While actin was discovered in the nucleus over 50 years ago, research lagged for decades due to strong skepticism. The revitalization of research into nuclear actin occurred after it was found that cellular stresses both induce the nuclear localization and alter the structure of nuclear actin. These studies provided the first hints that actin has a nuclear function. Subsequently, it was established that the nuclear import and export of actin is highly regulated. While the structures of nuclear actin remain unclear, it can function as monomers, polymers, and even rods. Furthermore, even within a given structure, distinct pools of nuclear actin that can be differentially labeled have been identified. Numerous mechanistic studies have uncovered an array of functions for nuclear actin. It regulates the activity of RNA polymerases, as well as specific transcription factors. Actin also modulates the activity of several chromatin remodeling complexes and histone deacetylases, to ultimately impinge on transcriptional programing and DNA damage repair. Further, nuclear actin mediates chromatin movement and organization. It has roles in meiosis and mitosis, and these functions may be functionally conserved from ancient bacterial actin homologs. The structure and integrity of the nuclear envelope and sub-nuclear compartments are also regulated by nuclear actin. Furthermore, nuclear actin contributes to human diseases like cancer, neurodegeneration, and myopathies. The work presented in this thesis aims to describe the nuclear localization and functions of actin during Drosophila oogenesis. Drosophila oogenesis, i.e. follicle development, provides a developmental system with which to study nuclear actin. Follicles are composed of roughly 1000 somatic follicle cells and 16 germline cells, including 15 nurse or support cells and a single oocyte. Follicles progress through a series of 14 morphological stages, from the germanium to Stage 14 (S14). Ovary staining using the anti-actin C4 antibody reveals one pool of nuclear actin during early oogenesis (germarium through S9), including in the germline and somatic stem cells, a subset of mitotic follicles cells, and a subset of nurse cells during S5-S9. Cofilin and Profilin, which regulate the nuclear import and export of actin, also localize to the nuclei. Expression of GFP-tagged actin results in nuclear actin rod formation. These findings indicate that nuclear actin is tightly regulated during oogenesis. One factor mediating this regulation is Fascin. Overexpression of Fascin enhances nuclear GFP-Actin rod formation, and Fascin colocalizes with the rods. Loss of Fascin reduces, whereas overexpression of Fascin increases, the frequency of nurse cells with high levels of C4 nuclear actin, but does not alter the overall nuclear level of actin within the ovary. These data suggest that Fascin regulates the ability of specific cells to accumulate C4 nuclear actin. Evidence indicates that Fascin positively regulates C4 nuclear actin through Cofilin. Indeed, loss of Fascin results in decreased nuclear Cofilin. In addition, Fascin and Cofilin genetically interact, as double heterozygotes exhibit a reduction in the number of nurse cells with high C4 nuclear actin levels. Thus, through Cofilin, Fascin positively regulates C4 nuclear actin. These studies identified Fascin as a novel means of nuclear actin regulation. Having established Drosophila oogenesis as an in vivo, developmental system to study nuclear actin, I sought to identify the functions of nuclear actin. To uncover the functions of nuclear actin, I manipulate nuclear actin levels by blocking its nuclear import (Importin 9) and export (Exportin 6). Knockdown of Importin 9, results in female sterility and defects within the germarium, supporting a role for nuclear actin in stemness. Additionally, reduced Importin 9 levels cause chromatin organization defects. Loss or knockdown of Exportin 6 causes reduced female fertility, abnormal nucleolar morphology, alterations in the nuclear envelope, and aberrant heterochromatin status. These data suggest several functions for nuclear actin in the ovary: nuclear actin is essential for stem cell differentiation, proper chromatin organization and dispersal, nucleolar structure and likely function, nuclear envelope morphology, heterochromatin status and likely gene expression. Ultimately, nuclear actin is absolutely required for the highly conserved process of follicle development. These studies provide insight into the regulation and function of nuclear actin in Drosophila oogenesis. The data presented here indicate that nuclear actin is critical for chromatin organization, nucleolar morphology, nuclear envelope shape, and heterochromatin status and suggest that nuclear actin ultimately impacts transcription, a process essential for all cells. Considering the high level of sequence and functional conservation of actin, studies in Drosophila oogenesis will provide insight into the conserved functions of nuclear actin in follicle development across higher organisms. The study of nuclear actin in the many cell types of the Drosophila ovary provide insight into the functions of nuclear actin for all cell types across evolution. Further, aberrant nuclear actin regulation has been implicated in several disease states. The studies in Drosophila provide insight into the regulation of nuclear actin and how misregulation contributes to disease states. Together, the data presented in this thesis advance our understanding of the nuclear localization and functions of actin.

Two phylogenies of Phalaenopsis (Orchidaceae) are presented, one from combined chloroplast DNA data and one from a nuclear actin gene. We used these phylogenies to assess and modify the classification of Phalaenopsis and to examine several morphological characters and geographical distribution patterns. Our results support Christenson’s (2001) treatment of Phalaenopsis as a broadly defined genus that includes the species previously placed in the genera Doritis and Kingidium. Some of Christenson’s subgeneric groups needed to be recircumscribed to reflect a natural classification. We recognized four subgenera and six sections, subgenera Aphyllae, Parishianae (with sections Conspicuum, Delisiosae, Esmeralda, and Parishianae), Phalaenopsis, and Polychilos (with sections Fuscatae and Polychilos). In order to find a set of universally amplifiable, phylogenetically informative, single-copy nuclear regions, we conducted a whole genome comparison of the rice (Oryza sativa) and Arabidopsis thaliana genomes. We constructed a database of both genomes and searched for pairs of sequences using criteria we felt would ensure primers that would reliably amplify using standard PCR protocols. We tested the most promising 142 primer pairs in the lab on eighteen taxa and found four potentially informative markers in Phalaenopsis and one in Helianthus. Our results indicated that it will be difficult to find universal nuclear markers, however our database provides an important tool for finding informative nuclear markers within specific groups. The full set of primer combinations is available online at, “The Conserved Primer Pair Project,” http://aug.csres.utexas.edu:8080/cpp/index.html. We used fourteen Phalaenopsis species and seven horticultural hybrids to create a real dataset with which to test phylogenetic network reconstruction methods. We tested the performance of Neighbor-Net, implemented in SplitsTree, under four different categories of complexity: one hybrid, two independent hybrids (hybrids with no parents in common), three independent hybrids, and two non-independent hybrids (one parent was shared between hybrids). Neighbor-Net was able to predict accurately the parents of hybrids in only about half of the datasets we tested, and there were so many false positives that it was impossible to distinguish the hybrids from the species. We plan to use this dataset to test methods, such as RIATA and RGNet, when they become available. / text