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Theses and Dissertations

Permanent URI for this collectionhttps://hdl.handle.net/10217/100356

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Browsing Theses and Dissertations by Subject "algae"

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    Alternative splicing and its regulatory mechanisms in photosynthetic eukaryotes
    (Colorado State University. Libraries, 2011) Link, Alicia, author; Reddy, A. S. N., advisor; Stack, Stephen, committee member; Lapitan, Nora, committee member
    In recent years, alternative splicing (AS) of pre-mRNAs, which generates multiple transcripts from a single gene, has emerged as an important process in general proteome diversity and in regulatory gene expression in multicellular eukaryotes. In Arabidopsis over 40% of intron-containing genes are alternatively spliced. However, mechanisms by which AS is regulated in plants are not fully understood, primarily due to the lack of an in vitro splicing system derived from plants. Furthermore, the extent of AS in simple unicellular photosynthetic eukaryotes from which plants have evolved is also not known. My research addresses these two attributes of splicing in plants. In Part 1 of my thesis, I have investigated an aspect of AS regulation in plants. We have previously shown that an SR-related splicing regulator called SR45 regulates AS of pre-mRNAs in Arabidopsis by altering splice site selection (Ali et al. 2007). In this work using bimolecular fluorescent complements, I have demonstrated that SR45 interacts with U2AF35, an important spliceosomal protein involved in 3' splice site selection in plant cells. This interaction takes place in the nucleus, specifically in the subnuclear domains called speckles, which are known to contain splicing regulators and other proteins involved in transcription. My work has shown that SR45 interacts with both paralogs of U2AF35 and I mapped the domains in SR45 that are involved in its interaction with U2AF35. In addition, my studies have revealed interaction of the paralogs as hetero- and homodimers. Interestingly, U2AF35 was found to interact with U1-70K, a key protein involved in 5' splice site selection. Based on this work and previous work in our laboratory, a model is proposed that explains the role of SR45 in splice site selection. In the second part of my work I studied the extent of alternative splicing (AS) in the unicellular green alga Chlamydomonas, that shares a common ancestor with land plants. In collaboration with Dr. Asa Ben Hur's lab, we have performed a comprehensive analysis of AS in Chlamydomonas reinhardtii using both computational and experimental methods. Our results show that AS is common in Chlamydomonas, but its extent is less than what is observed in land plants. However, the relative frequency of different splicing events in Chlamydomonas is very similar to higher plants. We have found that a large number of genes undergo alternative splicing, and together with the simplicity of the system and the use of available molecular and genetic tools. This organism is an experimental system to investigate the mechanisms involved in alternative splicing. To further validate predicted splice variants, we performed extensive analysis of AS for two genes, which not only confirmed predictions but also revealed novel splice variants, suggesting that the extent of AS is higher than we predicted. AS can also play a role in the regulation of gene expression through processes such as regulated unproductive splicing and translation (RUST) that involves nonsense-mediated decay (NMD), a mechanism of mRNA surveillance that degrades transcripts containing premature termination codons (PTCs). The basic mechanism of NMD relies upon many factors, but there are three critical proteins, termed the UP-frameshift (UPF) proteins due to their ability to up-regulate suppression of nonsense transcripts. UPF1, UPF2, and UPF3 appear to be conserved across animals and plants. Our analysis of AS has found that in Chlamydomonas, many splice variants have a premature termination codon (PTC). However, to date, the mechanism of NMD has not been investigated in Chlamydomonas. Analysis of the Chlamydomonas genome sequence shows that UPF1, 2, and 3 proteins are present, and we have shown that they share some sequence similarity with both plants and humans, indicating that the process of NMD may be present in this organism. To address the role of UPFs in NMD in Chlamydomonas, we have utilized the artificial miRNA approach. I have generated stably transformed Chlamydomonas cell lines that are expressing amiRNA for UPF1 and UPF3 that will be useful in analyzing NMD of selected genes as well as all PTC-containing transcripts globally.
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    Selection and fluorescence based screening of algal strains for temperature tolerance and increased productivity for industrial scale cultivation
    (Colorado State University. Libraries, 2025) Bertucci, Conor, author; Peers, Graham, advisor; Khakhar, Arjun, committee member; Peebles, Christie, committee member; Reddy, Anireddy, committee member
    Microalgae are emerging as a viable source of sustainable energy and bioproducts due to their rapid growth and capacity to produce valuable products. Their ability to grow in diverse, non-arable environments while minimizing resource use makes them a promising alternative to traditional crops for biofuel, feedstock, and other value-added products. Industrial-scale outdoor cultivation of microalgae subjects cells to dynamic environmental conditions such as fluctuating temperatures that can influence growth and biomass accumulation. This makes selecting a strain that can maintain high productivity crucial for industrial-scale cultivation. The first aim of this thesis was to compare the growth and biomass accumulation of ten strains that were known for their high productivities in temperatures ranging from 18°C to 30°C. Of the ten selected strains, Scenedesmus rubescens NREL 46B-D3 and Monoraphidium minutum 26B-AM were determined to have the best overall growth performance in both temperatures and the highest biomass accumulation when grown at 30°C. S. rubescens and M. minutum exhibited an 88.5% and 22.6% higher average total organic carbon accumulation compared to the next highest performing strain tested. The second aim of this thesis was to utilize gamma irradiation mutagenesis to generate mutants with improved biomass accumulation. Optimal LD90% dosages of 300 Gy and 75 Gy were determined for S. rubescens and M. minutum respectively. 3135 and 3356 putative mutants were characterized for S. rubescens and M. minutum, respectively. A fluorescence-based screening approach was used to screen for putative mutants with altered photophysiology traits correlated with photosynthetic efficiency. A total of 37 S. rubescens putative mutants and 14 putative M. minutum mutants demonstrated repeated photophysiological alterations and were selected for growth comparisons between their wild type counterparts. Only one putative M. minutum mutant, MRM J-325, demonstrated improvements in specific growth rate compared to wild type. This assumed mutant will be scaled up for biomass accumulation experiments at large scale.