ABSTRACT OF THE THESIS

Identifying the function of cGAS-Cap2b protein complex in Type-II Short CBASS Systems

by

David Garcia

Master of Science in BiochemistryUniversity of California San Diego, 2022 Professor Elizabeth A. Komives, Chair

Two sides, in an infinite battle for survival, as one side gets stronger, so does the other. As described by the Red Queen hypothesis, this is the dilemma between bacteria and bacteriophages (or phages), where both sides are constantly evolving to try and eliminate their opponent. Phages are simple organisms and outnumber all other organisms on earth, and their means of survival requires the destruction of bacteria. During infection, phages inject their DNA into bacterium and as a response bacterium will destroy itself but not before the phage has successfully replicated. This allows for further bacterial infection upon the destruction of bacterium from initial phage infection and can be recurrent until all bacteria has been destroyed. As a means for survival, bacteria have evolved a myriad of defense systems that allow it to fight phage infection. In this thesis I will cover some of the most well-known defense systems: the restriction-modification (R-M) systems that target phage sequences, the CRISPR-Cas systems where cells obtain immunity from previous phage infection, and the abortive infection systems (Abi) that upon phage detection will program cell death. Although each of these systems is unique and important for phage defense, I will focus on a type of abortive infection systems that were recently discovered in bacteria known as cyclic-oligonucleotide based antiphage signalling systems or ‘CBASS’. In this thesis, I also discuss the defense mechanisms of the different types of CBASS currently identified and discuss my study of a unique type of CBASS systems described as Type II (short) CBASS systems. Previously identified Type II (long) systems were discovered to have operons containing ancillary genes that share similarities with the non-canonical E1 and E2 signaling machinery described in the noncanonical autophagic ubiquitination pathway. Type II (short) CBASS systems have similar operons but with only one ancilliary gene related to ubiquitin E2 proteins. I was able to demonstrate how the bacterial E2-like protein, Cap2b, and the nucleotidyltransferase, cGAS, in Type II (short) systems using the artificial intelligence (AI) structural prediction tool, AlphaFold. Using the structural prediction, I focused on three strains to study and clone these proteins in order to isolate and purify them for structural determination using X-ray crystallography.

As a continuation of Mexican American education history and building on the research on undocumented students’ experiences in higher education, this dissertation uses history methods to examine the experiences of Latina/o undocumented students at the University of Texas Austin (UT Austin) between 2001 and 2013. During this time frame, important immigration and education legislation at state and federal levels was introduced or implemented, directly impacting undocumented students nationwide. Using a theoretical framework of critical race theory, Latina/o critical theory with a LatCrit Consciousness, this study centers race and immigration and challenges ahistoricism. Drawing on testimonios, institutional archives, newspapers, focus groups, personal collections, and social media posts, this dissertation’s findings reveal that (a) UT Austin did not foster an actively inclusive environment for Latina/o undocumented students; (b) Latina/o undocumented student activism at UT Austin evolved to a transformative resistance movement; and (c) the institution engaged in institutional neglect of Latina/o undocumented students, leading to significant challenges for this student population in navigating the university. Lastly, this dissertation explores Yosso’s Chicana/o Undergraduate “Stages of Passage” and adds dimensions to capture the experiences of Latina/o undocumented students in higher education.

This qualitative dissertation examined the role that mothers play in the educational success of Mexicana/Chicana working-class first-generation college students. Research has shown that Mexicana/Chicana mothers are integral to the educational achievement of their daughters, however few studies have explained why, or outlined specifically what it is that they do to inculcate educational success (Gándara, 1982, 1995, Gándara et al., 2013). Using Chicana/Latina feminist theory (Delgado Bernal & Elenes, 2011), this study explored the teaching and learning practices between Mexicana/Chicana immigrant working-class mothers and their first-generation college daughters. I focused on 10 mother-daughter dyads from Los Angeles County. The majority of the mothers are immigrants from México with an average of an elementary school education. Nine of the daughters are enrolled in Ph.D. programs in Los Angeles and one is an assistant professor at a local university. I facilitated 30 pláticas: 10 pláticas with the mothers, 10 pláticas with the daughters, and 10 mother-daughter pláticas. Additionally, I conducted 18 home visits, collected photographs and personal items, and kept a journal for self-reflexivity. I analyzed the data using modified grounded theory (Calderon, 2008; Malagón, Pérez-Huber, & Velez, 2009) with a Chicana/Latina feminist sensibility to generate theory from their lived experiences. During my analysis, I recognized the ways systems of oppression impact the lives of Women of Color. When preliminary themes were identified, I shared them with the mothers and daughters in the mother-daughter pláticas to discuss analysis, receive feedback, and engage in a collaborative data analysis process. Following this, I engaged in a final analysis stage where concluding themes were identified. My findings show that Mexicana/Chicana mothers use a pedagogy of the borderlands to raise muxeres truchas. A pedagogy of the borderlands encompasses the creative, defying, and empowering ways in which Mexicana/Chicana immigrant working-class mothers raise their daughters. It uses a bodymindspirit approach to instill a conocimiento of how to navigate, thrive in, and transform the physical and metaphorical borders that inform the everyday lives of Mexicanas/Chicanas. It is grounded in the epistemologies and ways of being of Mexicana/Chicana mothers and used to raise muxeres truchas. These are women who embody a unique form of intelligence of how to live in the world and transform it as Women of Color. Implications for this study point to the importance of improving the connection between Mexicana/Chicana mothers, daughters and schools, particularly for families of first-generation college students, reframing how we understand and measure success and achievement in schools, and the significance of continuing to develop Chicana/Latina feminist pedagogies and methodologies.

The mevalonate pathway is of critical importance to cellular function as it is the conduit for the production of terpenoids, hormones, and steroids. These molecules are also valuable on an industrial scale because they are used as antibiotics, flavoring agents, and fragrances, examples of which are clerocidin, cinnamon, and sandalwood, respectively. Because the biosynthetic route for the production of these compounds is the deoxyxylulose 5-phosphate pathway in E. coli, by engineering the heterologous mevalonate pathway into E. coli the native forms of pathway regulation were overcome to successfully create a bio-industrial route for the production of artemisinin, an antimalarial drug. Advanced biofuels can also be biosynthesized via the mevalonate pathway with some minor alterations to the final enzymatic conversions.

However, the toxicity of intermediates and products, as well as regulation internal to the pathway, limited our ability to increase production. The mevalonate pathway enzymes we used were native to S. cerevisiae, two of which had not been kinetically characterized. We set out to determine the nature of these enzymes and how they might be regulated by mechanisms internal to the mevalonate pathway, such as feedback inhibition. Because both mevalonate kinase (MK) and phosphomevalonate kinase (PMK) were derived from S. cerevisiae they had to be codon-optimized for production in E. coli, particularly because they had already been identified as expressing poorly in laboratory production strains through targeted protein studies. By cloning the codon-optimized sequences into high copy expression vectors with a six-histidine tag at the C- or N-terminus (PMK and MK, respectively) we were able to produce large enough amounts of the active proteins to purify on a Ni₂₊ resin column.

Kinetic characterization of MK revealed a K_M^ATP of 315 ± 21 μM and a v_max of 50 ± 3 μM/min/μgE (or K_cat = 41 ± 3 s^-1). Additionally, substrate inhibition of MK by mevalonate was determined to exist at concentrations above 2.5 mM, and K_i values for farnesyl pyrophosphate (79 ± 11 nM), geranyl pyrophosphate (147 ± 6 nM), geranylgeranyl pyrophosphate (303 ± 64 nM), dimethylallyl pyrophosphate, (29 ± 12 μM), and isopentenyl pyrophosphate (36 ± 5 μM) were determined.

Kinetic characterization of PMK revealed that maximum activity occurs at pH = 7.2 and [Mg²⁺] = 10 mM. K_M^ATP was determined to be 98.3 μM and 74.3 μM at 30 °C and 37 °C, respectively. K_M^mev-p was determined to be 885 μM and 880 μM at 30 °C and 37 °C, respectively. v_max was determined to be 45.1 μmol/min/μgE and 53.3 μmol/min/μgE at 30 °C and 37 °C, respectively. From the high K_M^mev-p value it appears as if PMK might have very poor activity at normal cellular concentrations of mevalonate-5-phosphate, indicating that MK and PMK might play a very coordinated role in balancing intermediate levels within the pathway.

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