Made available in DSpace on 2014-06-11T19:26:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-02-18Bitstream added on 2014-06-13T20:55:06Z : No. of bitstreams: 1 abla_ms_me_araca.pdf: 2846240 bytes, checksum: ffaac15bbba1c9145b215abe25edad01 (MD5) / Universidade Estadual Paulista (UNESP) / O presente estudo analisou o processo de reparo ósseo em cavidades ósseas experimentais realizadas em tíbias de coelho, quando implantado partículas de enxerto ósseo homógeno, tratadas com solução de tetraciclina. Foram realizadas análises histológicas e submetidas a tratamento estatístico após histometria. Os resultados mostraram que não houve, estatisticamente, diferenças significantes entre o grupo controle onde não houve nenhuma implantação, e o grupo tratado utilizando a tetraciclina. O processo de reparo ocorreu de maneira similar em ambos os grupos. Não houve reabsorção completa dos fragmentos ósseos implantados, permanecendo até o período estudado de 30 dias pós-operatórios. / The aim of the present study was to evaluate the bone repair process in experimental bone cavities which were realized in rabbit tibias when particles of homogenous bone graft were implanted and treated with tetracycline. Histolological analysis were carried out and undergone a statistical treatment after HISTOMETRIA. The results showed that the hydrochloric tetracycline didnþt interfered on the bone graft performance, which was used on the treatment of experimental bone defects. There wasnþt histological evidence of the decalcifier action of tetracycline; it could be the medicine exposion time of the bone graft. Evaluating the statistical results; it was observed that the tetracycline didnþt interfered on bone repair process, moreover its presence doesnþt cause delay or any significant interference that changes the healing process.

Synthetic biology consists of the design and construction of customized cell-based systems, and metabolic engineering is its co-discipline that aims to engineer these cells into biological factories for the production of drugs, chemical commodities and fuels. Together, these two disciplines continue to provide various innovative solutions to current problems of humanity in the areas of medicine, agriculture and energy. In this dissertation, we use synthetic biology and metabolic engineering approaches to explore the potential of engineered live yeasts as therapeutic platforms for treating inflammatory bowel disease (IBD). The vast majority of microbial-based therapeutics at the moment have focused on bacteria instead of yeasts, and all of these engineered live bacterial platforms use either proteins or peptides as therapeutic agents of choice. This dissertation seeks to enhance yeast’s beneficial properties to humans by genetically engineering them to produce TAN-1612, a small molecule tetracycline with therapeutic potential. We choose tetracyclines as our small molecule therapeutic agent because these compounds are one of the most impactful natural products that humanity has benefited from due to its significant antimicrobial and anti-inflammatory properties. We genetically engineer strains of baker’s yeast Saccharomyces cerevisiae and the probiotic yeast Saccharomyces cerevisiae var boulardii to produce in situ the fungal tetracycline TAN-1612, a natural product with anti-inflammatory properties (instead of anti-microbial so as to not disturb the gut microbiome), and to study the molecular mechanisms involved in their potential beneficial effects for IBD. Our engineered live yeast therapeutics would provide an effective, safe, and cheap alternative to treating IBD and other gastrointestinal tract disorders compared to the currently available but costly and laborious therapies. In Chapter 1, we review key milestones in the fields of synthetic biology and metabolic engineering that have enabled and inspired the generation of both engineered live microbial-based systems and small molecules as the therapeutic agents for the potential treatment of a wide array of human diseases such IBD, cancer, and pathogenic infections. In Chapter 2, we develop synthetic biology and metabolic engineering approaches for designing, building, and testing of the biosynthetic pathway of TAN-1612 in genetically engineered yeasts such as S. cerevisiae and S. boulardii. These approaches enable the production of TAN-1612 in yeasts with titers as high as ~61 mg/L which represent a 100-fold improvement from previous reported yeast strains. These engineering approaches hold great potential to advance the heterologous biosynthesis of other small molecule therapeutics in yeasts. In Chapter 3, we explore the role of TAN-1612 as an anti-inflammatory agent, inhibitor of tetracycline inactivating enzymes, and inducer of gene expression with the goal of identifying its best therapeutic or biological application that can be leveraged for the development of engineered live yeast-based systems for the in situ treatment of IBD. Advances in DNA synthesis and sequencing technologies have spurred the high-throughput construction of microbial strains for numerous applications in synthetic biology and metabolic engineering. Breakthrough technologies in our abilities to screen and select target molecule biosynthesis, however, are needed in order to realize the potential of both of these disciplines for drug discovery and production. Current state-of-the-art methods such as liquid/gas chromatography – mass spectrometry (LC/GC – MS) are applicable to screen or select a variety of target molecules but their throughput remains low (~102 samples/day). Other screening or selection methods available are highly dependent on the molecule of interest and generally inapplicable to other compounds. Therefore, in Chapter 4 we propose that the Fluorescence Polarization (FP) assay can be readily adapted as a general, medium-throughput (~104 samples/day) screen for synthetic biology and metabolic engineering applications. As a proof-of-principle, we develop an FP assay to detect the immunosuppressant polyketide FK506, use this assay to detect FK506 biosynthesized from Streptomyces tsukubaensis cultures in microtiter plates, and finally apply this FP assay to generate S. tsukubaensis strains with increased production of FK506. Lastly, we outline the experimental steps necessary to adapt the FP to screen different classes of natural products beyond polyketides such as FK506 or tetracyclines. The fungal polyketide TAN-1612 is an attractive chemical scaffold for the generation of novel tetracycline-based therapeutics using metabolic engineering approaches. Libraries of > 108 are usually required to generate chemical compound diversity to identify drugs with significant therapeutic activities. Compared to screening methods, genetic selections allow the detection of target molecules at a much higher throughput (> 108 samples/day) because the population of cells can be cultured and assayed in one-pot manner. Thus, in Chapter 5 we establish the yeast three hybrid (Y3H) assay as a general, high-throughput selection technology to detect tetracycline derivatives. We demonstrate the applicability of the Y3H assay to metabolic engineering by differentiating producer and non-producer yeast strains of TAN-1612. The Y3H assay can be used for the heterologous biosynthesis of tetracycline analogues in yeasts especially because the Y3H would enable the production and detection of these derivatives in the same yeast cell.

Bone discolouration of pig carcasses is a quality concern that has been observed in Ontario slaughter plants. The objectives of this study were to establish the prevalence of pig carcasses showing bone discolouration, its relationship with residues of tetracyclines in bones, and to investigate the use of tetracyclines in feeding programs for grower-finisher pigs as the main risk factor for discolouration. Abattoir data were examined to determine the extent of the problem and the prevalence of bone discolouration during 2006, 2008, 2009, and 2010 was found to be 0.13%, 0.22%, 0.26%, and 0.28%, respectively, indicating that the issue of bone discolouration was present at low levels over the entire period of the study. A controlled trial using feed, water, and injectable tetracycline products to investigate the effect of tetracyclines on residue and bone colour was conducted. Bones were assessed visually for signs of discolouration, and high performance liquid chromatography (HPLC) was used to measure the levels of tetracycline residues in the bones. Results from this trial demonstrated that discolouration could be produced with 660ppm of chlortetracycline (CTC) in feed for 12 weeks even when 33 days of withdrawal time was observed. It was also found that residues of tetracyclines can be present in bones in the absence of discolouration. A retrospective study was conducted to investigate tetracycline use in herds identified as having discoloured bones at slaughter. Positive shipments were associated with dosage and duration of CTC use as well as with length of withdrawal. In conclusion, discoloured bones of pig carcasses were identified at low levels in one large Ontario abattoir; however, further investigation is needed in order to determine the impact it may have on the swine industry. / Ontario Pork and the University of Guelph - Ontario Ministry of Agriculture (OMAFRA) Sustainable Production System Program

Thesis advisor: Daniel A. Kirschner / In the present study, the ability of small compounds to inhibit the fibrillogenesis of beta-amyloid 12-28 was explored. Beta-amyloid 12-28 is a synthetic fragment of Alzheimer's beta-amyloid, which contains the core hydrophobic residues thought to be significant for fiber formation. Using x-ray diffraction, preliminary screening of over sixteen compounds was performed. Cotinine, PTI-00703®, and tetracycline were chosen because of their ease of solubility, the effect on the coherent domain size of the beta-crystallite subunit in the presence of chosen small molecules as shown by x-ray diffraction, as well as their presence in previously published literature. This conformational-driven inhibition of fibrillogenesis was explored in the current research using circular dichroism spectroscopy and x-ray diffraction. Circular dichroism spectroscopy revealed the nascent beta-sheet structure of beta-amyloid12-28 when first dissolved and only cotinine, out of all three inhibitors, was able to shift the equilibrium away from the fibrillogenic beta-sheet structure toward a random coil secondary structure after 36 hours of incubation. X-ray diffraction in this study demonstrated no change in hydrogen bond spacing at ~4.7Å and intersheet spacing at ~10-12Å both alone and in the presence of all small molecules. With increasing concentration of inhibitor, however, the widths of these reflections increased, indicating a decrease in the coherent domain size. / Thesis (MS) — Boston College, 2010. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Tetracycline is a front line antibiotic for the treatment of chlamydial infections in both humans and animals, and the emergence of tetracycline resistant (tet[superscript R]) Chlamydia is of significant clinical importance. Recently, several tet[superscript R] chlamydial strains have been isolated from swine (Sus scrofa) raised in production facilities in Nebraska. Here, the intracellular development of two C. suis tet[superscript R] strains, R19 and R27, is characterized through the use of tissue culture and immunofluorescence. The strains grow in tetracycline up to 4 ��g/ml, while a tetracycline sensitive (tet[superscript S]) C. suis swine strain (S45) and a C. trachomatis strain of the human serovar L2 (LGV-434) grow in tetracycline up to 0.1 ��g/ml. Although inclusions form in the presence of tetracycline, many contain large aberrant RBs that do not differentiate into infectious EBs. The percentage of inclusions containing typical developmental forms decreases with increasing tetracycline concentrations, and at 3 ��g/ml of tetracycline, 100% of inclusions contain aberrant RBs. However, upon removal of the tetracycline, the aberrant RBs revert to typical RBs and a productive developmental cycle resumes. In addition, inclusions were found that contained both C. suis R19 and C. trachomatis L2 after sequential infection, demonstrating that two biologically distinct chlamydial strains could both develop within a single inclusion. / Graduation date: 2002

Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references. Also available on the World Wide Web.

Thesis (M.S.)--Ohio State University, 2008. / Title from first page of PDF file. Includes bibliographical references (p. 65-71).

published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

I. Efforts Toward the Synthesis of (–)-Tetracycline. A macrocyclic approach toward (–)-tetracycline is described. Traditional approaches towards the synthesis of tetracycline antibiotics employ the linear construction of the core structure starting with either the A- or D-rings. In contrast to this iterative annulation-based strategy, we have sought to employ a chiral macrocycle in our approach. Key to the success of our synthesis endeavor is the execution of two key steps: (1) a transannular Michael addition, which forms the A-ring and sets the C4a-stereogenic center; and (2) an isoxazole substitution reaction, which effects a ring contraction to produce both the B- and C-rings. This work describes our implementation of the strategy and focuses on the stereochemical interplay between the C4-, C4a-, C6-, and C12a-stereocenters within the context of the key steps. II. Analysis of the Peripheral Attack Model. The application of the peripheral attack model to 34 literature examples of intermolecular macrocyclic stereocontrol is described. While the peripheral attack model has been broadly applied in complex molecule synthesis, the validity of the model has not been subjected to analysis since being proposed in the early 1980’s. In order to assess the value of the model to organic chemists, we have developed a systematic method for probing the conformational profile of macrocycles. Using this tool, we then analyzed each of the 34 literature substrates and concluded whether the peripheral attack model predicts the correct stereochemical outcome in both a binary- and magnitude-based capacity. Analysis of both the bulk dataset and subsets of the dataset is included. / Chemistry and Chemical Biology

With water scarcity as a pressing issue in Saudi Arabia and other Middle Eastern countries, the treatment and reuse of municipal wastewater is increasingly being used as an alternative water source to supplement country water needs. Standards are in place to ensure a safe treated wastewater quality, however they do not regulate pathogenic bacteria and emerging contaminants. Information is lacking on the levels of risk to public health associated with these factors, the efficiency of conventional treatment strategies in removing them, and on wastewater treatment in Saudi Arabia in general. In this study, a municipal wastewater treatment plant in Saudi Arabia is investigated to assess the efficiency of conventional treatment in meeting regulations and removing pathogens and emerging contaminants. The study found pathogenic bacterial genera, antibiotic resistance genes and antibiotic resistant bacteria, many of which were multi-resistant in plant discharges. It was found that although the treatments are able to meet traditional quality guidelines, there remains a risk from the discussed contaminants with wastewater reuse. A deeper understanding of this risk, and suggestions for more thorough guidelines and monitoring are needed.