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We studied the microbial community structure of pilot two-stage membrane biofilm reactors (MBfRs) designed to reduce nitrate (NO[subscript 3]–) and perchlorate (ClO[subscript 4]–) in contaminated groundwater. The groundwater also contained oxygen (O[subscript 2]) and sulfate (SO[2 over 4]–), which became important electron sinks that affected the NO[subscript 3]– and ClO[subscript 4]– removal rates. Using pyrosequencing, we elucidated how important phylotypes of each “primary” microbial group, i.e., denitrifying bacteria (DB), perchlorate-reducing bacteria (PRB), and sulfate-reducing bacteria (SRB), responded to changes in electron-acceptor loading. UniFrac, principal coordinate analysis (PCoA), and diversity analyses documented that the microbial community of biofilms sampled when the MBfRs had a high acceptor loading were phylogenetically distant from and less diverse than the microbial community of biofilm samples with lower acceptor loadings. Diminished acceptor loading led to SO[2 over 4]– reduction in the lag MBfR, which allowed Desulfovibrionales (an SRB) and Thiothrichales (sulfur-oxidizers) to thrive through S cycling. As a result of this cooperative relationship, they competed effectively with DB/PRB phylotypes such as Xanthomonadales and Rhodobacterales. Thus, pyrosequencing illustrated that while DB, PRB, and SRB responded predictably to changes in acceptor loading, a decrease in total acceptor loading led to important shifts within the “primary” groups, the onset of other members (e.g., Thiothrichales), and overall greater diversity.
ContributorsOntiveros-Valencia, Aura (Author) / Tang, Youneng (Author) / Zhao, Heping (Author) / Friese, David (Author) / Overstreet, Ryan (Author) / Smith, Jennifer (Author) / Evans, Patrick (Author) / Rittmann, Bruce (Author) / Krajmalnik-Brown, Rosa (Author) / Biodesign Institute (Contributor) / Swette Center for Environmental Biotechnology (Contributor) / Julie Ann Wrigley Global Institute of Sustainability (Contributor) / School of Sustainability (Contributor)
Created2014-07-01
Description
Cleavage and polyadenylation is a step in mRNA processing in which the 3’UTR is cleaved and a polyA tail is added to create a final mature transcript. This process relies on RNA sequence elements that guide a large multimeric protein complex named the Cleavage and Polyadenylation Complex to dock on the 3’UTR and execute the cleavage reaction. Interactions of the complex with the RNA and specific dynamics of complex recruitment and formation still remain largely uncharacterized. In our lab we have identified an Adenosine residue as the nucleotide most often present at the cleavage site, although it is unclear whether this specific element is a required instructor of cleavage and polyadenylation. To address whether the Adenosine residue is necessary and sufficient for the cleavage and polyadenylation reaction, we mutated this nucleotide at the cleavage site in three C. elegans protein coding genes, forcing the expression of these wt and mutant 3’UTRs, and studied how the cleavage and polyadenylation machinery process these genes in vivo. We found that interrupting the wt sequence elements found at the cleavage site interferes with the cleavage and polyadenylation reaction, suggesting that the sequence close to the end of the transcript plays a role in modulating the site of the RNA cleavage. This activity is also gene-specific. Genes such as ges-1 showed little disruption in the cleavage of the transcript, with similar location occurring in both the wt and mutant 3’UTRs. On the other hand, mutation of the cleavage site in genes such as Y106G6H.9 caused the activation of new cryptic cleavage sites within the transcript. Taken together, my experiments suggest that the sequence elements at the cleavage site somehow participate in the reaction to guide the cleavage reaction to occur at an exact site. This work will help to better understand the mechanisms of transcription termination in vivo and will push forward research aimed to study post-transcriptional gene regulation in eukaryotes.
ContributorsSteber, Hannah Suzanne (Author) / Mangone, Marco (Thesis director) / Harris, Robin (Committee member) / LaBaer, Joshua (Committee member) / School of Life Sciences (Contributor, Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
The gastrointestinal (GI) tract is home to a complex and diverse microbial ecosystem that contributes to health or disease in many aspects. While bacterial species are the majority in the GI tract, their cohabitants, fungal species, should not be forgotten. Children with autism spectrum disorder (ASD) often suffer from GI disorders and associated symptoms, implying a role the bacterial and fungal gut microbiota play in maintaining human health. The irregularities in GI symptoms can negatively affect the overall quality of life or even worsen behavioral symptoms the children present. Even with the increase in the availability of next-generation sequencing technologies, the composition and diversities of fungal microbiotas are understudied, especially in the context of ASD. We therefore aimed to investigate the gut mycobiota of 36 neurotypical children and 38 children with ASD. We obtained stool samples from all participants, as well as autism severity and GI symptom scores to help us understand the effect the mycobiome has on these symptoms. By targeting the fungal internal transcribed spacer (ITS) and bacterial 16S rRNA V4 regions, we obtained fungal and bacterial amplicon sequences, from which we investigated the diversities, composition, and potential link between two different ecological clades. From fungal amplicon sequencing results, we observed a significant decrease in the observed fungal OTUs in children with ASD, implying a lack of potentially beneficial fungi in ASD subjects. We performed Bray-Curtis principal coordinates analysis and observed significant differences in fungal microbiota composition between the two groups. Taxonomic analysis showed higher relative abundances of Candida , Pichia, Penicillium , and Exophiala in ASD subjects, yet due to a large dispersion of data, the differences were not statistically significant. Interestingly, we observed a bimodal distribution of Candida abundances within children with ASD. Candida's relative abundance was not significantly correlated with GI scores, but children with high Candida relative abundances presented significantly higher Autism Treatment Evaluation Checklist (ATEC) scores, suggesting a role of Candida on ASD behavioral symptoms. Regarding the bacterial gut microbiota, we found marginally lower observed OTUs and significantly lower relative abundance of Prevotella in the ASD group, which was consistent with previous studies. Taken together, we demonstrated that autism is closely linked with a distinct gut mycobiota, characterized by a loss of fungal and bacterial diversity and an altered fungal and bacterial composition.
ContributorsPatel, Jigar (Author) / Krajmalnik-Brown, Rosa (Thesis director) / Kang, Dae Wook (Committee member) / Adams, James (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
The effect of an anaerobic reductive environment produced by the oxidation of zero valent iron (ZVI) on the microbial reductive dechlorination of trichloroethylene and its applicability to in-situ bioremediation processes was investigated using microcosms and soil column studies. I learned that microbial dechlorination requires a highly reductive environment, as represented by negative values for oxidation-reduction potential (ORP), which can be maintained through the addition of reducing agents such as ZVI, or to a lesser extent, the fermentation of added substrates such as lactate. Microcosm conditions represented distance from an in-situ treatment injection well and contained different types of iron species and dechlorinating bioaugmentation cultures. Diminishing efficacy of microbial reductive dechlorination along a gradient away from the injection zone was observed, characterized by increasing ORP and decreasing pH. Results also suggested that the use of particular biostimulation substrates is key to prioritizing the dechlorination reaction against competing microbial and abiotic processes by supplying electrons needed for microbial dechlorination.
ContributorsMouti, Aatikah (Author) / Krajmalnik-Brown, Rosa (Thesis director) / Delgado, Anca (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-12
Description
Cancer is one of the leading causes of death globally according to the World Health Organization. Although improved treatments and early diagnoses have reduced cancer related mortalities, metastatic disease remains a major clinical challenge. The local tumor microenvironment plays a significant role in cancer metastasis, where tumor cells respond and adapt to a plethora of biochemical and biophysical signals from stromal cells and extracellular matrix (ECM) proteins. Due to these complexities, there is a critical need to understand molecular mechanisms underlying cancer metastasis to facilitate the discovery of more effective therapies. In the past few years, the integration of advanced biomaterials and microengineering approaches has initiated the development of innovative platform technologies for cancer research. These technologies enable the creation of biomimetic in vitro models with physiologically relevant (i.e. in vivo-like) characteristics to conduct studies ranging from fundamental cancer biology to high-throughput drug screening. In this review article, we discuss the biological significance of each step of the metastatic cascade and provide a broad overview on recent progress to recapitulate these stages using advanced biomaterials and microengineered technologies. In each section, we will highlight the advantages and shortcomings of each approach and provide our perspectives on future directions.
ContributorsPeela, Nitish (Author) / Nikkhah, Mehdi (Thesis director) / LaBaer, Joshua (Committee member) / Harrington Bioengineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
Hydrogen is a key indicator of microbial activity in soils/sediments and groundwater because of its role as an electron donor for reducing sulfate and nitrate and carrying out other metabolic processes. The goal of this study was to quantitatively measure the total biological hydrogen demand (TBHD) of soils and sediments in anaerobic environments. We define the total biological hydrogen demand as the sum of all electron acceptors that can be used by hydrogen-oxidizing microorganisms. Three sets of anaerobic microcosms were set up with different soils/sediments, named Carolina, Garden, and ASM. The microcosms included 25g of soil/sediment and 75 mL of anaerobic medium. 10 mL of hydrogen were pulse-fed for 100 days. Hydrogen consumption and methane production were tracked using gas chromatography. Chemical analysis of each soil was performed at the beginning of the experiment to determine the concentration of electron acceptors in the soils/sediments, including nitrate, sulfate, iron and bicarbonate. An analysis of the microbial community was done at t = 0 and at the end of the 100 days to examine changes in the microbial community due to the metabolic processes occurring as hydrogen was consumed. Carolina consumed 9810 43 mol of hydrogen and produced 19,572 2075 mol of methane. Garden consumed 4006 33 mol of hydrogen and produced 7,239 543 mol of methane. Lastly, ASM consumed 1557 84 mol of hydrogen and produced 1,325 715 mol of methane. I conclude that the concentration of bicarbonate initially present in the soil had the most influence over the hydrogen demand and microbial community enrichment. To improve this research, I recommend that future studies include a chemical analysis of final soil geochemistry conditions, as this will provide with a better idea of what pathway the hydrogen is taking in each soil.
ContributorsLuna Aguero, Marisol (Author) / Krajmalnik-Brown, Rosa (Thesis director) / Delgado, Anca (Committee member) / Civil, Environmental and Sustainable Engineering Programs (Contributor) / School of Sustainability (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
Glioblastoma is the most aggressive and lethal brain tumor, due to its resistance to current conventional therapy. The resistance to chemo- and radiotherapy has been attributed to a special population of cells known as glioma stem cells. Previous literature has shown the importance of a Central Nervous System-restricted transcription factor OLIG2 in maintaining the tumor-propagating potential of these glioma stem cells. OLIG2's function was further elucidated, with its pro-mitogenic function due to its ability to negatively regulate the p53 pathway by suppressing the acetylation of the p53 protein's C terminal domain. Past work in our lab has confirmed that one of OLIG2's partner proteins is Histone Deacetylase 1 (HDAC1). In vitro experiments have also shown that targeting HDAC1 using hairpin RNA in glioma stem cells negatively impacts proliferation. In a survival study using a murine glioma model, targeting Hdac1 using hairpin RNA is shown to reduce tumor burden and increase survival. In this paper, we demonstrate that silencing Hdac1 expression reduces proliferation, increases cell death, likely a result of increased acetylation of p53. Olig2 expression levels seem to be unaffected in GSCs, demonstrating that the Hdac1 protein ablation is indeed lethal to GSCs. This work builds upon previously collected results, confirming that Hdac1 is a potential surrogate target for Olig2's pro-mitotic function in regulating the p53 pathway.
ContributorsLoo, Vincent You Wei (Author) / LaBaer, Joshua (Thesis director) / Mehta, Shwetal (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
CREB3L1 has been previously shown to auto-acetylate itself when prepared from HeLa cell based in vitro protein expression lysates. To circumvent the concerns of the contamination of co-purified human proteins from HeLa lysates, the protein was purified through insect cell transfection in vitro. The objective of this study was to assay the auto-acetylation activity of CREB3L1 prepared from insect cells using the baculovirus expression vector system (BEVS). To this end, His-tagged CREB3L1 was affinity purified from Hi5 cells using an IMAC column and used for acetylation assay. Samples were taken different time points and auto-acetylation was by western using antibodies specific to acetylated lysines. Auto-acetylation activity was observed after overnight incubation. Future experiments will focus on the improvement of purification yield and the identification of the substrates and interacting proteins of CREB3L1 to better understand the biological functions of this novel acetyltransferase.
ContributorsSchwab, Anna (Author) / LaBaer, Joshua (Thesis director) / Qiu, Ji (Committee member) / Barrett, The Honors College (Contributor)
Created2017-05
Description
A numerical study of wave-induced momentum transport across the tropopause in the presence of a stably stratified thin inversion layer is presented and discussed. This layer consists of a sharp increase in static stability within the tropopause. The wave propagation is modeled by numerically solving the Taylor-Goldstein equation, which governs the dynamics of internal waves in stably stratified shear flows. The waves are forced by a flow over a bell shaped mountain placed at the lower boundary of the domain. A perfectly radiating condition based on the group velocity of mountain waves is imposed at the top to avoid artificial wave reflection. A validation for the numerical method through comparisons with the corresponding analytical solutions will be provided. Then, the method is applied to more realistic profiles of the stability to study the impact of these profiles on wave propagation through the tropopause.
ContributorsCole, Alexandra Shea (Author) / Moustaoui, Mohamed (Thesis director) / Kostelich, Eric (Committee member) / Mahalov, Alex (Committee member) / School of International Letters and Cultures (Contributor) / School of Geographical Sciences and Urban Planning (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
Almost every form of cancer deregulates the expression and activity of anabolic glycosyltransferase (GT) enzymes, which incorporate particular monosaccharides in a donor acceptor as well as linkage- and anomer-specific manner to assemble complex and diverse glycans that significantly affect numerous cellular events, including tumorigenesis and metastasis. Because glycosylation is not template-driven, GT deregulation yields heterogeneous arrays of aberrant intact glycan products, some in undetectable quantities in clinical bio-fluids (e.g., blood plasma). Numerous glycan features (e.g., 6 sialylation, β-1,6-branching, and core fucosylation) stem from approximately 25 glycan “nodes:” unique linkage specific monosaccharides at particular glycan branch points that collectively confer distinguishing features upon glycan products. For each node, changes in normalized abundance (Figure 1) may serve as nearly 1:1 surrogate measure of activity for culpable GTs and may correlate with particular stages of carcinogenesis. Complementary to traditional top down glycomics, the novel bottom-up technique applied herein condenses each glycan node and feature into a single analytical signal, quantified by two GC-MS instruments: GCT (time-of-flight analyzer) and GCMSD (transmission quadrupole analyzers). Bottom-up analysis of stage 3 and 4 breast cancer cases revealed better overall precision for GCMSD yet comparable clinical performance of both GC MS instruments and identified two downregulated glycan nodes as excellent breast cancer biomarker candidates: t-Gal and 4,6-GlcNAc (ROC AUC ≈ 0.80, p < 0.05). Resulting from the activity of multiple GTs, t-Gal had the highest ROC AUC (0.88) and lowest ROC p‑value (0.001) among all analyzed nodes. Representing core-fucosylation, glycan node 4,6-GlcNAc is a nearly 1:1 molecular surrogate for the activity of α-(1,6)-fucosyltransferase—a potential target for cancer therapy. To validate these results, future projects can analyze larger sample sets, find correlations between breast cancer stage and changes in t-Gal and 4,6-GlcNAc levels, gauge the specificity of these nodes for breast cancer and their potential role in other cancer types, and develop clinical tests for reliable breast cancer diagnosis and treatment monitoring based on t-Gal and 4,6-GlcNAc.
ContributorsZaare, Sahba (Author) / Borges, Chad (Thesis director) / LaBaer, Joshua (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05