A previous study employed affinity purification and tandem mass spectroscopy to identify 142 SUMO-conjugation targets in the early Drosophila embryo. Multiple components of the Ras/MAPK signaling pathway are among these targets, and in accord with this discovery, SUMO was found to be required for activation of the Ras/MAPK pathway in Drosophila S2 cells. However, the question of what is SUMO’s role in the Ras/MAPK still remains. In this dissertation, I present experiments showing that SUMO is also required for Ras/MAPK signaling in the developing embryo. In addition, I explore the mechanism by which SUMO regulates Ras/MAPK signaling.

To determine if SUMO is required for Ras/MAPK signaling in developing embryos as it is in S2 cells, I investigated the role of SUMO in signaling through the Torso receptor tyrosine kinase, which signals through the Ras/MAPK pathway to specify the embryonic termini. I generated females containing SUMO mutant germline clones and performed immunoblotting and immunofluorescence on the resulting embryos to assess MAPK phosphorylation levels. In addition, I performed reverse transcriptase-qPCR to assess the expression of tailless, a target of the Torso pathway. The results show that reduction of the maternal SUMO contribution decreases both MAPK activation and tailless expression, consistent with a positive role for SUMO in Ras/MAPK signaling during embryogenesis.

Localization of Ras to the plasma membrane, which is required for Ras/MAPK signaling, depends on C-terminal prenylation as well as lysine residues in the Ras C-terminal hypervariable region. A published bacterial SUMOylation assay demonstrated that SUMO can be conjugated to the hypervariable region lysine residues suggesting a role for SUMO in membrane localization of Ras. However, my S2 cell SUMO knockdown studies revealed roles for SUMO in neither the lipid modification of Ras nor Ras trafficking. I saw no evidence for Ras SUMOylation in Drosophila cells, although I did detect a non-covalent complex that includes Ras and one or more SUMOylated proteins.

Included among the 142 SUMO-conjugated proteins detected in early embryos were protein phosphatase 2A (PP2A) and 14-3-3 family proteins, both of which modulate Ras/MAPK signaling through their interactions with Raf. These findings suggested that SUMO might modulate Ras/MAPK signaling via Raf, and I therefore determined how SUMO affects Raf phosphorylation and subcellular localization. An epitope-tagged form of Raf was expressed in wild-type and SUMO knockdown S2 cells followed by immunopurification and tandem mass spectroscopy to characterize the Raf phosphorylation state. Activation of the Ras/MAPK pathway promotes Raf dephosphorylation at S346, which is the counterpart of a residue in human C-Raf that needs to be dephosphorylated for efficient Ras/MAPK signaling. However, dephosphorylation of S346 did not occur upon activation of the Ras/MAPK pathway in SUMO knockdown cells. Furthermore, subcellular fractionation revealed that SUMO knockdown prevents Raf from localizing to the plasma membrane in insulin activated cells. By expressing a form of Raf that is constitutively targeted to the plasma membrane and that lacks S346, we were able to restore Ras/MAPK signaling in SUMO knockdown cells. These findings suggest that a primary role for SUMO in the Ras/MAPK pathway is to promote Raf dephosphorylation and targeting of Raf to the plasma membrane.

My studies have revealed a novel mechanism by which SUMO regulates Raf activity and therefore the Ras/MAPK pathway. Since mutations in the Ras/MAPK pathway are found in 30% of all cancers in humans, my findings may eventually lead to the development of novel therapies for cancer treatment that target the function of SUMO in Ras/MAPK signaling.

“[W]omen are…believers in muscular manhood. These lank, scrawny limbs…he was more like a woman than a man.”–1870

“What other men visit salons to get, the Asian gene pool provides for free…ladyboy fingers: soft and long.”–2004

Despite a difference of over a century in publication dates, these excerpts from popular periodicals evince remarkably similar imaginings of the Asian male body. This correspondence is indicative of the ways in which contemporary non-normative constructions of Asian American masculinity suggest a historical linearity rooted in early American conceptions of Asian immigrant men.

As the first group of Asian immigrants to warrant social visibility, an exploration of the social figuration of early Chinese immigrant laborers can serve as a basis for understanding this popularization of emasculation. Specifically, to the extent that this group also formed the foundational basis for a seemingly extant legacy of deviance today, an examination of how these pioneer Chinese men were descriptively bounded within popular spaces can expose both the parallels and points of departure from which we can more fully understand contemporary Asian American masculinity.

This paper analyzes the textual imagery of Chinese immigrant laborers as captured in popular periodicals and print media between 1850 and 1924. In exploring perceptions of Chinese masculinity during this period, the projection of deviance is examined through three lenses of Chinese male non-normative constitution: the diasporic attribution of otherness as non-White aliens, queer domesticity as defined by family and occupation, and most visibly within the perceived and material deficit of agency.

Background: Coronavirus disease 2019 (COVID-19) is a viral respiratory disease caused by SARS-CoV-2. Critical cases evolve into an acute respiratory distress syndrome (ARDS) with bilateral infiltrates, intrapulmonary shunting, and hypoxemia requiring intubation. A rescue treatment called prone positioning has been frequently utilized during the pandemic and known to improve dorsal lung ventilation, decreasing shunting, and improving mortality rates in non-COVID-19 ARDS literature. The clinical inquiry is whether a resource intensive procedure like pronation can benefit oxygenation, ICU length of stay, and days intubated in COVID-19 ARDS. Objective: To evaluate the effects of pronation and compare COVID-19 ARDS with a historical control group of non-COVID-19 ARDS. The specific aim is to assess whether there is a positive effect on oxygenation, ICU LOS, and days intubated. Methods: A retrospective descriptive chart review of COVID-19 ARDS versus non-COVID-19 ARDS adult patients ages 18 to 80 years at a quaternary academic center in Los Angeles, California. A convenient sample of intubated COVID-19 patients with moderate to severe ARDS based on the Berlin criteria. A historical control was age and gender matched of non-COVID-19 ARDS patients. Statistical analysis utilized Mann Whitney U, and the Wilcoxon Signed Rank Test. Results: A total of 41 patients met criteria in the COVID-19 ARDS group, and six patients in the non-COVID-19 ARDS group for a total sample size of 47 subjects. Pronation showed a positive impact on oxygenation (P/F ratios) at the end of pronation on day one (p < 0.01), day three (p < .042), and day four (p < 0.04) in the COVID-19 ARDS group compared to the non-COVID-19 ARDS group. The Wilcoxon Signed Rank Test found a positive impact on P/F ratios by pronation from day one through day six in the COVID-19 ARDS group while the non-COVID-19 group showed a positive impact on day two. Conclusion: Pronation impacted P/F ratios in COVID-19 ARDS compared to non-COVID-19 ARDS. ICU LOS and intubation days were not impacted by pronation. Oxygenation improvement was possibly related to assertive prone protocols instituted early during the exudative phase of ARDS. These results suggest there is an oxygenation benefit to pronating early.

Small ubiquitin-related modifier (SUMO) becomes covalently conjugated to many proteins regulating a variety of cellular and development processes. A previous study demonstrated that multiple components of the Ras/MAPK signaling pathway are directly conjugated to SUMO in the early Drosophila embryo, and that the Ras/MAPK signaling cascade requires SUMO for activation in insulin or Spitz-stimulated Drosophila S2 cells. SUMO knockdown leads to reduced MAPK phosphorylation in response to either signal. Here, we elucidate a mechanism by which SUMO positively regulates the Ras/MAPK signaling pathway by showing that the dephosphorylation of Raf at serine 346 to activate Ras/MAPK signaling requires SUMO. Furthermore, site-directed mutagenesis of serine 346 to alanine, preventing its phosphorylation, bypasses the requirement for SUMO in the activation of the Ras/MAPK pathway. Earlier studies have shown that serine 346 dephosphorylation is required for membrane localization of Raf, which is a pre-requisite for Raf activation. We show that fusing the Raf catalytic domain to the transmembrane and extracellular domains of Torso artificially tethering Raf to the plasma membrane restores Ras signaling in SUMO knockdown cells leading to SUMO independent Ras/MAPK signaling. We conclude that SUMO stimulates Ras/MAPK signaling by promoting the removal of an inhibitory phosphate group, which is a pre-requisite for Raf membrane localization and activation. While there is no evidence that Raf itself is sumoylated, several Raf interacting proteins, such as 14-3-3ζ and PP2A are members of the SUMO-ome suggesting an indirect role for SUMO in Raf localization.

Animal developmental patterning is a complex and intricate process, requiring the interpretation of multiple temporally and spatially regulated signals to define the transcriptional profile of each cell. In Drosophila, Groucho (Gro), a transcriptional corepressor, participates in these processes through long-range silencing of distant enhancers. Lacking any innate DNA-binding activity, Gro is targeted to these elements through interaction with multiple repressors.

Using a combination of ChIP-seq and RNA-seq techniques, I sought to characterize Groucho activity at multiple stages spanning the initial nine hours of embryonic development, and thereby gain insight into both the mechanisms and extent of Groucho-mediated repression. These data reveal that Groucho is recruited to thousands of sites at each stage of development. Most Gro peaks are < 1kb in width, consistent with recruitment of one or a small number of Gro complexes to a regulatory element, indicating that spreading of Gro along stretches of chromatin is not a common feature of repression, as previously thought. Gro binding is frequently observed as clusters of discrete peaks, which supports a model in which Gro self-association facilitates crosslinking of non-contiguous regions of chromatin. In some cases, Gro occupancy is observed at transcriptional start sites multiple kilobases from known Gro-binding silencing elements, suggesting that interactions with these sites via crosslinking and looping of chromatin is one method of Gro-mediated repression.

Both Gro gain- and loss-of-function embryos exhibit extensive perturbations in gene expression from the onset of zygotic transcription. Integration of these differentially expressed genes with ChIP-seq-derived Gro occupancy data facilitated the identification of Gro targets within each developmental stage, including known Gro-regulated genes and novel targets. Gro target genes are enriched for transcription factors involved in multiple developmental processes, as well as regulators of multiple signaling pathways. The activity of Gro in regulating both the inputs and outputs of signaling pathways suggests that Gro is utilized to regulate the cellular response to signaling on multiple levels by facilitating crosstalk between pathways.

Gro binds to many genes that it does not repress as exemplified by genes that are regulated by Dorsal, a bifunctional transcription factor that activates some targets and represses others. Gro is essential for repression, but dispensable for activation by Dorsal. However, data presented in this thesis suggest that Dorsal recruits Gro to both the repressed and the activated targets. This shows that Gro recruitment is not sufficient for repression.

Utilizing a technique to isolate and sequence the nascent transcripts of actively transcribed genes provides an accurate profile of the transcriptional activity of the Drosophila embryo at multiple stages of development. Nascent transcripts of Gro target genes are significantly enriched for promoter-proximal read density, indicating that Gro targets are enriched for stalled PolII. Coupled with the observation that Gro often binds overlapping or shortly downstream of start sites, we propose that Gro may repress genes by favoring the stalling of PolII, potentially through direct or mediated interaction with PolII or alteration of chromatin structure within transcribed regions.

Finally, I have contributed to a study characterizing the Gro interactome. This study showed that Gro interacts with U1 snRNP. I compared the effect of knocking down a U1 snRNP subunit with that of knocking down Gro on the S2 cell transcription profile. The results suggest that Gro-mediated repression of some targets may require U1 snRNP.