Identification of a second homolog of N-ethylmaleimide-sensitive fusion protein that is expressed in the nervous system and secretory tissues of Drosophila.

N-Ethylmaleimide-sensitive fusion protein (NSF) is an ATPase known to have an essential role in intracellular membrane transport events. Recently, cDNA clones encoding a Drosophila melanogaster homolog of this protein, named dNSF, were characterized and found to be expressed in the nervous system. We now report the identification of a second homolog of NSF, called dNSF-2 within this species and report evidence that this ubiquitous and widely utilized fusion protein belongs to a multigene family. The predicted amino acid sequence of dNSF-2 is 84.5% identical to dNSF (hereafter named dNSF-1), 59% identical to NSF from Chinese hamster, and 38.5% identical to the yeast homolog SEC18. The highest similarity was found in a region of dNSF-2 containing one of two ATP-binding sites; this region is most similar to members of a superfamily of ATPases. dNSF-2 is localized to a region between bands 87F12 and 88A3 on chromosome 3, and in situ hybridization techniques revealed expression in the nervous system during embryogenesis and in several imaginal discs and secretory structures in the larvae. Developmental modulation of dNSF-2 expression suggests that quantitative changes in the secretory apparatus are important in histogenesis.

Defining the Role of the Histone Methyltransferase, PR-Set7, in Maintaining the Genome Integrity of Drosophila Melanogaster

The complete and faithful duplication of the genome is essential to ensure normal cell division and organismal development. Eukaryotic DNA replication is initiated at multiple sites termed origins of replication that are activated at different time through S phase. The replication timing program is regulated by the S-phase checkpoint, which signals and repairs replicative stress. Eukaryotic DNA is packaged with histones into chromatin, thus DNA-templated processes including replication are modulated by the local chromatin environment such as post-translational modifications (PTMs) of histones.

One such epigenetic mark, methylation of lysine 20 on histone H4 (H4K20), has been linked to chromatin compaction, transcription, DNA repair and DNA replication. H4K20 can be mono-, di- and tri-methylated. Monomethylation of H4K20 (H4K20me1) is mediated by the cell cycle-regulated histone methyltransferase PR-Set7 and subsequent di-/tri- methylation is catalyzed by Suv4-20. Prior studies have shown that PR-Set7 depletion in mammalian cells results in defective S phase progression and the accumulation of DNA damage, which may be partially attributed to defects in origin selection and activation. Meanwhile, overexpression of mammalian PR-Set7 recruits components of pre-Replication Complex (pre-RC) onto chromatin and licenses replication origins for re-replication. However, these studies were limited to only a handful of mammalian origins, and it remains unclear how PR-Set7 impacts the replication program on a genomic scale. Finally, the methylation substrates of PR-Set7 include both histone (H4K20) and non-histone targets, therefore it is necessary to directly test the role of H4K20 methylation in PR-Set7 regulated phenotypes.

I employed genetic, cytological, and genomic approaches to better understand the role of H4K20 methylation in regulating DNA replication and genome stability in Drosophila melanogaster cells. Depletion of Drosophila PR-Set7 by RNAi in cultured Kc167 cells led to an ATR-dependent cell cycle arrest with near 4N DNA content and the accumulation of DNA damage, indicating a defect in completing S phase. The cells were arrested at the second S phase following PR-Set7 downregulation, suggesting that it was an epigenetic effect that coupled to the dilution of histone modification over multiple cell cycles. To directly test the role of H4K20 methylation in regulating genome integrity, I collaborated with the Duronio Lab and observed spontaneous DNA damage on the imaginal wing discs of third instar mutant larvae that had an alanine substitution on H4K20 (H4K20A) thus unable to be methylated, confirming that H4K20 is a bona fide target of PR-Set7 in maintaining genome integrity.

One possible source of DNA damage due to loss of PR-Set7 is reduced origin activity. I used BrdU-seq to profile the genome-wide origin activation pattern. However, I found that deregulation of H4K20 methylation states by manipulating the H4K20 methyltransferases PR-Set7 and Suv4-20 had no impact on origin activation throughout the genome. I then mapped the genomic distribution of DNA damage upon PR-Set7 depletion. Surprisingly, ChIP-seq of the DNA damage marker γ-H2A.v located the DNA damage to late replicating euchromatic regions of the Drosophila genome, and the strength of γ-H2A.v signal was uniformly distributed and spanned the entire late replication domain, implying stochastic replication fork collapse within late replicating regions. Together these data suggest that PR-Set7-mediated monomethylation of H4K20 is critical for maintaining the genomic integrity of late replicating domains, presumably via stabilization of late replicating forks.

In addition to investigating the function of H4K20me, I also used immunofluorescence to characterize the cell cycle regulated chromatin loading of Mcm2-7 complex, the DNA helicase that licenses replication origins, using H4K20me1 level as a proxy for cell cycle stages. In parallel with chromatin spindown data by Powell et al. (Powell et al. 2015), we showed a continuous loading of Mcm2-7 during G1 and a progressive removal from chromatin through S phase.

Knowing The Way: Scriptural Imagination and the Acts of the Apostles

In this dissertation, I offer a pedagogical proposal for learning the Christian Scriptures guided by respect for the nature of the reader and the integrity of the biblical text. Christian educators have profitably developed recent theoretical interest in the body’s role in human meaning with regard to worship and praxis methodologies, but the implications of this research for communal study of the biblical text merit further development. I make the case for adopting scriptural imagination as the goal of pedagogically constructed encounters with the Christian Scriptures. The argument proceeds through a series of questions addressing both sides of the text/reader encounter.

Chapter one considers the question “what is the nature of the reader and, subsequently, the shape of the reader’s ways of knowing?” This investigation into recent literature on the body’s involvement in human knowing includes related epistemological shifts with Christian education. On the basis of this survey, imagination emerges as a compelling designator of an incorporative, constructive creaturely capacity that gives rise to a way of being in the world. Teachers of Scripture who intend to participate in Christian formation should account for the imagination’s centrality for all knowing. After briefly situating this proposal within a theological account of creatureliness, I make the initial case for Scriptural imagination as a pedagogical aim.

Imagination as creaturely capacity addresses the first guiding value, but does this proposal also respect the integrity and nature of the biblical text, and specifically of biblical narratives? In response, in chapter two I take up the Acts of the Apostles as a potential test case and exemplar for the dynamics pertinent to the formation of imagination. Drawing on secondary literature on the genre and literary features of Acts, I conclude that Acts coheres with this project’s explicit interest in imagination as a central component of the process of Christian formation in relationship to the Scriptures.

Chapters three and four each take up a pericope from Acts to assess whether the theoretical perspectives developed in prior chapters generate any interpretive payoff. In each of these chapters, a particular story within Acts functions as a test case for readings of biblical narratives guided by a concern for scriptural imagination. Each of these chapters begins with further theoretical development of some element of imaginal formation. Chapter three provides a theoretical account of practices as they relate to imagination, bringing that theory into conversation with Peter’s engagement in hospitality practices with Cornelius in Acts 10:1-11:18. Chapter four discusses the formative power of narratives, with implications for the analysis of Paul’s shipwreck in Acts 27:1-28:16.

In the final chapter, I offer a two-part constructive pedagogical proposal for reading scriptural narratives in Christian communities. First, I suggest adopting resonance above relevance as the goal of pedagogically constructed encounters with the Scriptures. Second, I offer three ways of reading with the body, including the physical, ecclesial, and social bodies that shape all learning. I conclude by identifying the importance of scriptural imagination for Christian formation and witness in the twenty-first century.

dachshund Potentiates Hedgehog Signaling during Drosophila Retinogenesis

Proper organ patterning depends on a tight coordination between cell proliferation and differentiation. The patterning of Drosophila retina occurs both very fast and with high precision. This process is driven by the dynamic changes in signaling activity of the conserved Hedgehog (Hh) pathway, which coordinates cell fate determination, cell cycle and tissue morphogenesis. Here we show that during Drosophila retinogenesis, the retinal determination gene dachshund (dac) is not only a target of the Hh signaling pathway, but is also a modulator of its activity. Using developmental genetics techniques, we demonstrate that dac enhances Hh signaling by promoting the accumulation of the Gli transcription factor Cubitus interruptus (Ci) parallel to or downstream of fused. In the absence of dac, all Hh-mediated events associated to the morphogenetic furrow are delayed. One of the consequences is that, posterior to the furrow, dac- cells cannot activate a Roadkill-Cullin3 negative feedback loop that attenuates Hh signaling and which is necessary for retinal cells to continue normal differentiation. Therefore, dac is part of an essential positive feedback loop in the Hh pathway, guaranteeing the speed and the accuracy of Drosophila retinogenesis.

Dissecting signaling pathways that control glia migration in the developing Drosophila retina

The function of a complex nervous system relies on an intricate interaction between neurons and glial cells. However, as glial cells are generally born distant from the place where they settle, molecular cues are important to direct their migration. Glial cell migration is important in both normal development and disease, thus current research in the laboratory has been focused on dissecting regulatory events underlying that crucial process. With this purpose, the Drosophila eye imaginal disc has been used as a model. In response to neuronal photoreceptor differentiation, glial cells migrate from the CNS into the eye disc where they act to correctly wrap axons. To ensure proper development, attractive and repulsive signals must coordinate glial cell migration. Importantly, one of these signals is Bnl, a Fibroblast Growth Factor (FGF) ligand expressed by retinal progenitor cells that was suggested to act as a non-autonomous negative regulator of excessive glial cell migration (overmigration) by binding and activating the Btl receptor expressed by glial cells. Through the experimental results described in chapter 3 we gained a detailed insight into the function of bnl in eye disc growth, photoreceptor development, and glia migration. Interestingly, we did not find a direct correlation between the defects on the ongoing photoreceptors and the glia overmigration phenotype; however, bnl knockdown caused apoptosis of eye progenitor cells what was strongly correlated with glia migration defects. Glia overmigration due to Bnl down-regulation in eye progenitor cells was rescued by inhibiting the pro-apoptotic genes or caspases activity, as well as, by depleting JNK or Dp53 function in retinal progenitor cells. Thus, we suggest a cross-talk between those developmental signals in the control of glia migration at a distance. Importantly, these results suggest that Bnl does not control glial migration in the eye disc exclusively through its ability to bind and activate its receptor Btl in glial cells. We also discuss possible biological roles for the glia overmigration in the bnl knockdown background. Previous results in the lab showed an interaction between dMyc, a master regulator of tissue growth, and Dpp, a Transforming Growth Factor-β important for retinal patterning and for accurate glia migration into the eye disc. Thus, we became interested in understanding putative relationships between Bnl and dMyc. In chapter 4, we show that they positively cooperate in order to ensure proper development of the eye disc. This work highlights the importance of the FGF signaling in eye disc development and reveals a signaling network where a range of extra- and intra-cellular signals cooperate to non-autonomously control glial cell migration. Therefore, such inter-relations could be important in other Drosophila cellular contexts, as well as in vertebrate tissue development.

Bioecology of Spodoptera frugiperda (Smith, 1757) in different cover crops.

The objective of this work was to evaluate the biological variables of Spodoptera frugiperda on species of cover crops. The experiments were conducted in laboratory and greenhouse using the following species: sunflower (Helianthus annuus), sun hemp (Crotalaria juncea), brachiaria (Urochloa decumbens e Urochloa ruziziensis), millet (Pennisetum americanum), black oat (Avena stringosa), white lupin (Lupinus albus), forage turnip (Rafanus sativus) and maize (Zea mays). In laboratory the S. frugiperda larval survival varied from 57%, on L. albus, to 93% on H. annuus and the survival of the pre-imaginal phase varied from 45% on U. decumbens to 81.6% on Z. mays. On C. juncea the larval biomass was lower and the development period of the young and larval stage was higher. The adaptation index was less on C. juncea in greenhouse and laboratory. In greenhouse the larval survival at 14 days was similar for all plants and at 21 days was the lowest on C. juncea. There was less accumulation of biomass at 14 days on C. juncea and at 21 days on C. juncea and A. stringosa. Regarding damage, C. juncea presented less susceptibility to Spodoptera frugiperda attack, which together with the other evaluated parameters, indicated this plant as the most appropriate for soil cover before cultivation of maize.

Interventions for comorbid problem gambling and psychiatric disorders : advancing a developing field of research

Despite significant psychiatric comorbidity in problem gambling, there is little evidence on which to base treatment recommendations for subpopulations of problem gamblers with comorbid psychiatric disorders. This mini-review draws on two separate systematic searches to identify possible interventions for comorbid problem gambling and psychiatric disorders, highlight the gaps in the currently available evidence base, and stimulate further research in this area. In this mini-review, only 21 studies that have conducted post-hoc analyses to explore the influence of psychiatric disorders or problem gambling subtypes on gambling outcomes from different types of treatment were identified. The findings of these studies suggest that most gambling treatments are not contraindicated by psychiatric disorders. Moreover, only 6 randomized studies comparing the efficacy of interventions targeted towards specific comorbidity subgroups with a control/comparison group were identified. The results of these studies provide preliminary evidence for modified dialectical behavior therapy for comorbid substance use, the addition of naltrexone to cognitive-behavioral therapy (CBT) for comorbid alcohol use problems, and the addition of N-acetylcysteine to tobacco support programs and imaginal desensitisation/motivational interviewing for comorbid nicotine dependence. They also suggest that lithium for comorbid bipolar disorder, escitalopram for comorbid anxiety disorders, and the addition of CBT to standard drug treatment for comorbid schizophrenia may be effective. Future research evaluating interventions sequenced according to disorder severity or the functional relationship between the gambling behavior and comorbid symptomatology, identifying psychiatric disorders as moderators of the efficacy of problem gambling interventions, and evaluating interventions matched to client comorbidity could advance this immature field of study.