Human diseases such as spina bifida are caused by a failure in cell morphogenesis and tissue fusion. Dorsal closure in the Drosophila embryo is a model for these tissue closure processes where proper Jun N-terminal Kinase (JNK) signaling is necessary. JNK activity is required in the leading edge cells of the epithelial layer to modulate the cytoskeleton and cell shape, allowing the epidermis to close on the dorsal side of the embryo. The mixed lineage kinase (MLK), Slipper (Slpr), is the JNKKK which is responsible for activation of the pathway during dorsal closure. The pathway components that regulate Slpr, as well as upstream activation signals, are not yet identified. We have examined the involvement of the Ste20-like kinase Misshapen (Msn) to act as the JNKKKK in the JNK pathway during dorsal closure through a direct interaction between Msn and Slpr. By observing phenotypes of recombinant and heterozygous mutants of slpr and msn, we have examined the genetic interactions. Also, by using a non-biased screen, we have investigated unknown regulators of the Slpr-mediated JNK pathway which have an effect on dorsal closure. These techniques have begun to identify regulatory interactions of molecules within the JNK pathway, and have narrowed down regions of chromosome two which may contain new modifiers further regulating JNK signaling, in order to provide a robust and highly regulated tissue closure event.

Research has repeatedly demonstrated that herbivores can, at some times and in some places, control the distribution and abundance of plants. Consequently, explaining variation in herbivore control over plant communities is a central goal in ecology and evolutionary biology. Two major challenges have prevented theoretical progress in this area of research. First, although there are numerous hypotheses that attempt to explain variation in herbivore control over plant communities, theoretical reviews have focused on a single hypothesis. Thus, it has been unclear where these herbivore control hypotheses diverge in their predictions and rationale. Second, herbivore control hypotheses base their explanations on highly correlated vegetation characteristics, namely net primary productivity (NPP), plant vigor, plant apparency, plant tissue nitrogen, plant defenses, plant tolerance, and host plant concentration. Consequently, interpretations of field experiments and meta-analyses have been equivocal. To address the first problem, I simultaneously reviewed herbivore control hypotheses and their predictions and rationale. I demonstrate that these hypotheses can be synthesized into four central hypotheses based on NPP, plant size, resource availability, and host stem density. This provides researchers with few vs. many herbivore control hypotheses. To address the second problem, I simultaneously tested these hypotheses by experimentally manipulating resource availability, total stem density, plant species composition, and herbivore abundance under field conditions. I then monitored the response of herbivore abundance, damage to plants, and the reduction in plant mass due to herbivory. The experiments demonstrated that herbivory caused the strongest reductions in mean stem mass where per stem resource availability was lowest, regardless of where herbivore abundance and damage was greatest. This result supports the plant tolerance based resource availability hypothesis, which assumes that the ability of plants to tolerate herbivory increases as resource availability increases. In addition, herbivore control over both simple plant communities (i.e., monocultures) and complex plant communities (i.e., polycultures) was due to herbivory on the dominant plant species, Solidago canadensis. Together, these results suggest that future herbivore control hypotheses should focus on the effect of per-capita resource availability on the ability of dominant plants to tolerate herbivory.

Chromosomal alterations can arise from numerous events, including errors during cell division or repair of damaged DNA. Of these errors, segregational defects such as anaphase bridges and multipolar spindles play a major role in chromosomal instability, leading to tumorigenesis. Bridges can theoretically be produced by several mechanisms including telomere-telomere fusion, persistence of chromatid cohesion into anaphase or repair of broken DNA ends. DNA damage can induce anaphase bridges following exposure to agents such as hydrogen peroxide or ionizing radiation (IR). Our hypothesis is that while the majority of double strand breaks (DSBs) are repaired, to restore the original chromosome structure, incorrect fusion events also occur leading to bridging and that bridge formation allows cells to bypass the apoptotic pathways that are activated in response to DNA damage. To test this, we set out to determine what pathways the cells use to heal the damage and form bridges. Our data suggest that neither of the two major pathways used by the cell for repair of double strand breaks, homologous recombination (HR) and non-homologous end joining (NHEJ), is required for bridge formation. In fact, the NHEJ pathway seems to play a role in the prevention of bridges. When NHEJ is compromised, the cell appears to use HR to repair the break, resulting in increased anaphase bridge formation. Moreover, intrinsic NHEJ activity of different cell lines appears to be correlated with induction of bridges from DNA damage. Our preliminary data also suggest that cell lines with high levels of bridging are capable of apoptosis, yet further experiments are required to see if blocking bridging can enhance cell death. Multipolar spindles are aberrant mitotic figures when a cell divides into two or more poles, which can lead to uneven segregation of the chromosomes. In our studies, we found that IR treatment can lead to an increase in multipolarity shortly after treatment and changes the distribution of spindle pole components. Initial observations on the splitting of centrosomal proteins following IR treatment are presented.

Tbx6 was shown to be a T-box transcription factor expressed in the primitive streak and presomitic mesoderm of the developing mouse embryo; null mutations in Tbx6 resulted in ectopic neural tube formation in place of posterior somites and embryonic lethality. However nothing was known about expression, transcriptional activity, or downstream targets of Tbx6 protein. Using antibodies generated against Tbx6, Tbx6 was confirmed to be a 58 kDa protein in vivo, and Tbx6 protein and mRNA have similar spatial and temporal expression patterns. T-box proteins were defined by the presence of the T-domain, the DNA binding region. Given the conservation of the T-domain, it was not surprising that many of the characterized T-box binding sites were also somewhat conserved. Therefore, I first demonstrated that Tbx6 recognized the T palindromic consensus sequence and half-site sequence in vitro and then determined that the Tbx6 consensus binding site was 5- AGGTGTBRNNN -3 using a PCR-based binding site selection assay. Using luciferase reporter assays in cell culture, Tbx6 was determined to be a weak transcriptional activator. Lastly, initial studies with T and Tbx6 suggested that these T-box proteins do not synergistically activate a luciferase reporter. In other work, the spontaneous mutation rib-vertebrae was determined to be a Tbx6 hypomorphic mutation due to an insertion of 185 bp into a Tbx6 enhancer. Finally, I also sought to understand how the Notch signaling pathway and Tbx6 interacted. The Notch transcription factor, RBP-J?, was demonstrated to control the maintenance of Tbx6 in the PSM. Furthermore, Tbx6 bound to several putative binding sites in vitro within a previously known mesodermal Dll1 enhancer. These results suggested that Notch signaling functions upstream and downstream of Tbx6. The work described in this thesis used a variety of in vivo and in vitro approaches, molecular and biochemical, to gain further understanding into how Tbx6, a member of a large family of transcription factors, functions in the context of a developing embryo in the presence of other family members to regulate the expression of target genes.

The actin cytoskeleton is essential for a vast array of cellular processes and behaviors including migration, cell division, cell adhesion, intracellular trafficking, and maintenance of cell shape. Regulation of cytoskeletal dynamics is achieved through the actions of a diverse group actin-binding proteins. The actin-binding protein Apxl, is a member of the Shroom protein family, which also includes Apx and KIAA1202. Shroom, the most well-characterized member of this family, binds and bundles actin stress fibers and is required for apical constriction of the neuroepithelium during neural tube closure in mice and Xenopus embryos. Apxl was named for its similarity to Apical Protein Xenopus (Apx), a regulator of an amilioride-sensitive sodium channel. All Shrm family members possess at least two of three conserved domains; a N-terminal PDZ domain, a centrally located ASD1 (APX/Shroom Domain) and a C-terminal ASD2 domain. Because of its similarity to Shroom, mouse Apxl was sequenced and cloned in order to begin initial characterization of the protein. Western blot analysis has shown that mAxpl is expressed in the majority of adult tissues. Immunofluorescence analysis of frozen sections has demonstrated that Apxl is specifically expressed in multiple populations of polarized cells, such as the neuroepithelium, vascular endothelium, and the epithelium of renal tubules. The subcellular localization of Apxl was investigated and Apxl was found to reside at the plasma membrane of non-adherent cells and in the apical compartment of polarized cells, possibly through interactions with cortical actin or members of the apical junctional complex. Analysis of Apxl deletion proteins has revealed that the ASD1 domain is crucial for proper localization,while the requirement for the PDZ domain varies in different cell lines. Cytochalasin D treatment of Rat1 fibroblasts has indicated that disruption of the actin cytoskeleton perturbs Apxl localization. Additionally, Apxl directly binds actin through its ASD1 domain in F-actin cosedimentation experiments. Apxl is expressed in multiple polarized cell types where it binds cortical actin and localizes to the apical junctional complex. Although the biological function of Apxl is unknown, its expression pattern, subcellular localization, and similarity to Shroom suggest that Apxl may play a role in regulation of cellular architecture throughout development.

My dissertation work was focused to characterize three members of the Kinesin superfamily in vitro. Kinesins are required in the cell for the correct localization and directed transport of proteins, DNA, RNA and cellular organelles. The three molecular motors of the kinesin superfamily studied here are Kar3Cik1, Kar3Vik1 and Nod. These three kinesins are unconventional in that they do not motor to the microtubule plus end transporting cargo over long distances, like Kinesin-1. Kar3Cik1 and Kar3Vik1 are Saccharomyces cerevisiae Kinesin-14s, exhibiting minus end directionality characteristic of Kinesin-14s. Kar3Cik1 is essential for meiosis and for karyogamy, or mating in yeast. Kar3Cik1 also has non-essential roles in mitosis. Kar3Vik1 plays an important role at the spindle pole body during yeast mitosis. My work has shown that the two heterodimers interact with the microtubule in very different ways. Cik1 targets Kar3 to microtubule plus ends and enhances the Kar3-instrinsic depolymerizing ability. In contrast Vik1 binds the microtubule in addition to the Kar3 motor domain and depresses the Kar3 depolymerizing ability. Both of these functions correlate with the in vivo phenotypes and suggest different mechanisms of action for the two heterodimers. Nod is a Drosophila melanogaster orphan kinesin proposed to provide a polar ejection force to stabilize chromosomes at the metaphase plate in meiosis. Our work characterized Nod as a kinesin that regulates microtubule dynamics by binding to the microtubule plus end and promoting microtubule polymerization. These results provide a mechanistic explanation for the polar ejection force observed in vivo. My dissertation work has provided knowledge about the many ways in which different kinesins can interface with and regulate microtubule dynamics.

The effects of environmental variation on the phenotypes expressed by organisms have gradually gained interest in biology. This interest has been sparked by the realization that environment-dependent phenotypic expression (i.e. phenotype plasticity) can improve an organisms fitness when different environments are encountered. The challenge for researchers is to determine the importance of phenotypic plasticity to the various fields of biology. A major goal in community ecology is to understand predator-prey interactions within natural communities. Recently, ecologists have focused their attention towards the inducible defenses of prey with the realization that prey are not simply passive participants but instead express a variety of inducible defenses. This dissertation explores the ecology and evolution of inducible defenses using freshwater snails and their predators as a model system. My specific objectives were to identify the adaptive significance of induced responses against predators, to explore the importance of development for understanding inducible defenses, and to address the phenotypic and fitness consequences of spatial and temporal variation in predation risk on prey species. Snails were extremely flexible in their ability to respond to different predator environments. It was evident in each of my experiments that snails were able to alter a unique suite of traits with different predators and integrate their phenotypic responses to environments that contained multiple predator species. Moreover, phenotypic trade-offs resulting from internal resource competition among traits appear to be the underlying mechanism for the expression of inducible defenses. I also found induced defenses can come at the cost of reduced growth, delayed reproduction, or reduced fecundity. However, these costs were dependent on the identity of the predator. By incorporating a developmental perspective, I was able to document that snails have wide developmental windows for formation of defenses but narrow windows for the reversal of defenses. Moreover, the lag time associated with the formation of some defenses can limit the benefits of the defense. Lastly, responses to predators early in development can constrain future responses to different predators and have dramatic impacts on fitness.

Morphological differences in flowers have important evolutionary consequences; they influence the plants relationship with pollinators and are strongly correlated with sexual function in some breeding systems. Here, I explore the functional relationship between flower morphology and pollination dynamics (e.g. pollen receipt / export) in Solanum carolinense (Solanaceae) and evaluate whether this relationship varies with pollinator taxa. I also investigate if flower morphology determines fruit setting ability of flowers under different pollination regimes. Solanum carolinense has been characterized as having an andromonoecious sexual system where individual plants bear both hermaphroditic and male flowers. This species presents an ideal system to study the relationship between floral morphology, functionality and pollinators because flowers in natural populations vary in their style length and grow in diverse array of environments that vary in their pollinator fauna composition. I conducted a series of greenhouse experiments, pollinator observations and natural population surveys to test these relationships. My results demonstrate that long styled flowers serve as pollen recipients and short styled flowers as pollen donors. However, only bumblebees when (Bombus impatiens) are the pollinators I observe a positive relationship between style length and pollen deposition and a negative relationship with pollen removal. These findings support the female/male interference hypothesis and suggest that when plants are visited by species of species of Bombus, the differences in fitness could favor the evolution of andromonoecy. In contrast, when plants are visited either by Augochloropsis metallica or Lassioglossum spp. there is no selection for the dimorphism (or any particular style length). I also found that flower morphology, in particular style length, determines the fruit setting ability of the flowers in S. carolinense under different pollination regimes. However, in some flowers sexual functionality varies and does not accord with traditional classification of the flowers. The variation observed for style length, functionality and production of staminate flowers among individuals in natural populations of S. carolinense could be due to variation in abundance and visitation rate of pollinator taxa. Future studies should not neglect taxa-specific plant-pollinator interactions because the evolution of plant breeding systems can be determined by taxa specific interactions.

RNA Polymerase II (Pol II) transcription is a highly regulated process. Many factors associate with Pol II to ensure that transcription occurs as efficiently as possible. One of these factors is the Paf1 complex, which consists of the subunits Paf1, Ctr9, Rtf1, Cdc73, and Leo1. This complex has been shown to be important for the regulation of chromatin modifications that promote active transcription. Rkr1 was identified in a genetic screen to uncover factors that function in parallel with the Paf1 subunit Rtf1. My work has focused on characterizing a role for Rkr1 in transcription and chromatin function. I have shown that strains lacking RKR1 have transcription-related phenotypes. Genetic analysis has shown that Rkr1 functions in parallel with Rtf1-dependent histone modifications, particularly histone H2B ubiquitylation and histone H3 lysine 4 methylation. Strains lacking RKR1 have telomeric silencing defects, further connecting Rkr1 to chromatin function. Rkr1 is a nuclear protein that contains a RING domain at its extreme carboxy terminus. RING domain proteins often act as ubiquitin-protein ligases, which determine substrate specificity in the ubiquitylation pathway. Subsequent analyses have shown that Rkr1 does possess ubiquitin ligase activity in vitro, and mutational analysis shows that the RING domain of Rkr1 is required for in vivo activity. In an attempt to identify a functional process for Rkr1, a yeast two-hybrid screen was performed using an amino-terminal fragment of Rkr1 as bait. Twenty proteins were identified to interact with this region of Rkr1, many of which are functionally connected to transcription and chromatin. Microarray analysis shows that Rkr1 is required for proper expression of a subset of genes in yeast. Taken together, my work has identified a new ubiquitylation pathway within the nucleus that acts to regulate transcription and chromatin function.

Antitrypsin Deficiency is a primary cause of juvenile liver disease and arises from expression of the Z variant of the alpha-1 protease inhibitor (A1Pi). Whereas A1Pi is secreted from the liver, A1PiZ is retro-translocated from the endoplasmic reticulum (ER) and degraded by the proteasome, an event that may offset liver damage. To better define the mechanism of A1PiZ degradation, a yeast expression system was developed and a gene, ADD66, was identified that facilitates A1PiZ turn-over. I report here that ADD66 encodes an ~30 kDa soluble, cytosolic protein and that the chymotrypsin-like activity of the proteasome is reduced in add66Ä mutants. This reduction in activity may arise from the accumulation of 20S proteasome assembly intermediates or from qualitative differences in assembled proteasomes. Add66p also appears to be a proteasome substrate. Consistent with its role in ER associated degradation (ERAD), synthetic interactions are observed between the genes encoding Add66p and Ire1p, a transducer of the unfolded protein response, and yeast deleted for both ADD66 and/or IRE1 accumulate polyubiquitinated proteins. These data identify Add66p as a proteasome assembly chaperone (PAC) and provide the first link between PAC activity and ERAD.