Iron homeostasis is a vital process that balances access to free iron to fuel physiological processes with iron storage to reduce the deleterious aspects of excess free iron. Dysfunctional iron homeostasis can lead to iron overload, in which excess free iron can promote cellular injury, ferroptosis, and eventual organ damage as a result. The cellular consequences of long-term iron overload including the cellular pathways that lead to pathological changes or those that may provide protection against damage are incompletely defined. Here, we use dietary and genetically engineered mouse models of iron overload combined with mass spectrometry-based quantitative proteomics to determine the iron overload liver proteome. We identify and quantify over 8,000 proteins representing the most in-depth iron overload proteome evaluation to date. Using bioinformatics, we identify conserved upregulated pathways including ‘response to oxidative stress’ and downregulated pathways including ‘steroid homeostasis.’ Furthermore, we identify an unexpected role for NCOA4, an autophagy adaptor that targets ferritin for autophagic degradation during iron deprivation, during iron overload. This work highlights the importance of further investigation into NCOA4 and its role in diseases of iron overload. / 2025-01-30T00:00:00Z

A thesis submitted to the School of Graduate Studies in partial fulfillment of the requirements for the degree Master of Science. / Sprint interval training (SIT) is a time-efficient strategy to improve cardiorespiratory fitness; however, most protocols have been studied in a laboratory setting and require specialized equipment. We investigated the efficacy of brief intense stair climbing as a practical model of SIT to improve cardiometabolic health, with a key measure being cardiorespiratory fitness as indicated by peak oxygen uptake (VO2peak). Two separate studies, each consisting of an acute and chronic phase, were conducted in a total of 31 sedentary women (age=24±10 y; BMI=23±4 kg•m-2). The acute phase of Study 1 established that the heart rate (HR), blood lactate concentration (BLa), and rating of perceived exertion (RPE) responses were similar when participants (n=8) performed a SIT protocol that involved 3x20-s “all-out” efforts of either continuous stair climbing or cycling, interspersed with 2 min of recovery. The chronic phase demonstrated that when participants (n=12) performed the 3x20-s stair climbing protocol 3 d•wk-1 for 6 wk, absolute and relative VO2peak increased by 12%, or ~1 metabolic equivalent (1.80±0.25 to 2.02±0.27 L•min-1, p<0.001), as there were no changes in body mass (p=0.35), fat free mass (FFM; p=0.09) or % body fat (p=0.42). There were also no changes in resting systolic and diastolic blood pressure (BP; p=0.82 and p=0.97, respectively), resting HR (p=0.62), and fasting insulin sensitivity (p=0.52). The acute phase of Study 2 established that the HR and RPE responses were similar when participants (n=11) performed three different stair climbing protocols. The protocols investigated include the 3x20-s continuous ascent model used in Study 1 (protocol 1), as well as 3x60-s bouts of ascending and descending either one or two flights of stairs, with 60-s of recovery (protocol 2 and 3, respectively). The chronic phase demonstrated that when the same group of subjects performed the 3x60-s 1-flight protocol 3 d•wk-1 for 6 wk, absolute and relative VO2peak increased by 8 and 7%, respectively (1.79±0.36 to 1.93±0.39 L•min-1, p=0.001; 31.2±4.6 to 33.3±5.3 mL•kg-1•min-1; p=0.01). Despite no changes in % body fat (p=0.10), there was an increase 3% increase in FFM (p<0.001). There was no change in systolic (p=0.50) and diastolic BP (p=1.00), but resting HR improved by 8% after training (p=0.03). The change in insulin sensitivity derived from an OGTT was 7.1±11 mg I2•mmol-1•mIU-1•min-1 (p=0.056). These findings demonstrate that brief intense stair climbing is a practical, time-efficient strategy to improve cardiorespiratory fitness in previously untrained women. / Thesis / Master of Science (MSc) / Sprint interval training (SIT), involving brief bouts of very intense exercise separated by short periods of recovery, is a time-efficient alternative to traditional endurance training for improving fitness. This has largely been established in laboratory settings using specialized equipment, which is impractical for many individuals. This project examined whether brief intense stair climbing was a practical model of SIT to elicit adaptations previously shown with cycling protocols. Subjects performed either three 20-s ascents interspersed with 2 min recovery periods, or three 60-s bouts of ascending and descending one or two flights of stairs, with 60-s recovery periods. Both protocols were 10 min in duration including warm-up and cool-down, and subjects trained three days per week for six weeks. The main finding was that stair climbing is a practical, time-efficient model to improve fitness in previously sedentary individuals.

The aim of this study was to investigate the comparative effects of mindful meditation and cyclic hyperventilation with retention upon interoceptive ability and heart rate variability (HRV). Further studies comparing meditation and breathwork contribute to a greater understanding of their potential benefits and have the potential to enhance overall well-being in the general population. A pre-post randomized controlled trial design was employed. Interoceptive accuracy was assessed through heartbeat detection task. Interoceptive sensibility was measured through confidence judgments, and interoceptive awareness by the correlation between accuracy and sensibility. Participants were randomly assigned to perform mindful meditation or cyclic hyperventilation with retention once per day for 21 consecutive days. No differences in interoceptive capacity and HRV was observed between the groups, but a tendency towards improved accuracy and increased confidence for both groups. However, the mindful meditation group exhibited a stronger positive association between HRV and interoceptive accuracy after the intervention, while the opposite was true for the cyclic hyperventilation group. Additionally, interoceptive awareness remained stable throughout the study. Mindful meditation demonstrated an enhancement in parasympathetic activity, as evidenced by higher RMSSD values. In contrast, cyclic hyperventilation with retention resulted in a substantial reduction in parasympathetic activity. This study highlights the complexity of interoception and emphasizes the need for further investigation in the context of mindfulness-based and breathwork interventions. / Syftet med denna studie var att undersöka de jämförande effekterna av medveten meditation och cyklisk hyperventilation med bibehållande på interoceptiv förmåga och hjärtfrekvensvariabilitet (HRV). Fler studier som jämför meditation och andningsövningar leder till en ökad förståelse av dess potentiella fördelar och står att gynna det övergripande välbefinnandet i den breda befolkningen. En pre-post randomiserad försöksdesign användes. Interoceptiv noggrannhet bedömdes genom en uppgift för att känna hjärtslag. Interoceptiv känslighet mättes genom konfidenskattningar och interoceptiv medvetenhet genom korrelationen mellan noggrannhet och känslighet. Deltagarna tilldelades slumpmässigt medveten meditation eller cyklisk hyperventilation en gång om dagen i 21 dagar. Inga skillnader i interoceptiv förmåga och HRV observerades mellan grupperna, men det fanns en tendens till förbättrad noggrannhet och ökad sensibilitet för båda grupperna. Likväl visade medveten meditation en starkare positiv association mellan HRV och interoceptiv noggrannhet efter interventionen, medan det motsatta var sant för cyklisk hyperventilation. Dessutom förblev interoceptiv medvetenhet stabil under hela studien. Medveten meditation visade en förbättring av parasympatisk aktivitet, vilket bevisades av högre RMSSD-värden. Å andra sidan resulterade cyklisk hyperventilation med bibehållande en betydande minskning av parasympatisk aktivitet. Denna studie belyser interoceptionens komplexitet och betonar behovet av ytterligare undersökning inom ramen för interventioner baserade på mindfulness och andningsarbete.

The maintenance of protein homeostasis as a response to changing external conditions is crucial for cellular survival and proper function. Since plants cannot adapt by changing location, their need for a rapid intracellular response is accentuated. The AAA ATPase CDC48 maintains protein homeostasis in conjunction with NPL4 and UFD1 by coupling ATP hydrolysis with mechanical force to extract and unfold ubiquitylated proteins from organelle membranes, chromatin, or protein complexes. Our bioinformatic analysis revealed considerable domain and binding motif differences in A. thaliana NPL4 compared to its orthologs in animals and fungi. Using ITC, MST, and SEC-MALS, we found that NPL4 and UFD1 did not heterodimerize, NPL4 bound to CDC48A in the absence of UFD1, and the complex was not stable in vitro. Additionally, we provided the first medium-high-resolution reconstructions of CDC48A in both an AMP-PNP bound and apo state, using cryo-EM. AMP-PNP bound CDC48A was reconstructed in both a tense (3.3 Å) and relaxed (3.5 Å) conformation with the N domain was positioned above or coplanar with the D1 ring, respectively. Our heterogeneity analysis using CryoDRGN revealed continuous flexibility of the N domains between the two conformations. The apo state was reconstructed as a single conformation at 4.4 Å resolution. A cryo-EM reconstruction of the complex was also obtained at a resolution of ~6 Å, which showed expected cofactor stoichiometry and binding positions. Through our efforts, we have observed differences in the interaction between A. thaliana CDC48A and its cofactors UFD1 and NPL4 that may correspond to functional differences between kingdoms.

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Many bacterial species exist in nature as part of highly structured multicellular communities known as biofilms. Amyloids, proteins with a conserved β-sheet quarternary structure, show high resistance to many chemical and enzymatic processes including proteinase K and SDS treatments and are produced as essential adhesins during biofilm formation. Curli fibers expressed by Enterobacteriaceae family members including E. coli and S. Typhimurium are the most studied amyloids to date. Curli-like fibers are also produced by members of the predominant phyla found in the host gastrointestinal microbiota in environmental biofilms. Curli fibers are the predominant microbial-associated molecular pattern (MAMP) on enteric bacteria recognized by the Toll-like receptor (TLR) 2/1-heterodimer complex. Interestingly, the TLR2/1 complex has been implicated as a key player in modulating gastrointestinal homeostasis. The focus of the current studies centered on the innate immune recognition of curli fibers by cells of the gastrointestinal tract and how that contributes to gastrointestinal homeostasis. In the first phase of our studies, utilizing intestinal epithelial cells polarized on semi-permeable tissue culture inserts (Transwells®), we observed that the recognition of curli fibers on Salmonella enterica serovar Typhimurium by intestinal epithelial cells led to the augmentation of the intestinal epithelial barrier in a PI3K-dependent manner. We also observed that bacterial translocation of S. Typhimurium from the apical side to the basolateral side of the Transwell system was limited when curli fibers were present. Furthermore, infection of mice with S. Typhimurium showed that translocation of bacteria from the intestinal lumen into the cecal tissue and mesenteric lymph nodes was limited in C57BL/6 mice as compared to TLR2 knockout mice. In the second phase of our studies, we sought to further investigate the effect that curli fibers exert on gastrointestinal homeostasis through the induction of immunomodulatory cytokines such as Interleukin 10 (IL10) from subepithelial lamina propria cells. IL10 has been shown to contribute to the maintenance of the intestinal epithelial barrier and IL10-deficient mice develop lethal colitis within the first 2-3 months of life. 6-8 week-old female C57BL/6 and TLR2-/- mice were given 5mg/kg of curli fibers via intraperitoneal injection. Subsequent RT-PCR analysis of the small intestine showed a significant expression of Il10 in C57BL/6 that was absent in TLR2-/- mice. Interestingly, no changes in Ifnγ or Tgfβ mRNA were observed in these mice. This response was gut-specific, as Il10 was not detected at all in the spleen. Furthermore, in a chemically-induced colitis model, we observed that the administration of curli fibers to 8-week old Balb/c mice ameliorated disease severity as compared to colitic mice that received mock treatments. Interestingly, Il10 was also induced in the colons of colitic mice that received curli and which were euthanized 6 days after colitis was induced. Our results suggest that curli fibers induce IL10 production via a TLR2-dependent manner to dampen inflammation in the gastrointestinal tract. Overall, our results partially describe a novel role for curli amyloid fibers produced by commensal bacteria in modulating gastrointestinal health and homeostasis. We propose that the induction of immunomodulatory cytokine such as IL10 by amyloid fibers is an important mechanism utilized by commensal bacterial to confer beneficial effects that benefit both the host and microbe. We also propose curli fibers as a potential alternative in the treatment of inflammatory bowel disease. / Microbiology and Immunology

Most cellular proteins retain a stable concentration as cells grow and divide, but there are exceptions. Some cell cycle regulators change in concentration with cell size. In fission yeast, Cdc13 (cyclin B), an important activator of the core cell cycle kinase Cdc2 (CDK1), increases in concentration as cells grow. It has been proposed that the concentration of such cell cycle regulators serves as a proxy for cell size and makes cell cycle progression dependent on cell size, thereby contributing to cell size homeostasis. The underlying mechanisms for the size-dependent scaling of these cell cycle regulators are poorly understood. Here, I show that Cdc13 protein concentration, but not mRNA concentration, increases with cell size. Furthermore, only the nuclear, but not the cytoplasmic, fraction of Cdc13 increases in concentration as cell size increases. Computational modeling along with half-life measurements suggests that stabilization of Cdc13 in the nucleus plays an important role in establishing this pattern. Taken together, my results suggest that Cdc13 scales with time, and therefore only indirectly—not directly—with cell size. This leaves open the possibility that Cdc13 contributes to cell size homeostasis, but in a different way than originally proposed. / Master of Science / Cells maintain their size very efficiently, but how they manage to do so is not well characterized. It has been suggested that cells sense their size by the size-dependent concentration changes of cell cycle proteins. I have investigated how cyclin B may serve as such a proxy for cell size in fission yeast. My data suggest that fission yeast cyclin B indirectly scales with cell size through an unknown time-based mechanism.