During eukaryotic gene expression, non-coding intron sequences are removed from the protein-coding exon sequences of pre-messenger RNA (pre-mRNA) by the process of splicing. Splicing is carried out by the spliceosome, a macromolecular ribozyme that assembles de novo on every intron. It has been established that the spliceosome assembles co-transcriptionally while the nascent pre-mRNA is attached to the elongating RNA polymerase II (RNAP II). However, mechanisms that enforce the coupling of splicing to transcription and the chromatin landscape remain poorly understood.

Here, I describe our work to gain further insight into the regulation of co-transcriptional pre-mRNA splicing by employing genetic and molecular biology approaches. Using the traditional splicing model organism Saccharomyces cerevisiae, we interrogated the mechanism of a rapid splicing block at the ribosomal protein genes (RPGs) that occurs in response to amino acid starvation stress. By creating chimeric gene constructs, we show that an RPG promoter sequence element, and not the intron sequence, is both necessary and sufficient to confer a transcript-specific splicing response. This study reveals a mechanism whereby in response in stress RNAP II becomes de-enriched near the 3´splice site and early splicing factor recruitment is delayed, consistent with an environmentally-responsive splicing-dependent polymerase checkpoint, and underscoring the highly coordinated nature of splicing and transcription.

We further expanded our studies of splicing regulation into Schizosaccharomyces pombe, a emerging model organism that shares many splicing features with mammalian systems, such as abundant non-consensus splice sites, multi-intron containing genes, and conserved trans-acting splicing factors. In our work with the S. pombe system, we developed techniques for probing splicing factor association with chromatin. Starting from a genome-wide genetic screen, we characterized interactions between the spliceosome and Swr1 complex, which deposits the highly conserved histone variant H2A.Z. We identified H2A.Z’s requirement in efficient splicing at introns with weak splice sites, supporting a growing hypothesis of chromatin-dependent splicing checkpoints. Taken together, these studies reveal an unappreciated role of the promoter and its proximal nucleosome composition in transcript-specific pre-mRNA splicing regulation. We expect these results to provide foundational insights into the interconnectedness of gene expression processes and have important implications in human disease.

Background: No medications are approved by the FDA for the treatment of methamphetamine use disorders; yet, preclinical and clinical evidence advances naltrexone as a promising candidate. This dissertation investigated the effects of naltrexone on functional magnetic resonance imaging (fMRI) measures of functional connectivity during methamphetamine cue processing and subjective craving for methamphetamine.

Methods: A final sample of 23 non-treatment seeking individuals with methamphetamine use disorders (74% male, mean age=34.70 [SD=8.95]) were enrolled within a randomized, placebo controlled, within-subject design and underwent two blood-oxygen-level dependent (BOLD) methamphetamine cue-reactivity paradigms following three days of naltrexone treatment (50mg) and matched time for placebo. fMRI analyses tested naltrexone-induced differences in BOLD activation associated with methamphetamine and control cues, resting-state cerebral blood flow (CBF), and functional connectivity (using psychophysiological interaction analysis) during methamphetamine cue processing.

Results: Analyses revealed that (1) the novel methamphetamine cues task employed was successful in eliciting cue-induced craving and relevant regional activation, (2) greater subjective methamphetamine craving is related to enhanced recruitment of prefrontal regions, (3) naltrexone moderates methamphetamine cue-reactivity in sensorimotor regions, (4) the BOLD results are not a reflection of CBF changes induced by naltrexone, (5) reduced activation of sensorimotor regions during methamphetamine cue processing by naltrexone is related to functional connectivity of dorsal striatum, ventral tegmental area (VTA), periaqueductal gray, and precuneus with visual, sensory, and motor-related regions, (6) naltrexone enhances dorsal striatum, VTA, and sensorimotor region functional connectivity with the frontal cortex during methamphetamine cues processing , and (7) naltrexone weakens the associations between subjective craving and precuneus functional connectivity with sensorimotor regions and strengthens the associations between subjective craving and dorsal striatum and precuneus connectivity with frontal regions during methamphetamine cue processing, as compared to placebo.

Conclusions: The results of this study provide the first evidence of naltrexone-induced changes in BOLD measures of methamphetamine cue-induced craving. The results suggest that naltrexone may be functioning to reduce the salience of the methamphetamine cues by reducing sensorimotor processing and integration, and by engaging greater frontal regulation of salience attribution via pathways linking the dorsal striatum, midbrain, and precuneus to frontal, dorsal striatal, and sensorimotor regions during cue processing.

Coral reefs are among the most biodiverse and valuable ecosystems on Earth, providing food, coastal protection, and jobs for over a billion people worldwide. Coral reefs are also among the most imperiled by global climate change and local stressors that cause degradation. Disturbances that kill corals are becoming more frequent and intense and it has become increasingly important to understand how disturbances impact the recovery potential of coral reefs and which processes are most important for driving recovery. In my dissertation work I utilized time series data from the Moorea Coral Reef LTER along with observational approaches and field experiments to investigate the impacts of disturbances on coral communities, identify processes that drive recovery after disturbance, and understand the mechanisms shape early demographic processes in corals. I first investigated the impacts of a severe marine heatwave on coral communities in Moorea, French Polynesia, and found that coral bleaching caused disproportionate mortality of the largest, most fecund corals and near complete loss of newly-settled coral recruits. This work suggests that climate change may cryptically erode the recovery capacity of coral reefs by disproportionately impacting the coral life stages most important for recovery. Reefs in Moorea have a history of recovering from disturbance, and prior to the bleaching event reefs were recovering from an outbreak of the coral-eating sea star, Acanthaster planci, and a cyclone that killed nearly all corals on the outer reefs. I investigated the drivers of differences in recovery rates between deep and shallow reefs and found that slower recovery on deeper reefs was driven by lower rates of coral recruitment and post-settlement bottlenecks that limited the establishment of new corals. My work points to the importance of post-settlement processes that dictate rates of coral recruitment and ultimately reef recovery. Next, I investigated how benthic communities shape the settlement choices of coral larvae, and found that larvae made complex settlement choices that were strongly influenced by benthic community composition at the scale of millimeters. This work improves our ability to understand and predict how coral communities will respond to disturbances in the Anthropocene.

Vase-shaped microfossils (VSMs) are a group of early eukaryotic microfossils found globally in middle Neoproterozoic (800–730 Ma) marine strata and are interpreted as the earliest evidence for testate (shell-forming) amoebozoans. VSM tests are hypothesized to have been originally organic in life, and VSMs are occasionally reported preserved with carbonaceous tests. However, many of these reports are not confirmed. Bloeser (1985) reported carbonaceous VSMs from shales of the Walcott Member, Chuar Group, Grand Canyon, Arizona, USA, but Porter and Knoll (2000) suggested these specimens were organic-coated siliceous casts after analyzing stratigraphically equivalent VSM material. The goal of this study was to re-examine the microfossils reported by Bloeser (1985) and determine if these VSMs were actually preserved with organic tests. We identified VSMs from two samples of black shales from the Walcott Member in transmitted light microscopy and used scanning electron microscopy (SEM) to image VSMs. Energy dispersive x-ray spectroscopy was used to gain a qualitative understanding of VSM composition and wavelength dispersive x-ray spectroscopy was used to assess elemental spatial variation at a finer resolution. Raman spectroscopy was used to identify mineral components and to compare the thermal maturity of carbonaceous material within the samples. Of the VSM tests sampled, 29 possess carbonaceous tests and 37 have tests that are either partially or completely replaced by pyrite and an iron-phosphate mineral, possibly vivianite. Within some tests, the pyrite has been irregularly oxidized to jarosite. Carbonaceous material is found within the tests of all VSMs from both samples, which we suspect may be bitumen generated during the Phanerozoic (Cretaceous or later) within the Walcott Shales. Based on our observations and chemical analysis, we conclude that the VSMs are preserved with original organic test material, and speculate that sulfurization may have promoted organic preservation, consistent with observations of organic VSMs associated with sulfur, high total organic carbon content, and low concentrations of iron.

As decision- and policy-makers come to rely increasingly on estimates and simulations produced b y computerized models of the world, in areas as diverse and climate prediction, transportation planning, economic policy and civil engineering, the need for objective evaluation of the accuracy and utility of such models likewise becomes more urgent. This article summarizes a two-day workshop that took place in Santa Fe, New Mexico in December 1999, whose focus was the evaluation of complex computer models. Approximately half of the workshop was taken up with formal presentation of four computer models by their creators, each paired with an initial assessment by a statistician. These prepared papers are presented, in shortened form, in Section 3 of this paper. The remainder of the workshop was devoted to introductory and summary contents, short contributed descriptions of related models, and a great deal of floor discussion, which was recorded by assigned rapporteurs. These are presented in Sections 2 and 4 in the paper. In the introductory and concluding sections we attempt to summarize the progress made by the workshop and suggest next steps.

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