Theses and Dissertations from UMD
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Item Characterizing the Complex Spatial Patterns in Biological Systems(2015) Parker, Joshua; Losert, Wolfgang; Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Spatial point patterns are ubiquitous in natural systems, from the patterns of raindrops on a sidewalk to the organization of stars in a galaxy. In cell biology, these patterns can represent the locations of fluorescently-labeled molecules inside or on the surface of cells, or even represent the centers of the cells themselves. These patterns arise due to the signaling activity of the cells which are mediated by a broad range of chemicals, and understanding this activity is vital to investigating these complex systems. Luckily, though each pattern is unique, the statistical properties of the patterns embed information about the underlying pattern formation process. In this work, I demonstrate techniques to characterize the complex spatial patterns found in unicellular systems. Using topologically-derived measures, I demonstrated a technique to automatically classify sets of point patterns into groups to identify changes in higher order statistical moments due to experimental variation. This technique utilizes functional principal component analysis (FPCA) on the Minkowski functionals of a secondary pattern formed by imposing disks on each point center. I demonstrate that this better classifies a range of point pattern sets, and then applied this technique to pattern sets representing membrane-bound proteins in human immune cells, showing that this procedure correctly identifies non-interacting proteins. Further, I demonstrate a simulation-based technique to diminish the statistical impact of large-scale pattern features. In protein patterns, these represent the effects of membrane ruffling during pattern formation. These features dominate correlation measures, obscuring any hint of nanoscale clustering. Using heterogeneous Poisson null models for each cell to re-normalize their pairwise correlation functions, I found that patterns of LAT proteins ("linker for the activation of T-cells") do indeed cluster, with a characteristic length-scale of approximately 500 nm. By performing clustering analysis at this length scale on both the LAT patterns and their respective null models, I found that clusters are most commonly dimers, but that this clustering is strongly diminished upon T-cell activation. This loss of clustering may be due to the presence of unlabeled molecules that have been recruited to the cell membrane to form complexes with LAT. I also investigate both molecular and cell-center patterns in Dictyostellium discoideum cells, which are a model organism for amoeboid motion and G-protein receptor-mediated chemotaxis. These cells migrate using "autocrine" signal relay in that they both secrete and sense the same chemoattractant, cyclic adenosine monophosphate (cyclic AMP or cAMP). They also secrete phosphodiesterases that degrade the chemoattractant. This leads to streaming patterns of cells towards aggregation centers, which serve as sites of sporulation. To study these cells, I demonstrate an image analysis technique that statistically infers the local population of fluorescently-labeled mRNA units in fluorescent images of self-aggregating cells. The images were of experiments where two particular mRNAs were labeled along with their respective proteins, the first being adenylyl cyclase A (ACA), a molecule involved in the production of cAMP. ACA itself has already been seen to accumulate at the back of migrating cells. The location of these molecules were compared to that of the locations of cyclic AMP receptor 1 (cAR1), which is the cell's mechanism for gradient sensing. Using my analysis technique, I found that statistically significant proportions of ACA mRNA preferentially locate towards the rear of migrating cells, an assymetry that was also found to identically correlate with the asymmetry of ACA itself. This asymmetry was not seen in cAR1 mRNA, which tends to distribute uniformly. Further, the asymmetry in ACA was most exaggerated in cells migrating at the rear of streams, with the approach to the local aggregate center diminishing leading to more uniformly distributed molecules. This may suggest that ACA is locally translated at the back of migrating cells, a result requiring further investigation. I then construct a computational migration model of D. discoideum chemotaxis and use it to investigate how the streaming phase is effected by cell-cell adhesion as well as by the global degradation of cAMP. To classify the dynamics of the model with respect to cell density and external chemical gradient, the two relevant phase variables, I develop an order parameter based on the fraction of broken cell-cell contacts over time. This parameter successfully classifies the dynamic steady states of the model (independent motion, streaming, and aggregation), outperforming the often used "chemotactic index". I found that the elimination of degradation strongly diminishes any presence of streaming, suggesting that chemical degradation is vital to stream formation. In contrast, the addition of cell-cell adhesion expanded the streaming phase, stabilizing streams that were formed initially through signal relay.Item Messenger RNA Destabilization by -1 Programmed Ribosomal Frameshifting(2012) Belew, Ashton Trey; Dinman, Jonathan D; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Although first discovered in viruses, previous studies have identified programmed -1 ribosomal frameshifting (-1 PRF) signals in eukaryotic genomic sequences, and suggested a role in mRNA stability. This work improves and extends the computational methods used to search for potential -1 PRF signals. It continues to examine four yeast -1 PRF signals and show that they promote significant mRNA destabilization through the nonsense mediated (NMD) and no-go (NGD) decay pathways. Yeast EST2 mRNA is highly unstable and contains up to five -1 PRF signals. Ablation of the -1 PRF signals or of NMD stabilizes this mRNA. These same computational methods identified an operational programmed -1 ribosomal frameshift (-1 PRF) signal in the human mRNA encoding CCR5. A -1 PRF event on the CCR5 mRNA directs translating ribosomes to a premature termination codon, destabilizing it through the nonsense-mediated mRNA decay (NMD) pathway. CCR5-mediated -1 PRF is stimulated by at least two miRNAs, one of which is shown to directly interact with the CCR5 -1 PRF signal. Structural analyses reveal a complex and dynamic mRNA structure in the -1 PRF signal, suggesting structural plasticity as the underlying biophysical basis for regulation of -1 PRF.Item Development of Carbon Nanotube Field-Effect Transistor Arrays for Detection of HER2 Overexpression in Breast Cancer(2011) Aschenbach, Konrad Hsu; Gomez, Romel D; Electrical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)We developed a carbon nanotube biosensor platform that was deployed at the National Cancer Institute and successfully detected the HER2 oncogene in real cancer cells at clinically relevant levels. HER2 is a receptor protein that resides on the surface of certain cancer cells and is associated with higher aggressiveness in breast cancers. Overabundance of HER2 at the chromosomal, cell surface, and intermediate gene expression levels can all indicate a dangerous HER2 status. At the present, testing for HER2 status requires labor-intensive laboratory procedures using expensive reagents. Cost remains the major barrier to widespread screening. We propose an integrated electronic testing platform based on direct label-free gene detection. The system would integrate the various labor-intensive processes that are usually performed by skilled laboratory technicians. The heart of the system is an array of carbon nanotube field-effect transistors that can detect unlabelled nucleic acids via their intrinsic electric charges. We developed a scalable fabrication technique for carbon nanotube biosensor arrays, hardware and software for data acquisition and analysis, theoretical models for detection mechanism, and protocols for immobilization of peptide nucleic acid probes and hybridization of nucleic acids extracted from cells. We demonstrated detection of HER2 from real cell lines which express cancer genes, thereby lowering the technological barrier towards commercialization of a low-cost gene expression biosensor. The system is suitable for lab-on-a-chip integration, which could bring rapid, low-cost cancer diagnoses into the clinical setting.Item Characterization of Arabidopsis thaliana SR protein genes: mutations, alternative splicing, and ESE selection(2007-06-07) edmonds, jason matthew; Mount, Stephen M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)RNA processing in eukaryotes is a highly complex process requiring numerous steps and factors that can play roles in the regulation of functional protein production. SR proteins are a well-defined family of splicing factors identified by a conserved RNA Recognition Motif (RRM) and carboxyl-terminal arginine/serine (RS) repeats. SR proteins are known to bind to mRNA precursors via Exonic Splicing Enhancers, and to recruit U2AF and the U1 snRNP to promote splicing. I have identified mutations in five Arabidopsis thaliana SR protein genes that result in altered phenotypes. Two (scl28-1 and srp31-1) result in embryonic lethal phenotypes, while three others (sc35-1, sr45-1, and srp30-1) result in viable and fertile plants with a range of phenotypes. I have also found that mutations in individual SR protein genes can effect the ability of a specific sequence to act as an ESE and hence affect splicing efficiency. Because 16 of the 20 Arabidopsis thaliana SR proteins themselves are alternatively spliced, I have looked for cross regulation using RT-PCR analysis of isoform accumulation in alternatively spliced SR protein genes. I found that SR proteins do, in fact, regulate the alternative splicing of gene targets and do so in both a gene and a tissue specific manner. In order to begin to fully understand the relationship between individual SR proteins it is essential to know when and where they are expressed throughout development. I have studied the expression pattern of 16 of the 20 SR proteins in the roots of wild-type plants as well as sc35-1, srp30-1, and sr45-1 mutants. I have identified both spatial and temporal expression patterns for these 16 proteins relative to specific tissues that compose the root.