infections in Patients with Cystic Fibrosis

One in 3,000 people in the US are born with cystic fibrosis (CF), a genetic disorder affecting the reproductive system, pancreas, and lungs. Lung disease caused by chronic bacterial and fungal infections is the leading cause of morbidity and mortality in CF. Identities of the microbes are traditionally determined by culturing followed by phenotypic and biochemical assays. It was first thought that the bacterial infections were caused by a select handful of bacteria such as S. aureus, H. influenzae, B. cenocepacia, and P. aeruginosa. With the advent of PCR and molecular techniques, the polymicrobial nature of the CF lung became evident. The CF lung contains numerous bacteria and the communities are diverse and unique to each patient. The total complexity of the bacterial infections is still being determined. In addition, only a few members of the fungal communities have been identified. Much of the fungal community composition is still a mystery. This dissertation addresses this gap in knowledge. A snap shot of CF sputa bacterial community was obtained using the length heterogeneity-PCR community profiling technique. The profiles show that south Florida CF patients have a unique, diverse, and dynamic bacterial community which changes over time. The identities of the bacteria and fungi present were determined using the state-of-the-art 454 sequencing. Sequencing results show that the CF lung microbiome contains commonly cultured pathogenic bacteria, organisms considered a part of the healthy core biome, and novel organisms. Understanding the dynamic changes of these identified microbes will ultimately lead to better therapeutical interventions. Early detection is key in reducing the lung damage caused by chronic infections. Thus, there is a need for accurate and sensitive diagnostic tests. This issue was addressed by designing a bacterial diagnostic tool targeted towards CF pathogens using SPR. By identifying the organisms associated with the CF lung and understanding their community interactions, patients can receive better treatment and live longer.

Characterization of engineered human lung tissue

Characterizing engineered human lung tissue is an important step in developing a functional tissue replacement for lung tissue repair and in vitro analysis. Small tissue constructs were grown by seeding IMR-90 fetal lung fibroblasts and adult microvascular endothelial cells onto a Polyglycolic acid (PGA) polymer template. Introducing the constructs to dynamic culture conditions inside a bioreactor facilitated three-dimensional growth seen in scanning electron microscopy images (SEM). Characterization of the resultant tissue samples was done using SEM imagery, tensile tests, and biochemical assays to quantify extra-cellular matrix (ECM) composition. Tensile tests of the engineered samples indicated an increase in the mechanical properties when compared with blank constructs. Elastin and collagen content was found to average 3.19% and 15.49% respectively in relation to total mass of the tissue samples. The presence of elastin and collagen within the constructs most likely explains the mechanical differences that we noted. These findings suggest that the necessary ECM can be established in engineered tissue constructs and that optimization of this procedure has the capacity to generate the load bearing elements required for construction of a functional lung tissue equivalent.

Understanding the role of Dimethylsulfoniopropionate as a Potential Chemotactic Cue for Coral-Associated Bacteria

Most reef-building corals are known to engage in symbiosis not only with unicellular dinoflagellates from the genus, Symbiodinium, but they also sustain highly complex symbiotic associations with other microscopic organisms such as bacteria, fungi, and viruses. The details of these non-pathogenic interactions remain largely unclear. The impetus of this study is to gain a better understanding of the symbiotic interaction between marine bacteria and a variety of coral species representative of differing morphologies. Studies have shown that certain bacterial orders associate specifically with certain coral species, thus making the symbiotic synergy a non-random consortium. Consequently both corals and bacteria may be capable of emitting chemical cues that enables both parties to find one another and thus creating the symbiosis. One potential chemical cue could be the compound DMSP (Dimethylsulfoniopropionate) and its sulphur derivatives. Reef-building corals are believed to be the major producers of the DMSP and its derivatives during times of stress. As a result corals could potentially attract their bacterial consortium depending on their DMSP production. Corals may be able to adapt to fluctuating environmental conditions by changing their bacterial communities to that which may aid in survival. The cause of this attraction may stem from the capability of a variety of marine bacteria to catabolize DMSP into different metabolically significant pathways, which may be necessary for the survival of these mutualistic interactions. To test the hypothesis that coral-produced DMSP play a role in attracting symbiotic bacteria, this study utilized the advent of high-through sequencing paired with bacterial isolation techniques to properly characterize the microbial community in the stony coral Porites astreoides. We conducted DMSP swarming and chemotaxis assays to determine the response of these coral-associated bacterial isolates towards the DMSP compound at differing concentrations. Preliminary data from this study suggests that six out of the ten bacterial isolates are capable of conducting unidirectional motility; these six isolates are also capable of conducting swarming motility in the direction of an increasing DMSP concentration gradient. This would indicate that there is a form of positive chemotaxis on behalf of the bacteria towards the DMSP compound. By obtaining a better understanding of the dynamics that drive the associations between bacterial communities and corals, we can further aid in the protection and conservation processes for corals. Also this study would further elucidate the significance of the DMSP compound in the survival of corals under times of stress.

Understanding the role of Dimethylsulfoniopropionate as a Potential Chemotactic Cue for Coral-Associated Bacteria

Most reef-building corals are known to engage in symbiosis not only with unicellular dinoflagellates from the genus, Symbiodinium, but they also sustain highly complex symbiotic associations with other microscopic organisms such as bacteria, fungi, and viruses. The details of these non-pathogenic interactions remain largely unclear. The impetus of this study is to gain a better understanding of the symbiotic interaction between marine bacteria and a variety of coral species representative of differing morphologies. Studies have shown that certain bacterial orders associate specifically with certain coral species, thus making the symbiotic synergy a non-random consortium. Consequently both corals and bacteria may be capable of emitting chemical cues that enables both parties to find one another and thus creating the symbiosis. One potential chemical cue could be the compound DMSP (Dimethylsulfoniopropionate) and its sulphur derivatives. Reef-building corals are believed to be the major producers of the DMSP and its derivatives during times of stress. As a result corals could potentially attract their bacterial consortium depending on their DMSP production. Corals may be able to adapt to fluctuating environmental conditions by changing their bacterial communities to that which may aid in survival. The cause of this attraction may stem from the capability of a variety of marine bacteria to catabolize DMSP into different metabolically significant pathways, which may be necessary for the survival of these mutualistic interactions. To test the hypothesis that coral-produced DMSP play a role in attracting symbiotic bacteria, this study utilized the advent of high-through sequencing paired with bacterial isolation techniques to properly characterize the microbial community in the stony coral Porites astreoides. We conducted DMSP swarming and chemotaxis assays to determine the response of these coral-associated bacterial isolates towards the DMSP compound at differing concentrations. Preliminary data from this study suggests that six out of the ten bacterial isolates are capable of conducting unidirectional motility; these six isolates are also capable of conducting swarming motility in the direction of an increasing DMSP concentration gradient. This would indicate that there is a form of positive chemotaxis on behalf of the bacteria towards the DMSP compound. By obtaining a better understanding of the dynamics that drive the associations between bacterial communities and corals, we can further aid in the protection and conservation processes for corals. Also this study would further elucidate the significance of the DMSP compound in the survival of corals under times of stress.

Pseudomonas Aeruginosa AmpR Transcriptional Regulatory Network

In Enterobacteriaceae, the transcriptional regulator AmpR, a member of the LysR family, regulates the expression of a chromosomal β-lactamase AmpC. The regulatory repertoire of AmpR is broader in Pseudomonas aeruginosa, an opportunistic pathogen responsible for numerous acute and chronic infections including cystic fibrosis. Previous studies showed that in addition to regulating ampC, P. aeruginosa AmpR regulates the sigma factor AlgT/U and production of some quorum sensing (QS)-regulated virulence factors. In order to better understand the ampR regulon, the transcriptional profiles generated using DNA microarrays and RNA-Seq of the prototypic P. aeruginosa PAO1 strain with its isogenic ampR deletion mutant, PAO∆ampR were analyzed. Transcriptome analysis demonstrates that the AmpR regulon is much more extensive than previously thought influencing the differential expression of over 500 genes. In addition to regulating resistance to β-lactam antibiotics via AmpC, AmpR also regulates non-β-lactam antibiotic resistance by modulating the MexEF-OprN efflux pump. Virulence mechanisms including biofilm formation, QS-regulated acute virulence, and diverse physiological processes such as oxidative stress response, heat-shock response and iron uptake are AmpR-regulated. Real-time PCR and phenotypic assays confirmed the transcriptome data. Further, Caenorhabditis elegans model demonstrates that a functional AmpR is required for full pathogenicity of P. aeruginosa. AmpR, a member of the core genome, also regulates genes in the regions of genome plasticity that are acquired by horizontal gene transfer. The extensive AmpR regulon included other transcriptional regulators and sigma factors, accounting for the extensive AmpR regulon. Gene expression studies demonstrate AmpR-dependent expression of the QS master regulator LasR that controls expression of many virulence factors. Using a chromosomally tagged AmpR, ChIP-Seq studies show direct AmpR binding to the lasR promoter. The data demonstrates that AmpR functions as a global regulator in P. aeruginosa and is a positive regulator of acute virulence while negatively regulating chronic infection phenotypes. In summary, my dissertation sheds light on the complex regulatory circuit in P. aeruginosa to provide a better understanding of the bacterial response to antibiotics and how the organism coordinately regulates a myriad of virulence factors.