Thesis advisor: Marc-Jan Gubbels / The two type I genotype T. gondii strains, RH, a lab-adapted strain, and GT1, a non-lab-adapted strain, have a genetic difference of only 0.002%, but show remarkable phenotypic differences in vitro. For example, it has long been known that RH’s in vitro virulence (i.e. plaquing capacity) and extracellular survival is far superior to that of GT1, likely due to several decades of adaptation to the in vitro environment (i.e. lab-adaptation). The genetic basis of these phenotypes, however, remains largely unknown despite previous allele-swapping experiments, thus inspiring two hypotheses: 1) epistatic interactions between two or more alleles and/or 2) gene regulatory mechanisms are responsible for lab-adaptive phenotypes. Uncovering the molecular basis underlying lab-adaptive phenotypes will support our growing understanding of T. gondii virulence and suggest therapeutic targets that affect the parasites lytic cycle in a host-independent manner. To answer this question, we applied Evolve and Resequencing (E&R) of GT1 during the first 1500 generations of its lab-adaptation in order to chronologically identify emerging genotype-phenotype correlations. Indeed, lab-adaptation augmented GT1’s in vitro virulence by improving its extracellular survival and reinvasion capabilities- both extracellular phenotypes of the lytic cycle. DNA-sequencing of parallel GT1 populations at multiple evolutionary timepoints (i.e. passages) identified a polymorphic phospholipid flippase gene whose gene expression is critical for in vitro virulence but, unfortunately, the evolved mutations could not be functionally characterized due to technical limitations. RNA-seq of both intracellular and extracellular parasites across several passages identified hundreds of “pro-tachyzoite” differentially expressed genes (DEGs), but only in extracellular parasites, paralleling our phenotypic observations. Interestingly, several upregulated DEGs are connected to fatty acid biosynthesis. Lastly, genetic KO of five seemingly non-related DEGs indicates that GT1’s lab-adaptive in vitro virulence is a complex and polygenic phenotype that is largely controlled by mechanisms independent of genomic mutations. / Thesis (PhD) — Boston College, 2020. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis advisor: Marc-Jan Gubbels / Thesis advisor: Sarah McMenamin / Toxoplasma gondii, an obligate intracellular parasite, infects nearly one-third of the global population, causing the disease toxoplasmosis. Despite its significant health impact, the molecular mechanisms governing its lytic cycle and stress-induced adaptation remain incompletely understood. The unique asexual cell division mechanism, endodyogeny, used by T. gondii to expand its parasitic biomass in intermediate hosts, including humans, leads to severe pathological consequences through repeated rounds of the lytic cycle, resulting in acute toxoplasmosis. The parasite’s cell cycle is characterized by a prolonged G1 phase, with centrosome duplication marking the onset of the S phase, followed by a transient G2 phase and a near-simultaneous onset of mitosis and cytokinesis. These overlapping division processes, coupled with the challenges of synchronizing T. gondii, obscure the precise molecular mechanisms of its transcriptional programs. To address these challenges, we employed single-cell RNA sequencing (scRNA-seq) and single-cell ATAC sequencing (scATAC-seq), combined with advanced machine learning tools, to reveal ‘transition points’ in gene expression and chromatin accessibility that correspond to shifts in biological activity during the lytic cycle. RNA velocity and time-course clustering analyses uncovered a significant G1a transcriptional burst and identified specific AP2 family transcription factors (TFs) that peak during the C-to-G1a transition, likely driving this burst to regulate G1 progression. Further, we conducted an in-depth functional characterization of G1-specific TFs, focusing on AP2XII-8, which plays a critical role in activating a ribosome regulon to promote G1 progression. The study identified combinatorial binding motifs and suggested the existence of a large AP2XII-8 protein complex, involving other TFs and epigenetic factors, that reuglates the intricate processes of T. gondii cell cycle replication. Additionally, we examined stress-responsive AP2 TFs associated with enhanced virulence during in vitro evolution, providing insights into adaptive mechanisms that enable T. gondii to thrive under extracellular stress conditions. Collectively, these findings enhance our understanding of T. gondii’s complex regulatory networks, offering potential targets for therapeutic intervention against acute toxoplasmosis. This dissertation provides the time-resolved transcriptional and chromatin accessibility landscapes of T. gondii’s lytic cycle, resolves transcriptional programs to DNA motifs, and identifies key regulatory elements involved in its cell cycle progression and stress response. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.