Childhood maltreatment is one of the most common adverse events in children and has been associated with a variety of negative effects on children’s social adjustment and mental well-being later in life. One explanation is that maltreated children may develop maladaptive emotion regulation skills, which mediate the pathway between maltreatment and psychopathology. Accumulating literature suggests that attachment security may be a protective factor against maladaptive emotion regulation skills in maltreated children. Prior research has investigated attachment as a mediator between childhood maltreatment and poor behavior outcomes. However, little research has examined attachment as a protective factor associated with better emotion regulation skills in maltreated children, which in turn results in better behavioral and emotional adjustment. The current study hypothesizes that, childhood maltreatment will have a less negative effect on children who are securely attached to one of their care givers, compare to those who have insecure attachment. In other words, secure attachment will serve as a protective factor for children exposed to adverse experiences, leading to better emotional and behavioral outcomes. Data from two separately collected samples of maltreated and control children (n= 221 and n= 124) are analyzed based on multiple linear regression models. We expect to find a significant difference between the effect of maltreatment on emotion regulation in high and low attachment individuals, such that children with more secure attachment are less affected by maltreatment. These findings would suggest that secure attachment may protect maltreated children from developing maladaptive emotion regulation skills which contribute to better general behavior outcomes in children.

The transcription factor hypoxia inducible factor-1 (HIF-1) plays a central role in responding to changes in environmental oxygen levels in both mammals and nematodes. HIF-1 stabilization in Caenorhabditis elegans via knockout of the E3 ubiquitin ligase von Hippel-Lindau 1 gene (vhl-1) improves proteostasis and lifespan. In humans, however, stabilization of HIF-1 via loss of VHL results in von Hippel-Lindau syndrome, a disease characterized by angiomas and renal carcinomas. Because it is a transcription factor, HIF-1 activates multiple genes that are likely responsible for its effects – whether they are improved proteostasis and lifespan in worms or neoplastic disease in humans. Previously, my lab performed a screen to determine which genes downstream of hypoxic signaling are responsible for the beneficial effects of HIF-1 stabilization in worms. This screen yielded a list of genes which were subsequently tested for whether they were necessary and/or sufficient to promote health and longevity, leading to the isolation of flavin-containing monooxygenase-2 (FMO-2), as a protein that increases lifespan and improves multiple measures of healthspan in nematodes. My project involves first characterizing the sensitivity of the yeast FMO1 deletion strain (fmo1Δ) to specific forms of toxic stress. Having established these phenotypes, I am performing a screen to find genes that, when transgenically expressed, rescue the effects of the FMO1 deletion. My strategy is to overexpress a library of other yeast genes as well as orthologous FMO genes to isolate those that rescue the fmo1Δ phenotypes. Additionally, I will overexpress FMO1, and any genes of interest from the screen, for analysis in replicative lifespan assays, testing whether they also play a role in increasing yeast longevity. This screen and the associated replicative lifespan assays will help us characterize the role of yeast FMO1 and the conserved role of FMOs in general, furthering our understanding of the process of aging.

In order to successfully invade and survive within a host cell, pathogens such as the bacteria Shigella flexneri have to suppress the host cell’s immune response. To do this, Shigella flexneri inject bacterial effector proteins into the host cell that hamper the host cell’s ability to detect and respond to invasion. One such protein is IpaH4.5, a bacterial E3 ubiquitin ligase, which hijacks the host cell’s ubiquitylation machinery to transfer ubiquitin, a small signaling protein, to a substrate. Ubiquitin signaling impacts all aspects of eukaryotic cell biology. Often modification of a protein by ubiquitin serves to mark protein for destruction. IpaH4.5 targets proteins involved in the host cell’s immune response, dampening the host cell’s ability to respond to invasion by Shigella flexneri. I am investigating IpaH4.5 and its interactions with both host substrates and the host ubiquitin machinery. After sub cloning out the various domains of TbK1, a known substrate of IpaH4.5, I will perform ubiquitilation assays in order to determine which domain of TBK1 is polyubiquitilated, indicating that IpaH4.5 interacts with this domain. Then, this domain can be used for structural and mechanical analysis of IpaH4.5, such as determining how mutations in IpaH4.5 affect ubiquitilation through biochemical assays. Understanding how IpaH4.5 binds to and interacts with its substrates will increase our understanding of how Shigella flexneri dampens host immune response as well as help us better understand the mechanisms of bacterial E3 ubiquitin ligases. Since IpaH4.5 functions differently than eukaryotic E3 ubiquitin ligases, increasing our understanding of its mechanisms could allow us to use it as a target for future drug development. Finding new targets for drug development is becoming increasingly important with the spread of antibiotic resistant bacteria.

As the primary producers of energy (ATP) in a cell, the mitochondria are key players in controlling cell aging and death. Interestingly, mild oxidative stress, derived from mitochondrial dysfunction or exogenous agents, evokes adaptive stress response pathways that can increase healthspan and lifespan in the nematode worm Caenorhabditis elegans. One of the pathways that responds to mitochondrial stress is the mitochondrial unfolded protein response (UPR^mt). The UPR^mt responds to stress from unfolded proteins or oxidative damage by upregulating expression of chaperones that stabilize and assist folding of unfolded proteins as well as proteases that can degrade proteins within the mitochondria. To search for genes that regulate the UPR^mt, we performed a genome-wide RNAi screen and found that knockdown of the tald-1 gene activates the UPR^mt. The tald-1 gene encodes transaldolase, an enzyme in the pentose phosphate pathway (PPP). One role of the PPP is to generate NADPH, which is required for cellular redox homeostasis. Although transaldolase deficiency has been linked to mitochondrial dysfunction in humans and mice, how the PPP controls mitochondrial function is not well understood. The focus of our study was to determine the relationship between PPP inhibition, mitochondrial function, and longevity by investigating the role of transaldolase in redox homeostasis and regulation of redox sensitive signaling pathways. Here we report that transaldolase deficient worms induced a starvation-like metabolic response that requires JNK MAPK signaling, a pathway that is directly activated by oxidative stress, to increase lifespan. Since the UPR^mt and PPP are correlated with several age-related diseases such as cancer and neurodegenerative disease, understanding the underlying genetics and molecular mechanisms of these pathways and their role in aging is likely to be relevant for human health.
 

The mitochondrial unfolded protein response (UPR^mt) is a key cellular mechanism for maintaining mitochondrial function in the face of oxidative and proteotoxic stress. This pathway upregulates mitochondrial chaperones and proteases that aid in folding and degradation of aberrant proteins. Induction of the UPR^mt was observed to correlate with increased longevity in the nematode worm Caenorhabditis elegans, a model whose genetic system is widely used to study the molecular mechanisms of aging. Through a genome-wide RNAi screen for regulators of the UPR^mt, we found that deficiency in the transaldolase (tald-1) resulted in activation of the UPR^mt. TALD-1 is responsible for the production of NADPH in the oxidative phase of the pentose phosphate pathway (PPP). In subsequent studies, we have found that knockdown of transaldolase reduces in vivo mitochondrial respiration, changes the overall metabolism of lipids, and affects the activity of transcription factors that regulate a starvation response and expression of metabolic genes. Among the genes transcriptionally altered by transaldolase knockdown is the flavin-containing monooxygenase-2 (fmo-2) gene, which has been previously found to be regulated by starvation. Like starvation, tald-1(RNAi) induced nuclear localization of HLH-30, a transcription factor responsible for the expression of autophagy genes. HLH-30 is also required for fmo-2 up-regulation. Epistasis lifespan analyses using tald-1 (RNAi) and deletion mutant strains of C. elegans, quantitative gene expression studies, and fluorescence reporter assays were used to determine the mechanistic relationship between tald-1, hlh-30, and fmo-2 as regulators of longevity in C. elegans.

The models of the universe’s diffuse configuration of hydrogen and helium atoms, known as the Intergalactic Medium (IGM), have become increasingly accurate in predicting the mechanics of the universe. Simulations of the IGM can provide a foundation for cosmology to be built upon. The purpose of our research is to test the accuracy and precision of our latest model of the IGM. Using data from the Very Large Telescope (VLT) in Chile, we analyzed quasar spectra for absorption lines at the Lyman-Alpha wavelength, creating phenomena called the Lyman-Alpha forest. We then processed this data incorporating scripts written in the programming language, Python, in order to compare the probability distribution function of the Lyman-Alpha absorption of the observed quasar spectra to the simulated quasar spectra. Interpreting our data we found that our simulations fell within one standard deviation of the observed data. Further analysis of our data showed that the simulations were more accurate for quasars at a lower redshift - those which were moving away from us at a slower rate - than quasars at a higher redshift - those moving away from us most rapidly. The largest contributors to error stem from the effects of other material in the IGM causing metal lines, our methods of metal line removal, and the limits of the size and quality of our observational sample. Metal lines skew our understanding of the Lyman-Alpha forest and a larger sample would decrease the variance among the samples. We plan on continuing research and improving the techniques of comparing the observations to models. An increased understanding of an effective model of the IGM can be used as a cosmological map of time to predict the origins of the universe as well as its future.

High aspect ratio nanostructures have been targeted for use in nanoscale electronics and photonics integration, as well as energy storage and conversion due to quantum confinement effects and high curvature. For instance, 3rd generation solar cells based on Si semiconductor nanowires exhibit improved absorption and current generation due to high aspect ratio and quantum confinement, yet widespread adoption of these technologies is limited due to lack of scalable synthesis methods. Solution-liquid-solid growth of semiconductor nanowires is a cheap, potentially scalable, environmentally-benign process that uses bismuth nanocrystallites as a growth site. Synthesis of Bi nanoparticles, however, is currently achieved by using costly and lethal reagents, in a technically difficult process that is sensitive to O2 and H2O. Furthermore, storage of these reagents is a large concern due to their volatility and the particles themselves tend to oxidize extremely quickly.  In this work, we evaluate a one-pot reaction for the production of bismuth nanoparticles using benign Bi2O3 as a precursor. We observe that the size of these particles depends directly on reaction time, and we hypothesize particle synthesis occurs through nucleation-and-growth process. In addition, these Bi nanoparticles show a remarkable resistance to oxidation after storage for weeks in an atmospheric environment. We speculate surface is passivated with a fluoride shell as a synthesis byproduct, which inhibits oxidation. We have studied these particles in different ways including SEM and XPS to determine the size and composition respectively. Our synthesis uses safe precursors and produces particles which are much more stable over the long term; this has the potential to make the use of bismuth nanoparticles a much more viable process and increase our understanding of bismuth on the nanoscale.

Aging is characterized by time-dependent deterioration of cellular and physiological functions. Due to the numerous challenges and limitations of studying aging in humans, model organisms are used to further extend our understanding of basic mechanisms of aging. The Kaeberlein laboratory uses the single celled budding yeast Saccharomyces cerevisiae as one such model. Advantages of this model organism include the ability to measure the lifespan of great numbers of organisms under various environmental conditions. One method of measuring a yeast cell’s longevity is by assaying its replicative lifespan (RLS). The RLS model refers to the number of daughter cells that can be produced by a mother cell before senescence. The number of budded daughters can be determined by micro-dissection with fiber optic needles from mother cells after each round of incubation. This process is repeated until mother cells no longer generate daughters. Approximately 600 mother cells are assessed weekly and incubation cycles last one month. Yeast lifespan can be manipulated through genetic changes, altering media content, addition of pharmacological agents, and change of temperature. Data collected is compared to control groups and to past and expected results. These results clarify how senescence is regulated. RLS is thought to be a better representation of senescence in mitotically active cells, like certain tissues in multicellular organisms. By analyzing thousands of cells, a variety of genes and environmental conditions that impact aging have been identified. These genes and interventions can be further studied and applied to other organisms. Studying budding yeast enhances our understanding of the molecular basis of aging.​

Aging and severe mitochondrial disease are disparate, complex processes whose causes have yet to be fully understood. One protein involved in both is the mechanistic target of rapamycin (mTOR), which regulates cell growth and metabolism. mTOR protein is a component of two complexes, the mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Inhibiting mTORC1 with the drug rapamycin increases lifespan in model organisms and suppresses severe mitochondrial disease and shortened lifespan in the Ndufs4 knockout (KO) mouse model of Leigh Syndrome. In mouse models, KO of the S6K1 protein, a substrate of mTORC1, also increases lifespan. Therefore, we hypothesized that KO of S6K1 in the whole body and specific tissues (brain, liver, fat) would rescue the short lifespan of Ndufs4 KO mice. A breeding strategy utilizing Cre-recombinase mice, S6K1^Flox/Flox mice, and Ndufs4 +/- mice was employed to ultimately produce tissue-specific Cre +/- Ndufs4 -/- S6K1^Flox/Flox mice. PCR and Western blot analysis confirmed the whole body and tissue-specific deletion of S6K1 gene and depletion of the protein in each mouse. Our findings indicate that whole body and liver-specific S6K1 KO modestly increased lifespan and delayed the onset of neurological symptoms originating from the mitochondrial disease. However, S6K1 KO is still less effective in rescuing mitochondrial dysfunction than the optimal dose of rapamycin. We speculate that S6K1 partially contributes to the mitochondrial disease of Ndufs4 KO mice, and the liver may play an important role in regulating lifespan. In the long run, these findings may enable us to potentially produce drug treatments to extend the lifespan of healthy humans as well as Leigh Syndrome patients.

Flavin containing monooxygenases (FMO) are a highly conserved and ancient protein family. They have been suggested to have evolved pre-Last Universal Common Ancestor (LUCA) and to be present in all kingdoms of life. FMOs are known as catalysts for detoxification of xenobiotic compounds, and their activity in the nematode Caenorhabditis elegans, animal model par excellence for aging research, has recently been found to modulate stress resistance and aging. However, the mechanisms by which FMOs affect these processes remain vastly undercharacterized. Our project seeks to define the subcellular localization and endogenous substrates of the C. elegans FMO, FMO-2, as well as investigate its role in redox balance and iron homeostasis. To compare fmo-2 knockout worms, FMO-2 overexpressor worms, and wild-type worms, we crossed the three strains into various established reporters, biochemically stained worms for changes in iron and fat, and performed stress epistasis experiments feeding them RNAi. We also assessed subcellular localization via several fluorescent translational fusion reporters including crossing them into organelle-marked worms. With FMOs’ newly uncovered role as longevity- and health-promoting enzymes, characterizing them more fully may lead in time to human therapies making use of these enzymes.

In the context of the growing aging population and constantly improving medical advancement, studies aiming to delay or reverse age-related health issues and enhance longevity have become increasingly relevant, especially in pre-clinical trials with drug interventions. One pharmacological agent, rapamycin which inhibits mTOR (mechanistic target of rapamycin), has been shown to robustly promote longevity in multiple animal model systems. Previous studies in the Kaeberlein lab showed that transient rapamycin treatment in aged C57BL/6JNia mice dramatically extended life expectancy, improved measures of healthspan, and remodeled the gut microbiome. We were particularly interested in the last discovery—specifically whether the increased prevalence of segmented filamentous bacteria (SFB) or C. savagella, commensal microbes shown to modulate the gut immune response, plays a role in rapamycin’s robust effects on longevity and health. To test this hypothesis, we will transplant SFB into aged Balbc By mice, obtained from the National Institute of Aging (NIA), that do not harbor SFB in their gut. The effects of SFB inoculation, paired with transient rapamycin treatment in these mice, will be evaluated by measuring changes in their lifespan and healthspan. We will also assess the changes in their gut microbiome after rapamycin interventions. This set of experiments provides an avenue to evaluate two important aspects of rapamycin intervention. Firstly, since this drug has not been tested in Balbc By mice, we can assess the role of genetic background on rapamycin’s effect on longevity. Secondly, we can establish whether the remodeling of the gut microbiome is relevant to the drug’s role in longevity and health. Additionally, does SFB play a role in this process? Answers to these questions can have a significant impact on how we view the aging process and may help pave the way for possible clinical interventions that can attenuate the negative effects associated with human aging.

 Eukaryotic cells contain membrane-bound organelles that serve to compartmentalize certain cellular functions. Different organelles contain optimal pH levels necessary for the organelle function. The vacuolar ATPase (V-ATPase) is a highly evolutionarily conserved proton pump that is responsible for acidification of many organelles. This proton pump consists of various protein subunits and loss of any these subunits results in disruption of the entire protein complex, which in return is not compatible with life for most organisms. Within certain conditions, S.cerevisiae (yeast) is able to survive in the absence of a functional V-ATPase, which makes it an ideal model organism to study V-ATPase function. In yeast, the proteins that comprise the V-ATPase are encoded for by vacuolar membrane ATPase (VMA) genes. Loss of any individual VMA gene results in pleiotropic phenotypes including an inability to grow on nonfermentable carbon sources and shortened replicative lifespan. In order to understand more about how the V-ATPase functions, we have performed multiple screens to identify genes that when overexpressed or mutated can suppress the respiratory phenotype of VMA mutants. Our results implicate iron homeostasis as a process that is crucially disturbed when the V-ATPase is disrupted.

The budding yeast, Saccharomyces cerevisiae, has been an invaluable model organism in the study of aging. Yeast cells divide asymmetrically. During division, the “mother” cell retains any accumulated cellular damage and produces a rejuvenated “daughter” cell. Thus, each yeast cell has a finite replicative lifespan defined by the number of daughter cells it produces before senescence. The historical belief in the field of aging has been that the asymmetrical inheritance of damage is absolute; until the last few divisions of a mother cell’s lifespan, daughters are born with a full lifespan potential, completely independent of the mother’s remaining lifespan. In this project we measure the replicative lifespans of daughter cells that budded off from mothers at different ages. We found a loss of daughter lifespan potential and an increase in mother-daughter remaining lifespan correlation in middle-aged mother cells. By tracking the granddaughter lifespans of aged mother cells, we find that this effect is compounded across generations. The implications of this research are to improve our understanding of yeast replication so as to better interpret the data gained from experiments that, as an example, use yeast aging as a model for human cell aging.

A key goal of aging research is to identify cellular factors that slow aging in hopes of one day developing treatments to combat age related diseases. Caenorhabditis elegans is a roundworm that has been used extensively to study the biology of aging. Low oxygen (hypoxic) environments have been shown to increase the longevity of C. elegans significantly, but the specific mechanisms of hypoxia induced longevity are still not completely understood. Temperature is one of the key determinants of lifespan in cold-blooded (poikilothermic) animals and also greatly influences the physiological response to decreased oxygen. This study aims to identify the relationship between temperature, oxygen levels, and development in C. elegans. Using a temperature gradient within a sealed gas environment we analyzed C. elegans development, growth and reproduction over a wide range of temperatures and oxygen concentrations. Our results show that under normal atmospheric oxygen concentrations (21%) growth is fastest at 26.7C. Interestingly, at extremely low oxygen concentrations (0.1%), development was extremely slow and was fastest at 16C. We hypothesize that increased oxygen demand at normally optimal growth temperatures prevents growth in hypoxia leading to the observed decrease in maximum growth temperature. We are continuing our work with various mutant strains to identify the genetic factors controlling the responses to low oxygen across a range of temperatures. These findings will provide insight into the fundamentals mechanisms of development and aging, and may facilitate development of therapies to delay the progression of age related diseases in humans. 

The Ndufs4 gene knockout (Ndufs4-/-) mice show the phenotype of severe neurodegenerative mitochondrial disease analogous to human Leigh syndrome. Phosphoproteomics is a large-scale study of phosphorylated proteins, which are post-translational modifications that can turn on and off biological signaling pathways. Phosphoproteomics and proteomics experiments using mass spectrometry can allow us to quantify protein levels in mouse tissues. The broad goal of my project is to define the underlying mechanism of mitochondrial dysfunction using a phosphoproteomics approach in the Ndufs4-/- mouse model and translate the findings to improve the health of patients with severe mitochondrial disorders. Our lab previously showed that rapamycin, a drug that inhibits mTOR kinase, partially rescued the disease phenotype of Ndufs4-/- mice. However, the downstream biological pathways affected by rapamycin in this setting are not understood yet. Those may be discovered by analyzing differences in phosphorylated proteins among brain tissues of wildtype, Ndufs4-/-, and rapamycin treated Ndufs4-/- mice. For the initial analysis, protein data sets with quantitative measurements of more than 4,000 proteins were analyzed. Proteins that may be important and most affected by mitochondrial disease and rescued by rapamycin were pinpointed. After the analysis of the data was complete, the proteins that passed the filtering thresholds were quantified with Western blots, a biochemical technique to quantify protein level individually. We have already identified two signaling pathways from the phosphoproteomics and will test the effect of inhibitors of these pathways in the mice. It is our hope that a better mechanistic understanding of disease progression in this mitochondrial disease mouse model will yield new therapies to treat mitochondrial disease in patients for whom there is not currently any effective treatment.
 

The drug rapamycin is a specific inhibitor of the mechanistic target of rapamycin (mTOR) and has been previously shown to increase lifespan in yeast, worms, flies, and mice. Rapamycin inhibits mTOR by binding the Fpr1 protein (FKBP12 in mammals); the rapamycin-Fpr1 complex then dissociates the mTOR complex I. In order to identify new, natural product inhibitors of mTOR that function similarly to rapamycin we have developed a high resolution assay for growth inhibition in yeast using a Bioscreen C MBR machine. We are currently testing twenty-two natural product compounds, and their mixes in this assay to determine if they inhibit the TOR pathway, change mitochondrial function, or have any effect on replicative lifespan in yeast. Each compound and mixture is being assessed for its ability to alter the growth kinetics of four yeast strains: wild-type, TOR1 deletion, FPR1 deletion, and SIT4 deletion. Tor1p is a kinase in the TOR complex, and deletion of TOR1 leads to rapamycin hypersensitivity. The fpr1Δ mutant is resistant to rapamycin. The sit4Δ deletion is sensitive to multiple drugs. Compounds were dissolved in the solvent DMSO, or in some cases other solvents, and diluted to 1 µg/ml, 10 µg/ml, and 100 µg/ml in culture medium. All tests were performed in yeast extract peptone dextrose (YEPD) solution. We used a Bioscreen C MBR analyzer to measure the optical density of growing yeast in the YEPD with the compounds. The bioscreen analyzer generates an optical density curve over time that can be further analyzed by using the web tool, Yeast Outgrowth Data Analyzer (YODA), to selectively examine and compute the doubling time of the cultured yeast cells. The doubling time data was used to evaluate the effect of supplements on growth rate compared to rapamycin, in order to identify compounds of interest for further study.

Mussels are bivalve mollusks that construct a byssus – an assemblage of protein threads with a strong adhesive plaque that attaches to underwater substrates. A mussel’s survival relies on a strong byssal attachment. Research on this adhesive can help explain trends in mussel attachment and inform how to emulate this glue or design surfaces that mussels cannot adhere to. Our work seeks to understand how abiotic factors affect the properties of mussel attachment, particularly at the thread-substrate interface (plaque). Specifically, we wanted to understand how increased temperature, decreased _pCO₂, and maturity (age) of the plaque affect its mechanical properties. This study involved three local species of mussel, as well as hybrid mussels between two species. The degree of hybridization was determined through PCR analysis. Byssal threads were cut from the animal and allowed to soak in a variety of treatments, and then stress-strain curves were generated for individual plaques. Initial findings indicate no difference in plaque strength after treatment in high temperature seawater. The results of this work could help explain trends in seasonal dislodgement of mussels, which are relevant ecologically and for aquaculture businesses. It can also give us insight into how changing environmental factors associated with ocean acidification will affect mussel attachment, independently of how they affect the organism itself.