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Title: The Development of T7 Phage Based Imaging Probe and the Nature of Floating Cells in Human Embryonic Stem Cell Culture
Creator: Dasa, Siva Sai Krishna
Date: 2011-05-16, 2011-05
Description: Target specific nanoparticle based nuclear imaging probes have attracted significant attention recently. In most of the cases, these probes...
Show moreTarget specific nanoparticle based nuclear imaging probes have attracted significant attention recently. In most of the cases, these probes were synthesized by conjugating both affinity reagent and chelator molecules on the particle surface chemically. Subsequently, the isotope ions were attached to the particles via metalchelator interactions. Due to the nature of bio-conjugation process, the number of the chelator molecules and the geometry of the affinity reagent are hard to control. To overcome these limitations, a new approach of constructing an imaging probe was developed. In specific, T7 phage was used as a vehicle to synthesize a particle based imaging probe by displaying peptide that had both metal chelating (6 histidine) and targeting (RGD domains on the phage surface. It was demonstrated that the attachment of copper ions to the metal chelating domain does not interfere with target binding. Furthermore, by reducing the metal ions to metal, we were able to generate a very stable peptide templated metal hybrid T7 phage particle as potential target specific imaging probe. The behavior of the probes against both normal and tumor cells was investigated. The presence of floating cells is a common phenomenon in human embryonic stem (hES) cell cultures. Current assumption for the source of floating cells is that they are from apoptosis / senescence, cellular differentiation and other unavoidable imperfections in culture conditions. Inspired by recent studies on mitotic activities in stem cell colony, we believe the existence of floating cells is resulting from essential events required for hES cells proliferation. It was discovered that not all floating cells are dead and the percentage of floating cells over the attached cells was significantly increased with culture time possibly due to the space limitation for cells in the central part of the colonies. By providing more horizontal or vertical space through colony cutting and overlaid membrane insert in the Z direction, the percentages of floating cells were significantly reduced. All these results indicate that continuous cell division across the colonies is responsible for the emergence of floating cells during hES cell culture.
Ph.D. in Biology, May 2011
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Title: REGENERATIVE POTENTIAL OF HUMAN CD34+ STEM CELLS MOBILIZED AND ENRICHED FROM BONE MARROW
Creator: Cohen, Amy
Date: 2014, 2014-12
Description: This thesis provides an in-depth investigation of the heterogeneous population of CD34+ cells obtained through granulocyte-colony stimulating...
Show moreThis thesis provides an in-depth investigation of the heterogeneous population of CD34+ cells obtained through granulocyte-colony stimulating factor (G-CSF) mobilization, apheresis, and an immuno-magnetic selection process. The results provide a better understanding of the phenotypic characteristics, functional migratory capabilities and paracrine secretory performance when exposed to normal and low-oxygen environments in vitro. The overall composition of the population acquired through the cells selection process and the CD34+ cells were characterized for maturity and commitment to various cell types as well as their potential for migration, adhesion and proliferation. The detailed phenotypic characteristics included positivity for more primitive, undifferentiated hematopoietic stem cells (HSCs) as well as markers indicating enrichment of long term HSCs. A functional quantitative bioassay was used to measure the migratory ability of the CD34+ cells. On average, 18.7% of the CD34+ cells migrated after 3 hours of incubation in the presence of 200ng/mL of Stromal Cell-Derived Factor One alpha (SDF-1α) as compared to 0.6% of the CD34+ cells incubated without chemoattractant. The migration was successfully neutralized by using monoclonal antibodies to CXCR4 and a CXCR4 antagonist, AMD3100. An evaluation of the migratory cell population showed that the cells exhibited an enhanced commitment to a monocyte or lymphocyte lineage and a larger percentage were CXCR4+. CD34+ cells were incubated in normoxic and hypoxic conditions and the proliferation, viability, immunophenotypic profile, migratory potential and secreted cytokines were evaluated and compared to incubation in normoxic conditions. The CD34+ cells proliferated in both the normoxic and hypoxic conditions over three days of incubation. The proliferation was higher in the hypoxic condition after one day of incubation, but after three days of incubation the normoxic conditions resulted in more proliferation. An increase in CD164 and a decrease in CD99 in the Day 3 hypoxic condition suggest that hypoxia was stimulating an adhesion pathway and suppressing the functional migration of the CD34+ cells. The three hour hypoxia incubation resulted in a significant (63%) reduction in the migration of the CD34+ cells as compared to the 3 hour normoxia condition, but many of the cells continued to migrate in hypoxia over the 24 hour period while the normoxic cells continued to migrate, but at a much slower rate. Culture media from CD34+ stem cells that had been incubated in normoxic or hypoxic conditions for one day and three days was assayed for sixty cytokine proteins known for possessing angiogenic qualities. Twenty five angiogenesis cytokines were increased in the culture media incubated with CD34+ cells, including tissue inhibitor of metalloproteinases-1 and 2 (TIMP-1 and TIMP-2), angiopoietin-1 (ANG-1) and insulin-like growth factor-1 (IGF-1). Thirteen angiogenesis cytokines were elevated in hypoxia as compared to normoxia, including TIMP-1, IGF and growth-regulated oncogene-β (Gro-β).
Ph.D. in Biological and Chemical Sciences, December 2014
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Title: SCREENING AND COMPARING GENES OF INTEREST IN MICROBIAL SPECIES USING WHOLE GENOME SEQUENCING
Creator: Butler, Robert Raymond Iii
Date: 2016, 2016-12
Description: This study utilized the latest advances in whole genome sequencing, assembly and annotation to develop high quality curated genomes, which...
Show moreThis study utilized the latest advances in whole genome sequencing, assembly and annotation to develop high quality curated genomes, which were compared to related organisms with differential traits to identify or characterize the trait-associated genes. Additionally, we were able to infer potential origins of these traits, and present gene targets for further study. Here we examined two biological phenomena: the desulfurization capability of a Paenibacillus species, and the exceptionally high spore heat resistance of Clostridium sporogenes PA 3679. Microorganisms with the capability to desulfurize petroleum are in high demand with escalating restrictions currently placed on fuel purity. Thermophilic desulfurizers are particularly valuable in high temperature industrial applications. A culture containing Paenibacillus naphthalenovorans 32O-Y and Paenibacillus sp. 32O-W was isolated by repeated passage of a soil sample at up to 55°C in medium containing dibenzothiophene (DBT) as sulfur source. Only 32O-Y metabolized DBT, apparently via the 4S pathway, however 32O-W enhanced DBT metabolism by 32O-Y in a mixed culture. Genome sequencing identified desulfurization gene homologs in the strains consistent with their desulfurization properties, with 32O-W lacking homologs for two necessary components of the 4S pathway. Both 32O-Y alone and the 32O-Y/32O-W mixed culture may be useful in development of an improved thermophilic petroleum biodesulfurization process. Clostridium sporogenes PA 3679 is a nonpathogenic, nontoxic model organism for proteolytic Clostridium botulinum used in the validation of conventional thermal food processes due to its ability to produce highly heat-resistant endospores. Because of its public safety importance, the uncertain taxonomic classification and genetic diversity of PA 3679 are concerns. Therefore, isolates of C. sporogenes PA 3679 were obtained from various sources and characterized using pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing. The phylogenetic relatedness and genetic variability were assessed based on 16S rRNA sequence and whole-genome single nucleotide polymorphism (SNP) analysis. All C. sporogenes PA 3679 isolates were categorized into two clades. Clade I C. sporogenes isolates were genetically distinct from clade II isolates, and thermal destruction studies revealed that clade I isolates were more sensitive to high temperature than clade II isolates; clade II demonstrating the typical phenotype of PA 3679. A pan-genomic analysis of clade I and clade II isolates identified genes associated with PA 3679’s exceptional heat resistance. The most significant difference was the acquisition of a second spoVA operon, spoVA2, whose products are responsible for dipicolinic acid transport into the spore core during sporulation. The small acid-soluble spore protein ssp4 potentially plays a role in spore heat resistance, though further exploration is needed. spoVA2 was also found in some C. botulinum species clustering phylogenetically with PA 3679. Most other C. sporogenes examined both lack the spoVA2 locus and are phylogenetically distant within the group I Clostridia, adding to the understanding that C. sporogenes are dispersed C. botulinum strains lacking toxin genes. C. sporogenes strains are thus a very eclectic group, and few strains possess the characteristic heat resistance of PA 3679. Analysis from both Paenibacillus and Clostridium models revealed some interesting insights into genomic analysis that extrapolate to other projects. Each of the four generations of sequencing technology has remained a necessary component of genomics. The delineating factors for which sequencing tool to use depends heavily on the application they are being used for. New software for assembly and annotation are developed and released daily, and the challenge has become deciding which tools are actually an improvement over existing methodology. In order to best facilitate the large amount of genomic data in need of analysis, pipelines that are consistent and comprehensive are of higher value. Our studies identified many useful tools for future comparative analysis, and explored some novel ways to represent data in a visually appealing manner. As these tools and new ones continue to be developed, the value of genomics will increase with the new insights it provides.
Ph.D. in Biology, December 2016
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Title: ELASTIC ELEMENTS IN THE SARCOMERES OF STRIATED MUSCLE
Creator: Ma, Weikang
Date: 2016, 2016-07
Description: The flight muscle of the Hawkmoth, Manduca sexta, is an emerging model system for structure and function studies. M. sexta flight muscle shows...
Show moreThe flight muscle of the Hawkmoth, Manduca sexta, is an emerging model system for structure and function studies. M. sexta flight muscle shows several interesting properties such as its length tension curve is similar with cardiac muscle, but the detailed protein compositions of M. sexta flight muscle is not known. Here we identified proteins that might be responsible for the elastic properties of M. sexta flight muscle. 1% vertical SDS-agarose gel electrophoresis combined with western blot analysis was used to separate and identify high molecular weight proteins in M. sexta flight muscle. Two projectin isoforms as well as two kettin isoforms were found in M. sexta flight muscle. In addition, two high molecular weight proteins were seen in agarose gels which turned out to be Sallimus (Sls) proteins isoforms based on the sallimus (sls) gene map. The localization and orientation of projectin and Sallimus proteins were determined by immuno-localization using confocal microscopy. The thin and thick filament lengths were also determined, and shown to be consistent with the length tension curve data. Knowledge of myofilament compliance is critical in interpreting cross bridges kinetics interpretation and modeling. Here we used small angle X-ray diffraction to study thick filament compliance in intact mouse soleus muscle. The thick filament compliance was estimated by plotting the spacing changes of myosin based meridional reflections against tension generated by the muscle during contraction. A non-linear relationship of thick filament compliance was seen for the first time. Nebulin is a giant thin filament protein and has been proposed to play significant roles in muscle physiology, but the underlying mechanisms are largely unknown. A conditional nebulin gene knockout mouse model was used here to study any structural and functional changes caused by nebulin deficiency using small angle X-ray diffraction. The thin filament compliance was estimated, and the results showed that the thin filament compliance in nebulin deficient muscle was larger than muscle from control animals, whereas no difference was seen in thick filament compliance between knockout and control muscles. The inter-filament spacing was larger in knock out muscle than in control muscle, and less force was generated by each cross bridge. The larger interfilament spacing and less force per cross bridge might explain the muscle weakness seen in both the knock out mouse model, as well as in nemaline myopathy patients. Other structural changes caused by nebulin deficiency was also characterized by small angle X-ray diffraction.
Ph.D. in Molecular Biochemistry and Biophysics, July 2016
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Title: MEMBRANE-DRUG INTERACTION MECHANISMS OF PEPTOID-BASED ANTIMICROBIAL AGENTS
Creator: Andreev, Konstantin
Date: 2017, 2017-05
Description: Nature is a major source of inspiration for drug design. Bacteria are developing resistance towards conventional antibiotics. Utilizing...
Show moreNature is a major source of inspiration for drug design. Bacteria are developing resistance towards conventional antibiotics. Utilizing antimicrobial peptides (AMPs) – an essential component of innate immune system, as therapeutic agents, may be a viable alternative. Unfortunately, there are a number of serious hurdles on the way towards clinical application of AMPs, including their low bioavailability, costly manufacturing process and toxicity against host cells. To address this issues, current research is focused on the design of synthetic compounds mimicking natural peptides, among which oligo(Nsubstituted glycines), or peptoids, have shown great promise. Antimicrobial drug efficacy is defined by how it interacts with the membrane of invading pathogen. The physicochemical characteristics of peptoid molecule play a crucial role in these interactions, yet their detailed structure-activity relationships remain obscure. Herein, we have demonstrated that conformational flexibility, cationic charge or hydrophobicity, are critical for oligomeric peptoids to permeate bacterial cell membranes. The outer surface of membrane was modeled by Langmuir monolayers of desired lipid composition and subjected to the constant-pressure insertion assays, epifluorescence microscopy (EFM), synchrotron X-ray reflectivity (XR) and grazing incident-angle X-ray diffraction (GIXD). Our results shed light on the critical details in peptoid mode of action. We believe this will aid in the rational design and of novel anti-infective drugs. Additionally, we have applied our experimental system to model the processes occurring at the air-water interface in lungs. Alveoli are coated by a complex lipidprotein mixture referred to as pulmonary surfactant. This facilitates respiration and prevents alveolar collapse. Patients with respiratory distress receive surfactant replacement therapy that often has the serious drawbacks. X-ray scattering data shows that the structural organization of adsorbed films correlates with surfactant delivery methods onto the respiratory surface. We anticipate that our findings will contribute to the development of novel clinical approaches for treating respiratory diseases.
Ph.D. in Biology, May 2017
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