Organic matter (OM) production and decomposition is important to wetland health as it serves to cycle nutrients throughout the ecosystem. Current wetland monitoring programs in the Laurentian Great Lakes focus on structural indicators, such as fish, bird, and vegetation communities but lack the use of functional metrics such as organic matter processing. In this study we aim to create a new suite of functional indicators that focus on OM production and decomposition. Decomposition rates were measured using mesh litter bags containing dried bulrush tissue (Schoenoplectus acutus) as well as by the use of cotton strips incubated for 8 weeks at 20 coastal wetlands of Lakes Michigan and Huron. Nutrient limitation of decomposition was also measured using nutrient diffusing bags containing nitrogen, phosphorus, and a combination of nitrogen and phosphorus fertilizers. OM production rates were measured by periphyton biomass accrual on acrylic rods over an 8-week period. In addition, nutrient diffusing substrates were deployed to test the effects of nutrient limitation on algae growth. We predicted that decomposition rates would be positively associated with higher nutrient concentrations. We also predicted that algae growth rates would be greatest on substrates containing high nutrient concentrations. When developed, functional indicators could be used alongside current structural indicators to provide a better overall assessment of wetland health.

The longevity of an organism is influenced both by internal (genetic) and external (environmental) factors. With respect to internal factors, a significant effort is being made to identify novel pharmacological agents that extend lifespan by targeting genetic pathways with a defined role in the aging process. On the external side, the molecular mechanisms responsible for the positive influence of environmental interventions, such as dietary restriction and mild stress, are being widely explored. The environment experienced by humans in modern societies already contains countless compounds that may influence longevity. Understanding the role that the most prevalent of these compounds play in the aging process will be important to predicting and interpreting the outcome of introducing new interventions. Caffeine is the most widely used psychoactive drug worldwide. Prior studies indicate that caffeine may positively impact age-associated neurodegenerative diseases, such as Alzheimer’s. In the nematode, Caenorhabditis elegans, we observe that low concentrations of caffeine extend lifespan in worms in a temperature-dependent manner. Initial evidence from experiments combining caffeine with previously identified aging interventions suggests caffeine may increase life span by reducing signaling through the insulin/IGF pathway. We have also shown that caffeine delays pathology in a worm model of polyglutamine disease. Additionally, caffeine and dietary restriction by bacterial deprivation show non-additive life span extension, suggesting that they impact aging through a similar mechanism. Our initial effort is focused on characterizing the effect of caffeine on longevity and health span, and exploring the interplay between caffeine and other aging interventions.

The mitochondrial unfolded protein response (mitoUPR) is a protective mechanism that is activated when the mitochondria experience stress. mitoUPR signaling has been linked to systemic inflammation and the innate immune response in multicellular organisms as well as being an important factor involved in the etiology and progression of many diseases, especially mitochondrial diseases such as Leigh Syndrome, and in aging. We are using HSP60 and HSP6 GFP reporter expressing Caenorhabditis elegans (C. elegans), which express green fluorescent protein when the mitoUPR is activated. By specifically inhibiting and altering mitochondrial function with different drugs, we hope to identify the signaling components of the mitochondria that induce (or inhibit) the protective mechanism. In addition, we are constructing HSP6 and HSP60 expressing plasmids to be taken up by the bacteria that the C. elegans eat. By feeding HSP6 and HSP60 to the worms we can determine if the mitoUPR can be induced through exogenous heat shock protein uptake in the gut. We are also feeding the C. elegans different strains of bacteria to observe the effect of how different bacterial proteins can affect the strength of mitoUPR induction and whether the innate immune response can induce the mitoUPR. From this data we hope to gain a better understanding of the mitoUPR, and configure a model on how this mechanism is expressed.

Mitochondrial dysfunction and oxidative stress have been proposed to contribute to organismal aging. In the nematode C. elegans, reduction of mitochondrial function through knockdown of genes encoding components of the electron transport chain (ETC) is sufficient to increase life span. The increased longevity is not solely the by-product of decreased oxidative damage generated by the ETC, but also involves induction of the mitochondrial unfolded protein response (UPR^mt), as evidenced by the observation that life span extension requires an intact UPR^mt. The cytoplasm, endoplasmic reticulum, and mitochondria have each evolved compartment-specific signal transduction pathways to alter nuclear gene expression in response to perturbations in protein homeostasis. However, little is known about the UPR^mt and the mechanism by which unfolded proteins are recognized in the mitochondria and signal changes in gene expression. Previous studies demonstrate that knockdown of ETC components and a subset of mitochondrial proteins induce the GFP reporters for mitochondrial chaperones hsp-6 and hsp-60. To identify novel genes involved in the UPR^mt, we are performing a forward genome-wide RNAi screen using the hsp-6::GFP reporter and measuring UPR^mt activity by fluorescent microscopy. Upon completion of the screen, we expect positive hits to include mitochondrial proteins, signaling molecules, and transcription factors that regulate the UPR^mt. Consistent with previous studies, gene knockdowns that have robustly induced the UPR^mt thus far in our screen are largely components of the ETC. In addition, the screen has yielded many novel hits including genes outside of the mitochondria and of unknown function. After the screen is complete, we will validate the RNAi clones that induce the UPR^mt and perform lifespan experiments to understand how the UPR^mt is involved in the aging process. Our screen will further understanding of how the mitochondria and nucleus communicate with one another in order to maintain mitochondrial proteostasis and illuminate how mitochondrial stress contributes to aging.

Significantly reducing caloric intake without malnutrition (calorie restriction) has been shown to extend lifespan in many organisms including yeast, nematodes, and mammals. Since calorie restriction (CR) can also result in the extension of healthspan, CR or CR mimetics may be useful in the prevention of aging related disorders such as cancer, neurodegenerative disease, and cardiovascular disease. However, before long-term CR or CR mimetics can be considered safe for humans, more information is needed on the molecular mechanisms responsible for the effects of CR. In addition, since long-term voluntary CR may prove difficult to endure in humans, the consequences of reinstating a normal or ad libitum (AL) diet after prolonged CR must be investigated. We hypothesized that switching back to an AL diet after prolonged CR would reactivate pro-aging molecular pathways, such as the target of rapamycin (TOR) pathway. To test this, we obtained kidney tissue from aged F344 rats that had been subjected to long-term CR. Some of the long-term CR treated rats had been gradually switched to an AL diet and the rest had been maintained on the CR diet. Proteins were extracted from the kidney tissue and then analyzed by Western blot. Switching to an AL diet after long-term CR resulted in a significant increase in the phosphorylation of downstream signaling proteins in the TOR pathway in kidney tissue. In addition, after switching to an AL diet, there were significant changes in autophagy, which is regulated by the TOR pathway, as indicated by increases in the autophagy markers LC3 and p62. These results indicate that reinstating an AL diet after prolonged CR results in an increase in the activation of the pro-aging TOR pathway. This suggests that the TOR pathway may regulate some of the physiological effects of reinstating a normal or ad libitum (AL) diet after prolonged CR.

The study of chronological and replicative aging in Saccharomyces cerevisiae has provided both post-mitotic and mitotic aging models in this single-celled organism. In chronological aging, lifespan is determined by the length of time a yeast cell can survive in a non-dividing state, whereas in replicative aging, the lifespan is determined by the number of daughter cells produced by a mother cell prior to irreversible cell cycle arrest. The primary goals of this study were to observe how the chronological aging of yeast cells influences replicative lifespan (RLS) and to determine whether changes in the extracellular environment that influence chronological lifespan (CLS) would also influence subsequent RLS. By buffering the pH of the medium to 6.0 or reducing the concentration of available glucose (dietary restriction), we show that these manipulations were able to suppress the reduction in RLS associated with chronological aging, when compared with cells aged in synthetic complete medium with 2% glucose. The production of ethanol and acetic acid due to yeast fermentation may be responsible for cellular damage leading to reduced RLS, and it can be inferred that by buffering the pH to 6.0 or dietary restricting the cells, this damage is attenuated and replicative capacity is increased following chronological aging.

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has been used extensively to study the biology of aging, and several determinants of C. elegans longevity are conserved in higher order organisms. The specific mechanisms of longevity in C. elegans have not been completely identified and many pathways are still poorly understood. Dietary restriction is one promising pro-longevity intervention, though like other aspects of longevity, its mechanism is also poorly defined. DR, a reduction in caloric intake in the absence of malnutrition, extends lifespan and has been found to delay the onset of age-associated disease, not only in C. elegans, but in a wide range of species including yeast, flies, mice, and primates. We are performing a genome-wide screen of the Vidal RNA interference (RNAi) Library, which has a catalog of 11,511 clones. By knocking down specific genes within the C. elegans genome through RNAi, we are able to observe an extension or reduction in longevity in response to DR. We will follow up on our collected data by measuring the full lifespans of all mutants that respond strongly in the screen. By screening such a large number of genes, the widespread effect, and hopefully mechanism, of DR will be elucidated. We hope to see genes significantly affected by DR cluster into groups with common functionality, or along the same pathway, and then follow up on those specific pathways to clarify the mechanisms underlying DR. If these pathways are conserved, research findings relating to C. elegans may be applicable to other organisms. We hope to confirm these results and further investigate the interaction between DR and other potential longevity pathways.

Aging is a process that causes decline in normal biological function, eventually leading to disease and death. Aging is influenced by a number of factors, including stress-response pathways such as the hypoxic response. Over-expression of the hypoxic response pathway regulator, Hypoxia-inducible factor (HIF-1), increases lifespan in the nematode Caenorhabditis Elegans. Under atmospheric oxygen, HIF-1 is targeted for degradation by the Von Hippel Lindau (VHL-1) tumor suppressor protein, whose deletion increases nematode lifespan through increased HIF-1 activity. Since knocking out VHL-1 in mammals is known to cause disease, it is important to better understand the hypoxic pathway in nematodes to develop targeted therapies for age-related diseases in humans. The goal of this research is to determine the tissues and genes downstream of HIF-1 that modulate aging in C. elegans. To determine tissue specificity, we have developed vectors designed to reintroduce VHL-1 into VHL-1 knockout worms under the control of tissue specific promoters. Lifespan and other age-related parameters will be measured in these transgenic animals and compared to that of control groups. To further test tissue specificity, we are also developing vectors to overexpress a mutated non-degradable form of HIF-1 in specific tissues in wild-type worms, essentially the reverse of the reintroduction of VHL-1. To test for genes downstream of HIF-1 important for longevity, we have performed a screen and identified several genes that are important for HIF-1-induced lifespan extension. Our experiments will examine whether C. elegans lifespan is increased by the over-expression of these proteins, which would then suggest further characterization and a search for conserved proteins. Together, the results from these experiments will provide important information on how we might utilize the hypoxic response pathway to improve human health.

Over the past few decades the budding yeast Saccharomyces cerevisiae has emerged as a prominent model for aging research. One method for measuring aging in yeast is to determine the replicative life span (RLS) of mother cells, defined as the number of daughter cells produced prior to irreversible cell cycle arrest. The Kaeberlein Lab Dissection Team has measured RLS for hundreds of thousands of yeast mother cells over the past few years. In addition to RLS, we have also recently began recording the budding pattern at end of life as an additional phenotype of interest since these mutations have been known to lead to irregular cell cycle regulation. 4 different categories are used to define the terminal morphology of yeast cells: unbudded (U), small bud (S), long bud (L), and clumped (C). From the data gathered thus far, a positive correlation has been made between the terminal budding pattern of cells with reduced RLS compared to their unbudded counterparts. Meaning contructs that stop replicating mid cell cycle (S, L, or C) live shorter lives. We speculate that genomic instability contributes to terminal arrest in the budded state, but further research needs to be conducted to confirm this theory. It will also be of interest to determine how specific genetic defects lead to altered frequencies of the 4 different terminal morphologies and their relationship to replicative life span.

This project researches the garbage collection in the city of Tacoma. By looking at current routes, then determining the most efficient routes using GIS for collection in order to eliminate backtracking as much as possible. Is there a better system of routes than those that are currently being used by the city of Tacoma? What is the carbon footprint of the Municipal Solid Waste in Tacoma before and after the new routes? We have determined how many individual routes will be needed in order to service all the customers in the service area based on best routes, time needed to drive, and average number of customers per load. This information was used to determine a network of routes for each day of the week in order to determine how many routes were needed per day to evenly distribute the weekly pick-ups. The new routes were calculated using information and number of trucks the city plans on using. The network for the project was setup with parameters such as street pick-up only, where possible, road layers that connect, and starting and ending points. This is important because new tools such as GIS allow for a more sustainable system which will reduce the carbon footprint of not only Tacoma but many more municipalities and private companies.

The complexity of the factors underlying the aging process and difficulty in categorizing definitive aging phenotypes complicates efforts to elucidate the pathology of aging. One approach toward understanding human biology is to look to model organisms as simpler starting points. Specifically, we can ask which aging genes have counterparts in more complex organisms and, of those, which also influence longevity. The Kaeberlein lab is in the process of completing a genome-wide screen of the budding yeast Saccharomyces cerevisiae for genes that alter its lifespan. The more difficult challenge is to piece together the pathways connecting the myriad products of these genes into a cohesive model for their effect on the aging process. A pathway of particular interest is the mitochondrial unfolded protein response (mtUPR). This stress response pathway is activated by accumulation of unfolded or misfolded proteins in the mitochondrion. After stress, signaling from the mitochondria to the nucleus can increase mitochondrial chaperone presence to promote proper protein folding. This pathway might play a role in some of the lifespan extending effects of the genes identified through the genomic screen, and there is evidence that some mechanisms of longevity in other organisms also involve the mtUPR. Our goal is to develop a GFP reporter system for the yeast mtUPR and investigate mtUPR activation in the lifespan mutants already identified by the lab. By creating a yeast GFP-promoter construct, we will be able to conduct experiments using fluorescence microscopy and flow cytometry to quantitatively determine mtUPR activation for a variety of genotypes and environmental conditions.

Evolutionary conflicts involving mimicry are found throughout nature ranging in a multitude of levels from ecosystems to molecules. Moreover, structural mimicry is increasingly being recognized at the interface between viruses and hosts. The goal of my project is to provide structural and mechanistic insight into a newly discovered evolutionary ‘arms race’ between a viral protein and a host immunity protein. We have found that a poxviral protein IBP1 (IFIT Binding Protein 1) binds a host immunity protein, IFIT2, which represses viral mRNA translation. I hypothesize that IBP1 has convergently evolved to structurally mimic IFIT2’s binding partner, eIF3c, as a strategy to restore viral protein production. Because IBP1 has no known amino acid similarity to any other viral or host protein, the best way to test my hypothesis is to determine the molecular structure of IBP1 and compare it to eIF3c. I will biochemically engineer constructs to optimize the expression and amount of soluble IBP1 in E. coli and then utilize affinity columns to isolate IBP1, which will then be subjected to a myriad of crystallization conditions. Once optimized crystallization conditions have been found I will use X-ray diffraction techniques to solve the structure. Regardless of whether my hypothesis is correct, my work will provide atomic resolution detail for IBP1, which is a protein that is important for the fitness of the virus. If I find that IBP1 is indeed using molecular mimicry it will not only reveal the mechanism of IBP1 action, but it will also provide a case of molecular mimicry marked by the signature of convergent evolution. Furthermore, my work will make progress towards further understanding the interactions between hosts and viruses, which determine the results of potentially deadly infections in so many people.

Aging research seeks to determine the processes that cause degeneration and disease in aged organisms, and to discover ways to prevent and/or slow their progression. Previous studies have shown that aging can be slowed in several model organisms through various means; however much is still not understood. In the model organism Caenorhabditis elegans, previous work shows that decreasing oxygen levels (hypoxia) significantly increases lifespan. The hypoxia-induced lifespan increase is observed when worms are exposed to hypoxia either from a late larval stage for life or if they are deprived of oxygen for just one day at the same larval stage. The low oxygen environment activates the hypoxic response pathway, mediated by the hypoxia-inducible factor (HIF-1). Previous data show that an increase of the HIF-1 protein, via genetic mutation or alterations in atmospheric oxygen, is sufficient to extend lifespan. However, much about this pathway remains unknown, including the mechanism for increased longevity, the difference between genetic and environmental manipulations of HIF-1, and the downstream proteins involved. My project examines the genetic components responsible for extending lifespan in hypoxia. To test this, we measured whether hypoxia increases the lifespans of worm strains with deletions or transgenic overexpression of known aging genes. Our results suggest that disruption of most of the non-HIF-1-related pathways have no effect on hypoxia-induced lifespan extension. However, the data show that the forkhead transcription factor, DAF-16, is required for hypoxia to increase lifespan, and subsequent work has shown that hypoxia causes DAF-16 to enter the nucleus. Interestingly, results indicate that hypoxia decreases lifespan when the oxidative stress response factor, SKN-1, is overexpressed, suggesting that SKN-1 is somehow toxic to worms in low oxygen. Collectively, the results show that hypoxic treatment increases lifespan in worms, but that the hypoxic pathway is intertwined with other stress responsive pathways.

Organisms have evolved specific mechanisms to respond to molecular and environmental stress. These stress-response pathways are evolutionarily conserved and can modify lifespan in model organisms, making them popular targets of aging research. Using the nematode Caenorhabditis elegans (C. elegans), we have found the loss of important genes in stress-response pathways can cause a phenomenon called a vulval integrity defect (Vid). Worms with Vid show a small protrusion near their vulva that can eventually lead to rupture of the gonad and premature death. While many labs have noted this phenotype, it is unknown if Vid is a natural part of C. elegans aging or if other genetic and environmental factors induce this phenotype, leading to confusion as to whether worms with Vid should be censored in aging experiments. The goal of our project is to determine the mechanism of Vid and whether it is part of normal worm aging. To further define the mechanism of Vid, we are using three experimental approaches: (1) using mutant worm strains, we are testing the genetic pathways important for causing and preventing Vid, (2) because Vid is temperature dependent, with frequency increasing inversely with temperature, we are testing at what point in their lifespan worms can still be rescued from Vid, (3) beginning with the gonad and vulval muscle, we are looking for cellular death and/or structural weaknesses that could be the direct mechanism behind Vid. Our initial work has identified several genes and genetic pathways important for Vid, including the hypoxic response pathway, the oxidative stress response pathway, and the protein assembly pathway. Our current work is close to identifying when worms are most susceptible to Vid and has begun to describe the physical mechanism of Vid. We intend to combine the results from these experiments to develop a model for Vid.

Oxidative stress and aggregation of misfolded proteins have been linked to aging in a range of model organisms, including the nematode Caenorhabditis elegans. Reduced expression of genes involved in mitochondrial respiration, such as those coding for electron transport chain (ETC) components, have been found to ameliorate these decreases in lifespan. This rescue is thought to act through upregulation of the mitochondrial unfolded protein response (UPRmt) in a cell-non-autonomous manner. Induction of the UPRmt is essential for degradation of misfolded mitochondrial proteins, and has been correlated with longevity in ETC mutant worms. While the mechanism underlying the UPRmt and its effect on gene expression knockdown is not well understood, there is evidence that knockdown induces the UPRmt. Previous studies demonstrate that knockdown of ETC components, as well as knockdown of a subset of mitochondrial proteins both induce the GFP reporters for the mitochondrial chaperone, hsp-6. To identify novel genes involved in the UPRmt, we are performing a forward genome-wide RNAi screen using the hsp-6::GFP reporter and measuring the UPRmt activity by fluorescent microscopy. We expect genes induced by the UPRmt to cluster into groups with common functionality, or along the same metabolic pathways. These hits may include genes coding for the ETC or other mitochondrial proteins, as well as genes outside of the mitochondrion. We will follow up on our screen data results by sequence verifying RNAi mutants and completing full lifespans to elucidate the interaction between the UPRmt and gene knockdown.

Although aging has been studied extensively, the molecular mechanisms of longevity are unclear. Dietary restriction (DR), a reduction in calories without malnutrition, increases lifespan in most model organisms tested, including rats, flies, and the round worm, Caenorhabditis elegans. Although DR is the most proven longevity-promoting intervention, the mechanism by which it works has not been fully elucidated. A reduction of metabolic rate and reduction of insulin signaling have been implicated in longevity, though neither appear to be sufficient on their own. Another highly supported mechanism of longevity includes the link between TOR (Target of Rapamycin) signaling and DR. Recent data suggests that TOR and DR act along the same pathway in respect to longevity; however, it is widely accepted that DR influences other pathways in addition to TOR. There are many approaches to DR in C. elegans. Using a bacterium carrying an RNAi plasmid, we feed the worms from egg through the first four days of adulthood and are therefore able to knockout specific genes in temperature sterile nematodes. The worms are then transferred to a liquid medium in the absence of the bacterial food. This process allows us to test the effects of dietary restriction on specific genes and determine whether the mutants will respond synergistically or antagonistically to the intervention. These results will be followed up to determine common pathways relating to longevity. Caloric intake was believed to be the sole mechanism of lifespan extension in DR; however recent data supports the idea that different mechanisms appear to be involved. This screen will provide us with a basis of which knockouts extend or shorten lifespan allowing us to relate the biology of DR with the pathways recognized as determinants of aging. If these pathways are conserved, these research findings may be applicable to other organisms.

Model membranes are important tools for investigating physical properties of lipid bilayers. Unfortunately, current techniques for producing giant unilamellar vesicles (GUVs) with biologically and physically relevant properties (namely: inclusion of charged lipids, encapsulation of high salt buffer, and compositional asymmetry between the inner and outer leaflets) are difficult, unreliable, and/or low throughput. We examine a method called cDICE, developed by Abkarian et al., that has the potential to address all of these issues. Our results here for charged lipids in high salt buffers are promising, whereas asymmetry has proven difficult to measure.

 The research conducted by the Kaeberlein Lab is focused on identifying conserved longevity genes in evolutionarily divergent model organisms. The research methods discussed here concentrate particularly on the mechanism of aging in budding yeast. Aging in the budding yeast Saccharomyces cerevisiae was described more than fifty years ago with the observation that mother cells produce a finite number of daughter cells and then cease replication. Our poster describes the method utilized in our lab for quantifying replicative lifespan. In short, individual yeast cells are grown on solid media and monitored from their initial undivided state until they undergo senescence. Mother cells that produce more daughter cells are defined as having a longer replicative lifespan. Over the past several years, we have measured replicative lifespan on several hundred thousand mother cells, resulting in the identification of dozens of genes that modulate aging in yeast.  By identifying these genes and determining their mechanism of action it may be possible to one day develop therapies that delay the onset of aging and age-related disorders in people. 

Since political violence first broke out in 1999, Côte d’Ivoire has experienced a drastic loss of medical equipment, drugs, and skilled health personnel. Two civil wars in the past decade have posed significant challenges to outside intervention from international NGOs, and the weakening economy has provided little support for a state-funded health system. Currently, Côte d’Ivoire suffers from some of the highest maternal and neonatal mortality rates in the world, resulting from problems ranging from infection and hemorrhages to the compound effects of malaria, malnutrition, and HIV/AIDS. My research responds to these challenges by exploring what would be needed in a grant proposal to The Global Fund, a global funding agency that supports the fight against HIV/AIDS, malaria, and tuberculosis in developing countries. Of the thirteen Global Fund grants approved for Côte d’Ivoire in the past, none specifically address the problems of maternal and neonatal mortality. This project aims to understand why this gap in maternal and neonatal care persists and how it can be diminished in the context of political instability. To assess the efficacy of attempts to address health care gaps in Côte d’Ivoire, I analyzed the interventions currently in place to see if they were meeting their established goals and if not, why. Building upon this data and development theory, I created a proposal for improving maternal and neonatal mortality through a self-sufficient system that can remain in place should political violence persist. Preliminary research suggests that weak medical assistance is a leading cause of birth complications. Increased health monitoring during and immediately following pregnancy, as well as greater education for mothers on the crucial steps during this time period are vital to reducing mortality. Establishing self-sustaining health systems that are resilient in the face of violence and political uncertainty is invaluable across the developing world.