RELATIONSHIP BETWEEN MORPHOGENESIS AND SECRETION IN THE FILAMENTOUS FUNGUS \u3ci\u3eASPERGILLUS NIDULANS\u3c/i\u3e

Filamentous fungi have a long history in biotechnology for the production of food ingredients, pharmaceuticals and enzymes. The advancements made in recent years have earned filamentous fungi such as Aspergillus species a dominant place among microbial cell factories. Although the model fungus A. nidulans has been extensively studied, the genetic and regulatory networks that underlie morphogenesis and development have yet to be fully characterized. The Rho GTPases (Cdc42 and RacA) are one of the most important regulators of the morphogenetic processes among diverse eukaryotic organisms. Although the functions of these GTPases are relatively well-characterized, little is known about their downstream effectors. One likely effector is the formin SepA, which also belongs to a complex known as the polarisome that helps to stabilize and support hyphal growth. The uncharacterized gene ANID_05595.1 (ModB) possesses sequence features that suggest it also belongs to the polarisome. My genetic and functional characterization of ModB reveals some overlap with SepA, but also shows that ModB possesses distinct roles in the maintenance of hyphal polarity. In filamentous fungi, hyphal morphology requires the localized delivery of exocytic vesicles to the hyphal tip as well as to septation sites. The mechanisms that regulate vesicle trafficking to these locations are not yet well understood. In addition, because fungal hyphae presumably extend through a nutritionally variable environment, these mechanisms must be extremely sensitive to growth conditions. To begin to address these issues, I have investigated the effects of nutrient conditions on localization patterns of two different proteins that are trafficked to the cell surface; a glucose transporter (HxtB) and the enzyme β-glucosidase (BglA). Although the final localization of each protein differs, they display similar localization dynamics upon release from glucose repression. In parallel, I also used fluorescence microscopy to determine how shifts from glucose to a non-preferred carbon source (e.g., cellulose) affect hyphal extension and morphology. My results suggest that relief from glucose repression leads to the production of thinner hyphae after a transient delay in hyphal extension. Use of variety of signaling mutants further demonstrates that this response requires a functional protein kinase A (PKA) as well as proper down-regulation of heterotrimeric G protein signals. Collectively, these observations provide valuable new insight into how vesicle trafficking responds to variable growth conditions. Advisor: Steven D. Harri

Young CSF restores oligodendrogenesis and memory in aged mice via Fgf17

Recent understanding of how the systemic environment shapes the brain throughout life has led to numerous intervention strategies to slow brain ageing1-3. Cerebrospinal fluid (CSF) makes up the immediate environment of brain cells, providing them with nourishing compounds4,5. We discovered that infusing young CSF directly into aged brains improves memory function. Unbiased transcriptome analysis of the hippocampus identified oligodendrocytes to be most responsive to this rejuvenated CSF environment. We further showed that young CSF boosts oligodendrocyte progenitor cell (OPC) proliferation and differentiation in the aged hippocampus and in primary OPC cultures. Using SLAMseq to metabolically label nascent mRNA, we identified serum response factor (SRF), a transcription factor that drives actin cytoskeleton rearrangement, as a mediator of OPC proliferation following exposure to young CSF. With age, SRF expression decreases in hippocampal OPCs, and the pathway is induced by acute injection with young CSF. We screened for potential SRF activators in CSF and found that fibroblast growth factor 17 (Fgf17) infusion is sufficient to induce OPC proliferation and long-term memory consolidation in aged mice while Fgf17 blockade impairs cognition in young mice. These findings demonstrate the rejuvenating power of young CSF and identify Fgf17 as a key target to restore oligodendrocyte function in the ageing brain

RELATIONSHIP BETWEEN MORPHOGENESIS AND SECRETION IN THE FILAMENTOUS FUNGUS \u3ci\u3eASPERGILLUS NIDULANS\u3c/i\u3e

Filamentous fungi have a long history in biotechnology for the production of food ingredients, pharmaceuticals and enzymes. The advancements made in recent years have earned filamentous fungi such as Aspergillus species a dominant place among microbial cell factories. Although the model fungus A. nidulans has been extensively studied, the genetic and regulatory networks that underlie morphogenesis and development have yet to be fully characterized. The Rho GTPases (Cdc42 and RacA) are one of the most important regulators of the morphogenetic processes among diverse eukaryotic organisms. Although the functions of these GTPases are relatively well-characterized, little is known about their downstream effectors. One likely effector is the formin SepA, which also belongs to a complex known as the polarisome that helps to stabilize and support hyphal growth. The uncharacterized gene ANID_05595.1 (ModB) possesses sequence features that suggest it also belongs to the polarisome. My genetic and functional characterization of ModB reveals some overlap with SepA, but also shows that ModB possesses distinct roles in the maintenance of hyphal polarity. In filamentous fungi, hyphal morphology requires the localized delivery of exocytic vesicles to the hyphal tip as well as to septation sites. The mechanisms that regulate vesicle trafficking to these locations are not yet well understood. In addition, because fungal hyphae presumably extend through a nutritionally variable environment, these mechanisms must be extremely sensitive to growth conditions. To begin to address these issues, I have investigated the effects of nutrient conditions on localization patterns of two different proteins that are trafficked to the cell surface; a glucose transporter (HxtB) and the enzyme β-glucosidase (BglA). Although the final localization of each protein differs, they display similar localization dynamics upon release from glucose repression. In parallel, I also used fluorescence microscopy to determine how shifts from glucose to a non-preferred carbon source (e.g., cellulose) affect hyphal extension and morphology. My results suggest that relief from glucose repression leads to the production of thinner hyphae after a transient delay in hyphal extension. Use of variety of signaling mutants further demonstrates that this response requires a functional protein kinase A (PKA) as well as proper down-regulation of heterotrimeric G protein signals. Collectively, these observations provide valuable new insight into how vesicle trafficking responds to variable growth conditions. Advisor: Steven D. Harri

Associations of Prescription Opioid Use during Pregnancy with Low Birth Weight and Length of Hospital Stay: Analysis of PRAMS Data

Thesis (Master's)--University of Washington, 2023Background: Prescription opioid use among US pregnant women, primarily used for its analgesic properties, has increased substantially over the last decades. Findings from several studies investigating associations of prescription opioid use with adverse neonatal outcomes (including preterm birth, poor fetal growth, longer hospital stays, neonatal abstinence syndrome, and birth defects) were not consistent. We examined overall and infant-sex specific associations of maternal prescription opioid use in pregnancy with infant low birth weight and prolonged hospital stay.Methods: Data from the 2019-2020 Pregnancy Risk Assessment Monitoring System (PRAMS), a surveillance project of the CDC and state departments of health, was used for the analyses. Participants (N=35,404 mother-newborn pairs) with information on the exposure (maternal prenatal prescription opioid use) and outcomes (low infant birth weight defined as birth weight less than 2,500g, and, prolonged hospital stay defined as hospital stay longer than three days) were included in the analyses. We used crude and adjusted (for maternal age, race, household income, maternal smoking, depression, and infant sex) logistic regression models to estimate odds ratios (ORs) and corresponding 95% confidence intervals (CIs). We also evaluated infant sex as a potential effect modifier of the associations using stratified analyses and models with interaction terms. Results: Among study participants, 1,729 mothers (4.9%) reported prescription opioid use during pregnancy. Overall, 18.2% infants had low birth weight and 4.8% had prolonged hospital stay. In unadjusted models, mothers exposed to prescription opioids during pregnancy had 2-fold (95%CI:1.67-2.44) higher odds of delivering a low birth weight infant compared to mothers who were not exposed to prescription opioids during pregnancy. This association was attenuated but remained statistically significant after adjustment for all covariates (adjusted OR:1.70; 95%CI:1.40-2.07; p-value0.05). In unadjusted models, infants of mothers who reported prescription opioid use during pregnancy had 2.07-fold (95%CI:1.65-2.61) higher odds of prolonged hospital stay compared to infants of mothers who did not report prescription opioid use during pregnancy. This association remained statistically significant after adjustment for covariates (adjusted OR:1.86; 95% CI:1.47-2.36, p-value0.05). Conclusion: In the PRAMS cohort, prescription opioid use during pregnancy was associated with both low birth weight and prolonged hospital stay. We also found that the associations were similar among male and female infants. Our findings highlight the potential consequences of opioid use during pregnancy for further research

Small molecule C381 targets the lysosome to reduce inflammation and ameliorate disease in models of neurodegeneration.

SignificanceNeurodegenerative diseases are poorly understood and difficult to treat. One common hallmark is lysosomal dysfunction leading to the accumulation of aggregates and other undegradable materials, which cause damage to brain resident cells. Lysosomes are acidic organelles responsible for breaking down biomolecules and recycling their constitutive parts. In this work, we find that the antiinflammatory and neuroprotective compound, discovered via a phenotypic screen, imparts its beneficial effects by targeting the lysosome and restoring its function. This is established using a genome-wide CRISPRi target identification screen and then confirmed using a variety of lysosome-targeted studies. The resulting small molecule from this study represents a potential treatment for neurodegenerative diseases as well as a research tool for the study of lysosomes in disease

Exercise plasma boosts memory and dampens brain inflammation via clusterin.

Physical exercise is generally beneficial to all aspects of human and animal health, slowing cognitive ageing and neurodegeneration1. The cognitive benefits of physical exercise are tied to an increased plasticity and reduced inflammation within the hippocampus2-4, yet little is known about the factors and mechanisms that mediate these effects. Here we show that 'runner plasma', collected from voluntarily running mice and infused into sedentary mice, reduces baseline neuroinflammatory gene expression and experimentally induced brain inflammation. Plasma proteomic analysis revealed a concerted increase in complement cascade inhibitors including clusterin (CLU). Intravenously injected CLU binds to brain endothelial cells and reduces neuroinflammatory gene expression in a mouse model of acute brain inflammation and a mouse model of Alzheimer's disease. Patients with cognitive impairment who participated in structured exercise for 6 months had higher plasma levels of CLU. These findings demonstrate the existence of anti-inflammatory exercise factors that are transferrable, target the cerebrovasculature and benefit the brain, and are present in humans who engage in exercise