THE ROLE OF FIRE AND A FIRE-FREE INTERVAL IN THE RESTORATION OF UPLAND OAK COMMUNITIES ON THE CUMBERLAND PLATEAU, KENTUCKY

The decline of upland oak (Quercus spp.) communities in our eastern forests has been attributed to the loss of periodic disturbance after decades of fire suppression. As land managers have begun to reintroduce fire, effects on oak regeneration and species composition have varied widely, making it apparent that our understanding of how fire can aid in oak forest management needs refinement. Restoring upland oak communities requires decreasing stand density and opening of the canopy to release shade-intolerant oaks in the understory. This necessitates an extended fire-free interval to allow these oaks to be recruited into larger size classes and develop resistance to future fires. The ability of prescribed fire alone to create these structural changes is uncertain due to the low intensity of prescribed burns which for the most part do not kill larger diameter trees. In this work, I examined the utility of a fire-free interval following repeated fire alone as a management tool, as well as the combined effects of fire and mechanical removal in the form of midstory mastication. Where forest structure is significantly reduced by fire or mechanical removal, restoration of oak communities is complicated by both prolific sprouting and ingrowth of competitor species and the introduction of invasive species. The results of this study suggest that, in the absence of mechanical removals, reductions in stem density necessary to restore conditions for oak regeneration might be limited to sites that experience higher fire severity and/or drier landscape positions. Additionally, the rapid response of competing non-oak stems such as maple (Acer spp.), yellow-poplar (Liriodendron tulipifera) and sassafras (Sassafras albidum) during the fire-free interval and the increasingly severe invasion of Japanese stiltgrass (Microstegium vimineum) following disturbance are severe hindrances to successful restoration of upland oak ecosystems. Despite these management concerns, results of the research reported in this thesis indicate that restoring disturbance regimes slows the process of mesophication, improves size and stature of oak regeneration, and increases community diversity across the landscape

Translation-dependent mRNA localization in the Caenorhabditis elegans embryo

Includes bibliographical references.2022 Fall.Though each animal cell contains the same genetic information, cell-specific gene expression is required for embryos to develop into mature organisms. Embryos rely on maternally inherited components during early development to guide cell fate specification. In animals, de novo transcription is paused after fertilization until zygotic genome activation. Consequently, early embryos rely on post-transcriptional regulation of maternal mRNA to spatially and temporally regulate protein production. Caenorhabditis elegans has emerged as a powerful developmental model for studying mRNA localization of maternally-inherited transcripts. We have identified subsets of maternal mRNAs with cell-specific and subcellular patterning in the early C. elegans embryo. Previous RNA localization studies in C. elegans focused on maternal transcripts that cluster in the posterior lineage and showed mRNA localization occurs in a translation-independent manner through localization sequence elements in the 3'UTR. However, little is known about the mechanisms directing RNA localization to other subcellular locales in early embryos. Therefore, we sought to understand the localization of maternal transcripts found enriched at the plasma membrane and nuclear periphery, erm-1 (Ezrin/Radixin/Moesin) and imb-2 (Importin Beta), respectively. In this thesis, I characterize two different translation-dependent pathways for mRNA localization of maternal transcripts at the plasma membrane and nuclear periphery. I identified the PIP2-membrane binding region of the ERM-1 proteins is necessary for erm-1 mRNA localization while identifying additional membrane localized maternal transcripts through the presence of encoded PIP2-membrane binding domains. Additionally, I observed that mRNA localization patterns can change over developmental time corresponding to changes in translation status. For imb-2 mRNA localization, I found localization to the nuclear periphery is also translation-dependent. Through recoding the imb-2 mRNA sequence while maintaining the translated peptide sequence using alternative codons, I found both localization and transcript stability additionally depends on mRNA sequence context. These findings represent the first report of a translation-dependent localization pathway for two maternally-inherited transcripts in C. elegans and demonstrate the utility of C. elegans as a model for studying translation-dependent mRNA localization during development

Function of Lysosomes and Lysosomal Enzymes in the Senescing Corolla of the Morning Glory (Ipomoea purpurea)

The rapid senescence of the Ipomoea corolla is characterized by the breakdown of protein and nucleic acids. At the onset of wilting the activities of deoxyribonuclease (DNase), ribonuclease (RNase), and β-glucosidase are increased dramatically, while other hydrolytic activities such as the actions of protease, aminopeptidase, α-glucosidase, phosphatase, esterase, and α-amylase are only slightly changed. Isolated corolla discs show a course of senescence similar to that of the intact organ. When floating on solutions of cycloheximide the activities of DNase, RNase, and β-glucosidase do not increase. Actinomycin D inhibits the increase in RNase activity. It is concluded that protein synthesis is a prerequisite for the changes in these enzyme activities in the senescing corolla. The function of the lysosomal compartment in the process of senescence is illustrated by electron micrographs showing the autophagic activity of vacuoles. The last phase of senescence is characterized by the breakdown of the tonoplast and complete digestion of the cytoplasmic constituents in the autolysing cell

Examining the Human Relevance of a New Mouse Model of ALS and Observations on the Incidence in Human Populations

Amyotrophic Lateral Sclerosis (ALS) is a relatively rare neurodegenerative disorder that is fatal 2-4 years after the onset of the disease. Population studies are being done in order to help reveal candidates for further studies of disease etiology, disease progression, as well as to further understand racial and ethnic variations. Population-based studies have occurred mostly in the United States and Europe, with few to no studies conducted in other regions. One of the most widely used ALS animal models is the SOD^G93A mouse model. Up until now there has been no effective treatment significantly expanding the lifespan of this model. The Beckman lab has demonstrated that the copper ligand CuATSM effectively halts the progression of ALS symptoms in a variant of the SOD^G93A mouse model co- expressing the Human Copper Chaperone for SOD, hCCS. However, this SOD^G93AxhCCS mouse model is not well-characterized in terms of relative and absolute protein expression levels of SOD compared to CCS. The research done for this thesis aimed to fill this knowledge gap. We demonstrated that the relative expression ratio of SOD1 to hCCS in the SOD^G93AxhCCS mouse model is 1.5:1. These preliminary results will provide insights when comparing this new mouse model to humans. Key Words: Amyotrophic Lateral Sclerosis (ALS), SODG93A mouse model, CuATSM, SOD1, hCC

Relationship between petal abscission and programmed cell death in Prunus yedoensis and Delphinium belladonna

Depending on the species, the end of flower life span is characterized by petal wilting or by abscission of petals that are still fully turgid. Wilting at the end of petal life is due to programmed cell death (PCD). It is not known whether the abscission of turgid petals is preceded by PCD. We studied some parameters that indicate PCD: chromatin condensation, a decrease in nuclear diameter, DNA fragmentation, and DNA content per nucleus, using Prunus yedoensis and Delphiniumbelladonna which both show abscission of turgid petals at the end of floral life. No DNA degradation, no chromatin condensation, and no change in nuclear volume was observed in P. yedoensis petals, prior to abscission. In abscising D.belladonna petals, in contrast, considerable DNA degradation was found, chromatin was condensed and the nuclear volume considerably reduced. Following abscission, the nuclear area in both species drastically increased, and the chromatin became unevenly distributed. Similar chromatin changes were observed after dehydration (24 h at 60°C) of petals severed at the time of flower opening, and in dehydrated petals of Ipomoea nil and Petunia hybrida, severed at the time of flower opening. In these flowers the petal life span is terminated by wilting rather than abscission. It is concluded that the abscission of turgid petals in D. belladonna was preceded by a number of PCD indicators, whereas no such evidence for PCD was found at the time of P. yedoensis petal abscission. Dehydration of the petal cells, after abscission, was associated with a remarkable nuclear morphology which was also found in younger petals subjected to dehydration. This nuclear morphology has apparently not been described previously, for any organism

Proteomic analysis of pollination-induced corolla senescence in petunia

Senescence represents the last phase of petal development during which macromolecules and organelles are degraded and nutrients are recycled to developing tissues. To understand better the post-transcriptional changes regulating petal senescence, a proteomic approach was used to profile protein changes during the senescence of Petunia×hybrida ‘Mitchell Diploid’ corollas. Total soluble proteins were extracted from unpollinated petunia corollas at 0, 24, 48, and 72 h after flower opening and at 24, 48, and 72 h after pollination. Two-dimensional gel electrophoresis (2-DE) was used to identify proteins that were differentially expressed in non-senescing (unpollinated) and senescing (pollinated) corollas, and image analysis was used to determine which proteins were up- or down-regulated by the experimentally determined cut-off of 2.1-fold for P <0.05. One hundred and thirty-three differentially expressed protein spots were selected for sequencing. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine the identity of these proteins. Searching translated EST databases and the NCBI non-redundant protein database, it was possible to assign a putative identification to greater than 90% of these proteins. Many of the senescence up-regulated proteins were putatively involved in defence and stress responses or macromolecule catabolism. Some proteins, not previously characterized during flower senescence, were identified, including an orthologue of the tomato abscisic acid stress ripening protein 4 (ASR4). Gene expression patterns did not always correlate with protein expression, confirming that both proteomic and genomic approaches will be required to obtain a detailed understanding of the regulation of petal senescence