published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Molecular biology techniques were used in this research to assess the changes that occur in aquatic microbial communities when exposed to chemical wastes and the response of genetically engineered microorganisms (GEMs) in irradiated soils. Different molecular and microbial approaches to monitoring both types of microbial community changes can provide information about those systems at less complex and more sensitive levels than can be achieved with more traditional methods. Traditionally, aquatic microbial communities are evaluated by taxonomic identification and enumeration. Changes in species richness (diversity of taxa) and density of representative taxa were evaluated by DNA-DNA hybridization of prokaryotic communities at stations above and below points of industrial effluent release. Preliminary studies in 1990 indicated a positive correlation (0.95) between traditional identification and enumeration and DNA-DNA hybridization. This correlation was approaching significance but limited by a small sampling size. Study sites in 1991 did not have high correlation (0.04 and 0.36). Low correlation values were the result of methodological difficulties in the hybridization process rather than actual disparities between the two approaches. Graphically, the trends in protozoan species richness and densities of represented taxa were reflected in the DNA-DNA hybridization over time. Improvements in a molecular method include the use of radioisotopes for membrane bound hybridizations or liquid hybridizations (COT curves). In irradiated clay and loam microcosms, genetically-engineered Erwinia carotovora and wildtype declined significantly (p ≤ 0.05) over a 60 day period. Interestingly, in the clay soil, both the GEM and wildtype remained at decreased but detectable levels (using the MPN technique) after 60 days. In the loam soil, the GEM declined below levels of detection while the wildtype persisted and displayed a highly variable growth die-off pattern before declining in numbers. No significant (p ≥ 0.05) differences between the GEM and wildtype were observed in the clay soil. In the loam soil, wildtype survived at significantly (p ≤ 0.05) greater densities than the GEM over 60 days. Differences in soil characteristics as well as the type of GEM used resulted in novel patterns of persistence. An alternative to the MPN method for determining low densities of organisms is the polymerase chain reaction (PCR) in conjunction with DNA probes and hybridization techniques. For environmental probes to be used, the DNA sequence or a portion of the sequence must be determined to serve as a template for PCR amplification. Both ends of a probe specific for 63 serologically distinct strains of E. carotovora were sequenced in the initial phase of the PCR amplification and detection process. This research employs several molecular and microbial methods for the determination of changes in microbial community structure and function in response to chemical or irradiation perturbations. / Master of Science

Advanced laboratory techniques for Microbial and Molecular Biology at the graduate level are presented in this thesis. The procedures for the laboratory experiments are set forth in detail. This laboratory is conducted as two parts, each by a different professor. Part 1 covers the experiments conducted by Dr G.A.O. Donovan. These experiments include an introduction, staining procedures, biochemical reactions, mutagenesis experiment, essays,. preparation and analysis of plasmid DNA and various other topics. Part 2 covers the experiments conducted by Dr. Daniel Kunz and includes various topics like media preparation, phenotyping strains, conjugative transfer of plasmids, SDS-PAGE, induction and measurement of enzyme and transposon mutagenesis

In this dissertation a computer-aided automation design methodology for biotechnology applications is proposed that leads to several design guidelines. Because of the biological nature of the samples that propagate in the automation line, a very specific set of environmental and maximum allowed shelf time conditions have to be followed to obtain good yield. In addition all biotechnology protocols require precise sequence of steps, the samples are scarce and the reagents are costly, so no waste can be afforded. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2014. / FAU Electronic Theses and Dissertations Collection

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Infectious bursal disease virus (IBDV) causes an acute and highly contagious disease affecting young chickens, which is responsible for significant losses in the poultry industry world-wide. The virus specifically infects and destroys B-cell precursors within the bursa of Fabricius, an avian lymphoid organ, leading to immunosuppression. IBDV has a bi-segmented, double-stranded RNA genome. The larger segment encodes a 110-kDa precursor polyprotein, designated NH₂-VPX-VP4-VP3-COOH, in a single open reading frame. The autocatalytic processing of this precursor into mature proteins is a critical step in viral replication and VP4 is the putative protease responsible for this cleavage. This study concerns the development of a strategy to clone and express recombinant VP4 and describes the use of VP4 as a marker for rapid and effective detection of IBDV. VP4 cDNA was produced and amplified by optimisation of a reverse transcription coupled to the polymerase chain reaction (RT-PCR), providing a clear and sensitive assay. Anti-peptide antibodies were raised against a selected peptide from VP4 and were used to probe homogenates of infected bursae for the native protein to assess their potential for immunological detection. These antibody-related results are promising though inconclusive, due to the complex nature of the assayed sample. Amplified VP4 cDNA from KwaZulu-Natal strains of IBDV isolated from 1989 to 1997 was also examined by restriction fragment length polymorphism (RFLP) analysis to determine the relatedness of local IBDV to global strains. All KwaZulu-Natal samples produced identical patterns, which were most similar to one of ten international strains examined, namely, the British strain UK661. Samples infected with IBDV were also probed for VP4 activity. Double basic amino acid cleavage sites have been proposed for the putative protease and infected samples were assayed for activity against the fluorogenic peptide Cbz-Arg-Arg-AMC. Demonstrably higher activity was found in infected versus uninfected samples, although the origin of this activity is unclear. The findings in this study suggest that VP4 warrants further attention, both as a marker for infectious bursal disease, and as a novel viral protease. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.

High-throughput time series data characterizing magnitudes of gene expression, levels of protein activity, and the accumulation of select metabolites in vivo are being generated with increased frequency. These time profiles contain valuable information about the structure, dynamics and underlying regulatory mechanisms that govern the behavior of cellular systems. However, extraction and integration of this information into fully functional, computational and explanatory models has been a daunting task. Three types of issues have prevented successful outcomes in this inverse task of system identification. The first type pertains to the algorithmic and computational difficulties encountered in parameter estimation, be it using a genetic algorithm, nonlinear regression, or any other technique. The second type of issues stems from implicit assumptions that are made about the system topology and/or the functional model representing the biological system. These include the choice of intermediate pathway steps to be accounted for in the model, decisions on the irreversibility of a step, and the inclusion of ill-characterized regulatory signals. The third type of issue arises from the fact that there is often no unique set of parameter values, which when fitted to a model, reproduces the observed dynamics under one or several different sets of experimental conditions. This latter issue raises intriguing questions about the validity of the parameter values and the model itself. The central focus of my research has been to design a workflow for parameter estimation and system identification from biological time series data that resolves the issues outlined above. In this thesis I present the theory and application of a novel framework, called Dynamic Flux Estimation (DFE), for system identification from biological time-series data.

Indiana University-Purdue University Indianapolis (IUPUI) / Phosphatases of regenerating liver, consisting of PRL-1, PRL-2 and PRL-3, belong to a novel protein tyrosine phosphatases subfamily, whose overexpression promotes cell proliferation, migration and invasion and contributes to tumorigenesis and metastasis. However, although great efforts have been made to uncover the biological function of PRLs, limited knowledge is available on the underlying mechanism of PRLs’ actions, therapeutic value by targeting PRLs, as well as the physiological function of PRLs in vivo. To answer these questions, we first screened a phage display library and identified p115 RhoGAP as a novel PRL-1 binding partner. Mechanistically, we demonstrated that PRL-1 activates RhoA and ERK1/2 by decreasing the association between active RhoA with GAP domain of p115 RhoGAP, and displacing MEKK1 from the SH3 domain of p115 RhoGAP, respectively, leading to enhanced cell proliferation and migration. Secondly, structure-based virtual screening was employed to discover small molecule inhibitors blocking PRL-1 trimer formation which has been suggested to play an important role for PRL-1 mediated oncogenesis. We identified Cmpd-43 as a novel PRL-1 trimer disruptor. Structural study demonstrated the binding mode of PRL-1 with the trimer disruptor. Most importantly, cellular data revealed that Cmpd-43 inhibited PRL-1 induced cell proliferation and migration in breast cancer cell line MDA-MB-231 and lung cancer cell line H1299. Finally, in order to investigate the physiological function of PRLs, we generated mouse knockout models for Prl-1, Prl-2 and Prl-3. Although mice deficient for Prl-1 and Prl-3 were normally developed, Prl-2-null mice displayed growth retardation, impaired male reproductive ability and insufficient hematopoiesis. To further investigate the in vivo function of Prl-1, we generated Prl-1-/-/Prl-2+/- and Prl-1+/-/Prl-2-/- mice. Similar to Prl-2 deficient male mice, Prl-1-/-/Prl-2+/- males also have impaired spermatogenesis and reproductivity. More strikingly, Prl-1+/-/Prl-2-/- mice are completely infertile, suggesting that, in addition to PRL-2, PRL-1 also plays an important role in maintaining normal testis function. In summary, these studies demonstrated for the first time that PRL-1 activates ERK1/2 and RhoA through the novel interaction with p115 RhoGAP, targeting PRL-1 trimer interface is a novel anti-cancer therapeutic treatment and both PRL-1 and PRL-2 contribute to spermatogenesis and male mice reproductivity.

Indiana University-Purdue University Indianapolis (IUPUI) / Fanconi anemia (FA) is a heterogenous genetic syndrome characterized by progressive bone marrow failure, aneuploidy, and cancer predisposition. It is incompletely understood why FA-deficient cells develop gross aneuploidy leading to cancer. Since the mitotic spindle assembly checkpoint (SAC) prevents aneuploidy by ensuring proper chromosome segregation during mitosis, we hypothesized that the FA signaling network regulates the mitotic SAC. A genome-wide RNAi screen and studies in primary cells were performed to systematically evaluate SAC activity in FA-deficient cells. In these experiments, taxol was used to activate the mitotic SAC. Following taxol challenge, negative control siRNA-transfected cells appropriately arrested at the SAC. However, knockdown of fourteen FA gene products resulted in a weakened SAC, evidenced by increased formation of multinucleated, aneuploid cells. The screen was independently validated utilizing primary fibroblasts from patients with characterized mutations in twelve different FA genes. When treated with taxol, fibroblasts from healthy controls arrested at the mitotic SAC, while all FA patient fibroblasts tested exhibited weakened SAC activity, evidenced by increased multinucleated cells. Rescue of the SAC was achieved in FANCA patient fibroblasts by genetic correction. Importantly, SAC activity of FANCA was confirmed in primary CD34+ hematopoietic cells. Furthermore, analysis of untreated primary fibroblasts from FA patients revealed micronuclei and multinuclei, reflecting abnormal chromosome segregation. Next, microscopy-based studies revealed that many FA proteins localize to the mitotic spindle and centrosomes, and that disruption of the FA pathway results in supernumerary centrosomes, establishing a role for the FA signaling network in centrosome maintenance. A mass spectrometry-based screen quantifying the proteome and phospho-proteome was performed to identify candidates which may functionally interact with FANCA in the regulation of mitosis. Finally, video microscopy-based experiments were performed to further characterize the mitotic defects in FANCA-deficient cells, confirming weakened SAC activity in FANCA-deficient cells and revealing accelerated mitosis and abnormal spindle orientation in the absence of FANCA. These findings conclusively demonstrate that the FA signaling network regulates the mitotic SAC, providing a mechanistic explanation for the development of aneuploidy and cancer in FA patients. Thus, our study establishes a novel role for the FA signaling network as a guardian of genomic integrity.