Potential Application of Phage Therapy Against Pseudomonas aeruginosa Biofilm Infection in Cystic Fibrosis Patients

Majority of the microbial activity in humans is in the form of biofilms i.e. an Exopolysaccharide-enclosed bacterial mass. Unlike planktonic cells and the cells on the surface of the biofilm, the biofilm-embedded cells are more resistant to the effects of the antibiotics and the host cellular defense mechanisms. A combination of biofilm growth and inherent resistance prevents effective antibiotics treatment of Pseudomonas aeruginosa infections including those in patients with cystic fibrosis. This has lead to an increasing interest in alternative modalities of treatment. Thus, phages that multiply in situ, only in the presence of susceptible hosts can be used as natural, self-limiting, and deeply penetrating antibacterial agents. The objective of this study is to identify effective phages against a collection of P. aeruginosa isolates (PCOR strains) including the prototype PAOl and the isogenic constitutively alginate-producing PD0300 strains.These PCOR strains were tested against six phages (P105, P134, P140, P168, P175B and P182). Analysis shows 69 % of the PCOR isolates are sensitive and the rest are resistant to all six phages. These phages were then tested for their ability to inhibit biofilm formation using a modified biofilm assay. The analysis demonstrated that the sensitive strains showed increased resistance but none of the sensitive strains from the initial screening were resistant. Using the minimum biofilm eradication concentration (MBEC) assay for biofilm formation, the biofilm eradication ability of the phages was tested. The data showed that a higher volume of phage was required to eradicate preformed biofilms than the volume required to prevent colonization of planktonic cells. This data supports the idea of phage therapy more as a prophylactic treatment.

Microspheres for liver radiomicrospheres therapy and planning

Liver cancer accounts for nearly 10% of all cancers in the US. Intrahepatic Arterial Radiomicrosphere Therapy (RMT), also known as Selective Internal Radiation Treatment (SIRT), is one of the evolving treatment modalities. Successful patient clinical outcomes require suitable treatment planning followed by delivery of the microspheres for therapy. The production and in vitro evaluation of various polymers (PGCD, CHS and CHSg) microspheres for a RMT and RMT planning are described. Microparticles with a 30±10 µm size distribution were prepared by emulsion method. The in vitro half-life of the particles was determined in PBS buffer and porcine plasma and their potential application (treatment or treatment planning) established. Further, the fast degrading microspheres (≤ 48 hours in vitro half-life) were labeled with 68Ga and/or 99mTc as they are suitable for the imaging component of treatment planning, which is the primary emphasis of this dissertation. Labeling kinetics demonstrated that 68Ga-PGCD, 68Ga-CHSg and 68Ga-NOTA-CHSg can be labeled with more than 95% yield in 15 minutes; 99mTc-PGCD and 99mTc-CHSg can also be labeled with high yield within 15-30 minutes. In vitro stability after four hours was more than 90% in saline and PBS buffer for all of them. Experiments in reconstituted hemoglobin lysate were also performed. Two successful imaging (RMT planning) agents were found: 99mTc-CHSg and 68Ga-NOTA-CHSg. For the 99mTc-PGCD a successful perfusion image was obtained after 10 minutes, however the in vivo degradation was very fast (half-life), releasing the 99mTc from the lungs. Slow degrading CHS microparticles (> 21 days half-life) were modified with p-SCN-b-DOTA and labeled with 90 Y for production of 90Y-DOTA-CHS. Radiochemical purity was evaluated in vitro and in vivo showing more than 90% stability after 72 and 24 hours respectively. All agents were compared to their respective gold standards (99mTc-MAA for 68Ga-NOTA-CHSg and 99m Tc-CHSg; 90Y-SirTEX for 90Y-DOTA-CHS) showing superior in vivo stability. RMT and RMT planning agents (Therapy, PET and SPECT imaging) were designed and successfully evaluated in vitro and in vivo.

A novel method for rapid and selective extraction of male DNA from rape kits using alkaline lysis and pressure cycling technology (PCT)

There is an increasing demand for DNA analysis because of the sensitivity of the method and the ability to uniquely identify and distinguish individuals with a high degree of certainty. But this demand has led to huge backlogs in evidence lockers since the current DNA extraction protocols require long processing time. The DNA analysis procedure becomes more complicated when analyzing sexual assault casework samples where the evidence contains more than one contributor. Additional processing to separate different cell types in order to simplify the final data interpretation further contributes to the existing cumbersome protocols. The goal of the present project is to develop a rapid and efficient extraction method that permits selective digestion of mixtures. ^ Selective recovery of male DNA was achieved with as little as 15 minutes lysis time upon exposure to high pressure under alkaline conditions. Pressure cycling technology (PCT) is carried out in a barocycler that has a small footprint and is semi-automated. Typically less than 10% male DNA is recovered using the standard extraction protocol for rape kits, almost seven times more male DNA was recovered from swabs using this novel method. Various parameters including instrument setting and buffer composition were optimized to achieve selective recovery of sperm DNA. Some developmental validation studies were also done to determine the efficiency of this method in processing samples exposed to various conditions that can affect the quality of the extraction and the final DNA profile. ^ Easy to use interface, minimal manual interference and the ability to achieve high yields with simple reagents in a relatively short time make this an ideal method for potential application in analyzing sexual assault samples.^

Developing brownfields ranking models using decision analytic methods

The redevelopment of Brownfields has taken off in the 1990s, supported by federal and state incentives, and largely accomplished by local initiatives. Brownfields redevelopment has several associated benefits. These include the revitalization of inner-city neighborhoods, creation of jobs, stimulation of tax revenues, greater protection of public health and natural resources, the renewal and reuse existing civil infrastructure and Greenfields protection. While these benefits are numerous, the obstacles to Brownfields redevelopment are also very much alive. Redevelopment issues typically embrace a host of financial and legal liability concerns, technical and economic constraints, competing objectives, and uncertainties arising from inadequate site information. Because the resources for Brownfields redevelopment are usually limited, local programs will require creativity in addressing these existing obstacles in a manner that extends their limited resources for returning Brownfields to productive uses. Such programs may benefit from a structured and defensible decision framework to prioritize sites for redevelopment: one that incorporates the desired objectives, corresponding variables and uncertainties associated with Brownfields redevelopment. This thesis demonstrates the use of a decision analytic tool, Bayesian Influence Diagrams, and related decision analytic tools in developing quantitative decision models to evaluate and rank Brownfields sites on the basis of their redevelopment potential.

Theorizing suburban public space in Kendall

The purpose of this study was to determine whether public space in the suburbs has the same settings as that of a central city or if it has its own characteristics. In order to approach this problem the area of Kendall was thoroughly studied by examining aerial maps, historic images and writings of local historians such as Donna Knowles Born. Heavy emphasis was placed on the transformation of the original one-mile grid characteristic of the city of Miami. As the area of Kendall was being developed, the grid was transformed into an irregular and organic method of laying out a street system that directly affected pedestrian life. It became evident, therefore, that Kendall is primarily geared toward automobile movement, thus affecting the setting of public space. This also restricted social events forcing them to concentrate in specific places like the malls. These findings demonstrated that malls are centers of social interaction concentrating many social activities in one place. In other words, a mall serves as a common meeting place in the otherwise vast spread of the suburbs. This thesis also explains how public spaces in a suburban context can affect the community by working as filtering agents between the immediate context of a particular site and the overall city. The project, a "Wellness Center and Park" for the Kendall area, was an exploration of these filtering agents and the transitions they engendered. The research upon which this project was based recognized the important role of the site's history as well as extrapolating as to its future potential.

A Novel Method for Rapid and Selective Extraction of Male DNA from Rape Kits using Alkaline Lysis and Pressure Cycling Technology (PCT)

There is an increasing demand for DNA analysis because of the sensitivity of the method and the ability to uniquely identify and distinguish individuals with a high degree of certainty. But this demand has led to huge backlogs in evidence lockers since the current DNA extraction protocols require long processing time. The DNA analysis procedure becomes more complicated when analyzing sexual assault casework samples where the evidence contains more than one contributor. Additional processing to separate different cell types in order to simplify the final data interpretation further contributes to the existing cumbersome protocols. The goal of the present project is to develop a rapid and efficient extraction method that permits selective digestion of mixtures. Selective recovery of male DNA was achieved with as little as 15 minutes lysis time upon exposure to high pressure under alkaline conditions. Pressure cycling technology (PCT) is carried out in a barocycler that has a small footprint and is semi-automated. Typically less than 10% male DNA is recovered using the standard extraction protocol for rape kits, almost seven times more male DNA was recovered from swabs using this novel method. Various parameters including instrument setting and buffer composition were optimized to achieve selective recovery of sperm DNA. Some developmental validation studies were also done to determine the efficiency of this method in processing samples exposed to various conditions that can affect the quality of the extraction and the final DNA profile. Easy to use interface, minimal manual interference and the ability to achieve high yields with simple reagents in a relatively short time make this an ideal method for potential application in analyzing sexual assault samples.

Women Creators: Artistry and Sacrifice in the Novels of Virginia Woolf

This thesis examines different facets of feminine artistry in Virginia Woolf's novels with the purpose of defining her conception of women artists and the role sacrifice plays in it. The project follows characters in "Mrs. Dalloway," "To the Lighthouse," and "Between the Acts" as they attempt to create art despite society's restrictions; it studies the suffering these women experience under regimented institutions and arbitrary gender roles. From Woolf’s earlier texts to her last, she embraces the uncertainty of identity, even as she portrays the artist’s sacrifice in the early-to-mid twentieth century, specifically as the creative female identity fights to adapt to male-dominated spaces. Through a close-reading approach coupled with biographical and historical research, this thesis concludes that although the narratives of Woolf's novels demand the woman artist sacrifice for the sake of pursuing creation, Woolf praises the attempt and considers it a crueler fate to live with unfulfilled potential.

Microspheres for Liver Radiomicrospheres Therapy and Planning

Liver cancer accounts for nearly 10% of all cancers in the US. Intrahepatic Arterial Radiomicrosphere Therapy (RMT), also known as Selective Internal Radiation Treatment (SIRT), is one of the evolving treatment modalities. Successful patient clinical outcomes require suitable treatment planning followed by delivery of the microspheres for therapy. The production and in vitro evaluation of various polymers (PGCD, CHS and CHSg) microspheres for a RMT and RMT planning are described. Microparticles with a 30±10 µm size distribution were prepared by emulsion method. The in vitro half-life of the particles was determined in PBS buffer and porcine plasma and their potential application (treatment or treatment planning) established. Further, the fast degrading microspheres (≤ 48 hours in vitro half-life) were labeled with 68Ga and/or 99mTc as they are suitable for the imaging component of treatment planning, which is the primary emphasis of this dissertation. Labeling kinetics demonstrated that 68Ga-PGCD, 68Ga-CHSg and 68Ga-NOTA-CHSg can be labeled with more than 95% yield in 15 minutes; 99mTc-PGCD and 99mTc-CHSg can also be labeled with high yield within 15-30 minutes. In vitro stability after four hours was more than 90% in saline and PBS buffer for all of them. Experiments in reconstituted hemoglobin lysate were also performed. Two successful imaging (RMT planning) agents were found: 99mTc-CHSg and 68Ga-NOTA-CHSg. For the 99mTc-PGCD a successful perfusion image was obtained after 10 minutes, however the in vivo degradation was very fast (half-life), releasing the 99mTc from the lungs. Slow degrading CHS microparticles (> 21 days half-life) were modified with p-SCN-b-DOTA and labeled with 90Y for production of 90Y-DOTA-CHS. Radiochemical purity was evaluated in vitro and in vivo showing more than 90% stability after 72 and 24 hours respectively. All agents were compared to their respective gold standards (99mTc-MAA for 68Ga-NOTA-CHSg and 99mTc-CHSg; 90Y-SirTEX for 90Y-DOTA-CHS) showing superior in vivo stability. RMT and RMT planning agents (Therapy, PET and SPECT imaging) were designed and successfully evaluated in vitro and in vivo.