Molecular and Cellular Biology Masters Theses

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  • Publication
    The Effect of TGF-Beta and FGF2 on Endothelial Cells and Breast to Brain Metastasis
    (2024-05) Panagiotou, Alexi A.
    Metastasis, the spread of cancer cells from the initial, or primary, site to another region of the body, accounts for 90% of cancer-related mortalities1. Of the secondary sites of metastasis, the brain is one of the hardest to treat. Treatments are complicated because of the blood-brain barrier (BBB) and the sensitivity of the brain. Our team builds upon the seed and soil hypothesis which suggests that metastasis is not random and is driven by secondary tissue remodeling in response to signals from the primary tumor which “prepare” the tissue for the eventual arrival of cancer cells. This “preparation” of the secondary site is often referred to as a pre-metastatic niche. In the brain extracellular matrix (ECM), there are many proteins that play an important role in the pre-metastatic niche and are upregulated to support breast cancer metastasis. The focus of thisproject is on transforming growth factor beta (TGF-β) and fibroblast growth factor 2 (FGF2). In literature, TGF-β and FGF2 are two growth factors that are highly discussed as important upregulated proteins to support breast to brain metastasis2,3. FGF2 supports proliferation of the disseminated tumor cells at the secondary site through angiogenesis4. TGF-β promotes endothelial to mesenchymal transition (EndMT) in endothelial cells which decreases the tight junctions in the BBB and allows for tumor cells to more easily extravasate into the brain ECM5. Despite these two proteins being heavily researched, what is not discussed as much, is how they affect each other and how this could affect the pre-metastatic niche. In endothelial cells, when FGF2 binds to its receptor, it increases miRNA-20a expression which inhibits TGF-β receptors6. I hypothesize that their interactions are important to the pre-metastatic niche by altering the BBB. By designing and applying a brain specific microvascular niche assay, I can analyze how the timing of addition for these two growth factors affects the endothelial cells, astrocytes, and the corresponding niche. Understanding brain metastasis and how circulating tumor cells extravasate through the BBB can give rise to new therapies for treating brain metastases.
  • Publication
    Exploration of the Association between Muscle Volume and Bone Geometry Reveals Surprising Relationship at the Genetic Level
    (2020-09) Subba, Prakrit
    The evolution of jaws in cichlid fishes of the East African Great Lakes is a textbook example of adaptive radiation in vertebrates. Karl Liem postulated that this adaptive radiation has been possible due to the functional decoupling of two cichlid functional units – the pharyngeal jaw (PJ) and the oral jaw (OJ). This functional decoupling of the jaws has enabled the OJ to be relieved of its dual role of prey capturing and processing and has allowed the PJ to take on the role of prey processing. As a result, African cichlids have adapted the morphology of their functional units (i.e., oral jaws) to specialize in a feeding mechanism best suited for their habitat. However, global morphological changes (across the OJ and PJ) are understudied, especially, at the genetic level. In this study, two rock dwelling species of African Cichlids from Lake Malawi were used - Labeotropheus fuelleborni (LF) and Tropheops “red cheek” (TRC). Both species have distinct craniofacial morphologies for specialized benthic feeding (LF) and for generalized feeding (TRC). This morphological variation allowed us to investigate the functional decoupling of the jaws by studying differences in bone shape and muscle volumes in an F5 hybrid population. Strong phenotypic correlations were observed between and within the tissues of the OJ and PJ. Further, to identify phenotype to genotype associations, a Quantitative Locus (QTL) analysis and a fine mapping analysis was conducted. The results show some evidence of overlapping genetic control (i.e., pleiotropy) suggesting some genetic coupling between the two jaws.
  • Publication
    The Association Between Sperm DNA Methylation and Sperm Mitochondrial DNA Copy Number
    (2020-05) Houle, Emily
    Background: Infertility has become a growing concern across the world as cases continue to increase each year. Research has now shifted to identifying novel biomarkers to predict male fertility. While mtDNAcn has recently been found to show promising results as potential biomarker, its regulation remains unclear. Method: Triplex probe-based PCR was used to quantify mtDNA levels, while 850K Array was used to measure methylation levels. A-clustering algorithm followed by generalized estimating equations (GEE) lead to clustering of individual CpG sites, containing a minimum of 2 CpGs within 1000 base pairs of each other. These clusters were used for analysis of the association between mtDNAcn and DNA methylation within sperm. Metascape1 was used to annotate gene ontology terms. Result: Generalized estimating equation model analysis produced 6,038 FDR significant (q Conclusion: Thus, we show that sperm mtDNAcn is strongly associated with sperm DNA methylation and the associated implicates mtDNAcn as an influence on infertility.
  • Publication
    Sperm Mitochondrial DNA Biomarkers as a Measure of Male Fecundity and Overall Sperm Quality
    (2020-05) Rosati, Allyson
    Introduction. Sperm parameter analysis is the standard method of male fecundity testing; however, minimal evidence supports associations between individual sperm parameters and reproductive outcomes. Our previous work shows strong associations between sperm mitochondrial DNA copy number (mtDNAcn) and time-to-pregnancy (TTP) in general populations, and between mtDNAcn and fertilization outcomes in clinical populations. Thus it is possible for sperm mtDNA biomarkers to act as summary measures of semen quality. In this study, we developed a sperm quality index (SQI) from semen parameters and compared its ability to measure fecundity to sperm mtDNAcn. Methods. We received 384 semen samples from the Longitudinal Investigation of Fertility in the Environment Study. Sperm mtDNAcn and mtDNA deletions (mtDNAdel) were quantified using a triplex probe-based qPCR method. The SQI was developed by ranking and summing select sperm parameters within the study population, including sperm concentration, sperm count, normal morphology, high DNA stainability, and DNA fragmentation to create a cumulative index. Discrete-time proportional hazards models were used to determine fecundability odds ratios (FOR), indicating associations between mtDNAcn, SQI, and TTP. Receiver operating characteristic (ROC) analyses determined the validity of the SQI and mtDNAcn as predictors of pregnancy within 12 months. Results. The SQI was highly associated with mtDNAcn, both continuously (Spearman Rho: -0.487; p-value: <0.001) and in deciles (ANOVA p-value: <0.001). The SQI (FOR: 1.25; 95% confidence interval (CI): 1.09, 1.43) and mtDNAcn (FOR: 0.754; 95% CI: 0.657, 0.866) performed similarly in discrete-time survival models and indicated a significant decrease and increase in TTP, respectively. MtDNAcn more effectively predicted pregnancy within 12 months (AUC: 0.703; 95% CI: 0.617, 0.789) than the SQI (AUC: 0.642; 95% CI: 0.531, 0.753). With multiple predictors, mtDNAcn outperformed summary models, with addition of the SQI and percent normal morphology minimally increasing model efficacy (AUC: 0.718, 95% CI: 0.617, 0.819). Conclusion. The association between the SQI and mtDNAcn suggest that mtDNAcn may serve as a summary biomarker for overall sperm quality. Neither individual nor summed sperm parameters are useful indicators of couple fecundity and reproductive outcomes compared to mtDNAcn. These results suggest that mtDNAcn has potential for use as a biomarker of fecundity.
  • Publication
    Gene Expression Regulation in the Mouse Liver by Mechanistic Target Of Rapamycin Complexes I and II
    (2020-05) Poluyanoff, Anthony
    The mechanistic target of rapamycin (mTOR) is a key serine/threonine protein kinase that functions in complexes mTORC1 and mTORC2. mTORC1, originally discovered due to its sensitivity towards the mTOR inhibitor rapamycin, responds to extracellular growth factor signaling, WNT signaling, and nutrient abundance via glucose and amino acid-triggered signaling. Downstream effectors of mTORC1 include autophagy, mitochondrial metabolic function, protein synthesis, and ribosome biogenesis. mTORC2, initially discovered as a rapamycin-insensitive complex of mTOR, responds to insulin, growth factor signaling, and inflammatory signaling such as tumor necrosis factor-alpha, with its downstream effectors being Akt, a key serine/threonine kinase that functions in cell division and is frequently dysregulated in many types of cancer, the NFkB pathway, and cytoskeletal reorganization and protein synthesis. Much research has been devoted to mTORC1 signaling, with mTORC2 receiving significantly less attention, despite both complexes’ regulation of key cellular activities and response to rapamycin, as well as to other rapamycin-derived drugs (rapalogs). We have targeted both mTORC1 and mTORC2 for hepatocyte-specific deletion during the gestational period of mice, with the goal of describing mTORC1 and mTORC2 signaling and its perturbation in the adult mouse hepatocyte. Our model has shown that deletion of RAPTOR, the regulatory associated protein of mTOR, and RICTOR, the rapamycin insensitive component of mTOR, in mTORC1 and mTORC2 respectively, leads to widespread effects on the hepatocyte transcriptome. We have found that a subset of genes responds both to Raptor and Rictor knockout, and an analysis of these genes indicates their function in key disorders of the liver, such as non-alcoholic fatty liver disease and hepatocellular carcinoma. Bioinformatic analysis following hepatocyte RNA sequencing of mTORC1 and mTORC2 knockout mice has revealed an unexpected upregulation of genes known to be regulated by these respective complexes. We have also found that cross talk exists between both complexes, in which the knockout of one yields the activation of the other. We have additionally found translationally relevant enrichments following Ingenuity Pathway Analysis (IPA) of RNA sequencing data. These results provide a key mechanistic discovery of mTOR signaling activity, and allow for a better understanding of the potential physiological effects of mTOR inhibition in human patients.
  • Publication
    Investigating The Role Of LBH During Early Embryonic Development In Xenopus Laevis
    (2019-09) Weir, Emma
    LBH is a highly conserved protein whose role during vertebrate development is relatively under-studied. In collaboration with the Albertson lab, our lab has previously shown that it is necessary for cranial neural crest cell migration in the zebrafish and in Xenopus laevis. The molecular mechanisms through which it acts are not well understood. In Xenopus, LBH is a maternally deposited protein. As such, studying its role in early development has not been feasible through the morpholino-mediated knockdown techniques that prevent translation of target genes. Recently, a technique for degrading endogenous proteins was developed, called Trim-Away. This was developed in mammalian systems and utilizes the E3 ubiquitin ligase Trim21 in conjunction with an antibody against a protein of interest in order to degrade the protein. In order to observe the effects of a knockdown of LBH during early embryonic development, we sought to modify the technique for use in Xenopus. We injected embryos with mRNA encoding the human form of trim21 along with a monoclonal antibody against LBH that our lab developed (2B8) and tracked degradation of the protein over time, monitoring embryos for any phenotypes arising during early development. Our results demonstrate that Trim-Away can be utilized in Xenopus. LBH depleted embryos display a variety of defects during gastrulation, the process by which the three germ layers are properly organized. These appear to be mainly due to defects in fibronectin fibrillogenesis and mesodermal migration.
  • Publication
    Studies on the Interaction and Organization of Bacterial Proteins on Membranes
    (2019-05) Brena, Mariana
    Bacteria have developed various means of secreting proteins that can enter the host cell membrane. In this work I focus on two systems: cholesterol-dependent cytolysins and Type III Secretion. Cholesterol is a molecule that is critical for physiological processes and cell membrane function. Not only can improper regulation lead to disease, but also the role cholesterol plays in cell function indicates it is an important molecule to understand. In response to this need, probes have been developed that detect cholesterol molecules in membranes. However, it has been recently shown that there is a need for probes that only respond to cholesterol that is accessible at the membrane surface. Perfringolysin O (PFO) is a toxin secreted by Clostridium perfringens that has been developed into a probe capable of detecting accessible cholesterol. Recently, researchers have been expanding the capabilities of this probe by substituting residues, modifying residues, truncating the probe, or a combination of the three. However, lack of characterization of these new probes has led to controversial results. To understand the role of a conserved Cys residue, here we perform cholesterol binding assays and measure the pore formation activity of a Cys modified PFO derivative. The Type III Secretion (T3S) system is a syringe-like apparatus used by various pathogens to inject effector proteins into target cells. The apparatus spans both the inner and outer bacterial membrane, extending to make contact with the host cell where it forms a pore known as the translocon. In Pseudomonas aeruginosa, the translocon is made up of two proteins, PopB and PopD. While recent advances have been made on the structure of the needle and injectisome, information on the translocon remains sparse. In this work, the P. aeruginosa T3S translocon is analyzed using both in vivo and in vitro methods.
  • Publication
    Exploring the Influence of PKC-theta Phosphorylation on Notch1 Activation and T Helper Cell Differentiation
    (2018-09) Trombley, Grace
    The T cell-specific kinase, Protein Kinase C theta (PKCq) is essential to T cell activation and differentiation. PKCq integrates T cell receptor (TCR) and CD28 signaling, and ultimately activates transcription factors necessary for full T cell activation, proliferation, survival, and differentiation into T helper (Th) subsets. Th1, Th2, Th17 and Treg cells compose the four major lineages of T helper cells, differentiated from CD4 T cells, and each have different requirements for PKCq. PKCq, itself, is regulated through phosphorylation of specific resides, including tyrosine (Y)90 and threonine (T)538. Following T cell stimulation, PKCq is phosphorylated on Y90 by the kinase, LCK, and translocates to the cell membrane. There it remains associated with LCK in a structural complex known as the immunological synapse. Loss of PKCq in T cells produces a phenotype that is similar to loss of another important T cell protein, Notch1, suggesting these two proteins may function in the same signaling pathway. Our lab has shown that PKCq can interact with Notch1, but how this interaction regulates Notch1 function is not known. Due to the strong overlap between cellular functions regulated by PKCq and Notch1, understanding how these two proteins might function, cooperatively, can provide better insight into autoimmune diseases and may be useful in developing novel therapies We hypothesized that phosphorylation of a specific residue of PKCq (T538) is required for Notch1 cleavage and nuclear translocation. We also hypothesize that the phosphorylation status of PKCq (T538) will influence the ability of T cells to differentiate into specific T helper subsets. We used two means of inhibiting PKCq function to evaluate its regulation of Notch1 in differentiated T cells: 1) we blocked the association of PKCq and LCK, thereby preventing its movement to the immunological synapse and 2) we prevented PKCq phosphorylation on T538. We found that by preventing the phosphorylation of PKCq, we also decreased the level of cleaved Notch1 in Th1, Th2, and Th17 cells. We further determined that inhibiting PKCq decreased the amount of Notch1 that translocated to the nucleus in Th1 and iTreg cells. Understanding how PKCq interacts with and regulates Notch1 to influence T cell differentiation may lay the foundation for specifically modulating T cell responses.
  • Publication
    Characterization of Calcium Homeostasis Parameters in TRPV3 and CaV3.2 Double Null Mice
    (2017-09) Mehregan, Aujan
    In mammals, calcium influx is required for oocyte maturation and egg activation, as it supports the persistent calcium oscillations induced by fertilization. These oscillations are required for the initiation of embryo development. The molecular identities of the plasma membrane calcium-permeant channels that underlie calcium influx are not established. Among these channels, Transient Receptor Potential Vanilloid, member 3 (TRPV3) allows divalent cations, namely strontium (Sr2+) and calcium (Ca2+) with high permeability, into cells, and its expression pattern seems to predict an essential role in the initiation of development. Another channel that was identified to be expressed in oocytes/eggs is the low-voltage-activated T-type channel, CaV3.2. However, the ability to accurately probe the expression and function of these channels on Ca2+ homeostasis in mouse eggs is hindered by the lack of specific and known pharmacological agents and antibodies for these channels. Here, we simultaneously knockout out these two Ca2+ influx channels in the mouse to explore the effects on Ca2+ homeostasis. We examined fertility rates, development, and morphological defects that arose from the double null pups. Next, we investigated the consequences on Ca2+ store content in immature and mature oocytes and eggs. We also examined the effects on fertilization-induced Ca2+ oscillations in response to in vitro fertilization and PLCz cRNA microinjection. We found that female mice null for these channels display drastic subfertility compared to the single knockout mice for these channels. Additionally, the Ca2+ store content is significantly diminished in double knockout eggs versus controls, as was the frequency of the fertilization-induced Ca2+ oscillations. These results suggest that these channels play a crucial role in Ca2+ influx during maturation and contribute to maintain Ca2+ oscillations post-fertilization. These null oocytes and eggs will be an important tool to perform electrophysiological studies to accurately measure the native current(s) of a specific channel(s) in eggs, and to identify the channel(s) that mediate Ca2+ during fertilization.
  • Publication
    Regulation of Katanin Activity on Microtubules
    (2017-09) Tyler, Madison A.
    The cytoskeleton is a dynamic network of microtubules constantly being reorganized to meet the spatiotemporal demands of the cell. Microtubules are organized into subcellular highways to control cell processes such as cell division, cargo transport, and neuronal development and maintenance. Reorganization of this intricate network is tightly regulated by various stabilizing and destabilizing microtubule-associated proteins that decorate the network. Katanin p60 is a microtubule destabilizing enzyme from the ATPases Associated with various Activities (AAA+) family. It can both sever and depolymerize microtubules. In order to sever microtubules, katanin recognizes the tubulin carboxy-terminal tails (CTTs) and hydrolyzes ATP. Using super-resolution microscopy and image analysis, we find that the tubulin CTTs are not required for katanin to depolymerize microtubules. We also characterize the regulation of microtubule severing and depolymerization by katanin in various nucleotide states. A better understanding of how CTTs and nucleotides regulate microtubule severing and depolymerization by katanin will help future research aimed to correct katanin activity when these processes goes awry as in improper chromosome segregation during mitosis or loss of microtubule integrity in neuronal diseases.
  • Publication
    Partial Craniofacial Cartilage Rescue in ace/fgf8 Mutants from Compensatory Signaling From the Ventricle of Danio Rerio
    (2017-09) Calenda, Douglas A
    Examples of asymmetric organs are found throughout the animal kingdom. Whether it is superficial like the fiddler crab’s claw or within an organism like our visceral organs, asymmetries have repeatedly evolved in nature. However, the genetic and developmental origins for asymmetric organ development remain unclear, especially for superficially paired structures. Within zebrafish, a striking example of asymmetry occurs within the ace/fgf8 mutant. The pharyngeal cartilages of these mutants develop asymmetrically 35% of the time, with more cartilages developing on the left or right side of the head, but the origins of this asymmetry are unknown. A significant proportion of mutants also exhibit situs inversus, whereby the visceral organs develop on the opposite side of the body. Here we seek to understand the temporal window most sensitive to giving rise to this asymmetry, and to understand if there is a correlation between the developing heart field and pharyngeal cartilage with respect to the direction of the asymmetry. Wild type (WT) zebrafish were exposed to SU5402 during different periods of development, and heart position as well as cartilage development was observed within the developing larvae. The direction of asymmetry (i.e., left or right biased) was also recorded in ace/fgf8 mutant heart position and cartilage number to observe if there was a correlation between the two developing fields. SU5402 experiments revealed that the time window most sensitive to the development of cartilage asymmetries was during heart looping and pharyngeal arch segmentation. Furthermore, ace/fgf8 mutants exhibited a robust correlation between ventricle position and the side of cartilage asymmetry, with more cartilages forming on the side where the ventricle is located. Given the close proximity of the heart and pharyngeal cartilage fields we suggest that the heart field is influencing the developing cartilage, with signaling permeating from the developing heart to the pharyngeal mesoderm to provide a buffer on the side of the developing ventricle.
  • Publication
    Microtransplantation of Rat Brain Neurolemma into Xenopus Laevis Oocytes to Study the Effect of Environmental Toxicants on Endogenous Voltage-Sensitive Ion Channels
    (2017-05) Murenzi, Edwin
    Microtransplantation of mammalian neurolemma into Xenopus laevis oocytes has been used to study ion channels in terms of their structure and function in the central nervous system. Use of microtransplanted neurolemma is advantageous in that tissue can be obtained from various sources, ion channels and receptors are present in their native configuration and they can be used to evaluate numerous channelpathies caused by environmental toxicants. Here we show that Xenopus oocytes injected with fragments of rat brain neurolemma successfully express functional native ion channels that are assembled in their own plasma membrane. Using a high throughput two electrode voltage clamp (TEVC) electrophysiological system, currents that were sensitive to tetrodotoxin (TTX), omega-conotoxin MVIIC, and tetraethylammonium (TEA) were detected, indicating the presence of multiple voltage-sensitive ion channels (voltage-sensitive sodium, calcium and potassium channels, respectively). In this current research, a “proof-of-principle” experiment was conducted where TTX-sensitive voltage-sensitive sodium channel (VSSC) currents were measured. VSSCs are a well-established site of action for 1,1,1-trichloro-2,2-di(4-chlorophenyl)ethane (DDT) but not for its non-toxic metabolite 1,1-bis-(4-chlorophenyl)-2,2-dichloroethene (DDE). A differential sensitivity of DDT versus DDE on TTX-sensitive sodium current in neurolemma-injected oocytes was determined. DDT elicited an increase in depolarization-dependent, TTX-sensitive sodium current while DDE had no significant effect. Additionally, DDT resulted in a slowing of sodium channel inactivation kinetics whereas DDE has no similar effect. These results are consistent with the findings obtained using heterologous expression of single isoforms of rat brain VSSCs by injecting cRNA into Xenopus oocytes. By demonstrating the classic structural activity relationship of DDT and DDE on mammalian voltage-gated sodium channels isolated in rat brain neurolemma, this study supports the use of automated high-throughput electrophysiology to study the effects of various environmental toxicants on multiple mammalian cellular targets. More importantly, using rat brain neurolemma ensures that the proteins of interest have been transcribed and have undergone all the necessary post-translational modifications before they were injected and expressed in the Xenopus oocytes which is not the case for traditional heterologous expression.
  • Publication
    THE FAR C-TERMINUS OF TPX2 CONTRIBUTES TO SPINDLE MORPHOGENESIS
    (2017-02) Estes, Brett
    A cell must build a bipolar mitotic spindle in order to faithfully segregate replicated DNA. To do so, multiple microtubule nucleation pathways are utilized to generate the robust spindle apparatus. TPX2, a microtubule binding protein, holds crucial roles in both the Ran-dependent and Augmin-dependent pathways where microtubules are nucleated near the chromosomes and from pre-existing microtubules. However, the exact role TPX2 plays in branching microtubules is less understood. Here, we explored the effect of truncating the essential TPX2 C-terminal 37 amino acids on Augmin localization and branching microtubule activity. First, we depleted LLC-Pk1 cells of the Augmin subunit HAUS6 and show that microtubule nucleation around the chromosomes following a nocodazole washout is strongly reduced leading to exaggerated kinetochore microtubule growth. Next, we depleted endogenous TPX2 in LLC-Pk1 cells harboring full length or truncated TPX2 bacterial artificial chromosome (BAC) DNA. Results show that TPX2 710 LAP cells have reduced Augmin localization on the spindle fibers, which correlates with reduced microtubule regrowth in the chromosomal region. In TPX2 710 LAP cells, regrowth was like Augmin depleted cells. Therefore, we provide evidence that the far C-terminus of TPX2 is required for branching microtubule nucleation and that kinetochore microtubule growth is Augmin-independent. In addition, we investigated cell cycle regulation of TPX2 by mutating the S738 phosphosite in the C-terminal motor interacting region. We utilized BAC recombineering to create phospho-mimetic and phospho-null mutants. In combination with plasmid DNA knockdown/rescue, overexpression and spindle assembly assays, we show that the phosphorylation of the C-terminal domain contributes to early mitotic events. LLC-Pk1 cells showed a significant increase in aberrant spindle morphology and reduced spindle stability in the presence of 738A and absence of endogenous TPX2. While rescue with the alanine mutant caused in an increase in multipolar spindles, overexpression resulted in a strong dominant negative monopolar phenotype. Therefore, S738 appears to contribute to mitotic force regulation during mitosis. In conclusion, the far C-terminus of TPX2 and its regulation play a role in the formation of a proper mitotic spindle.
  • Publication
    Modulation of Notch in an Animal Model of Multiple Sclerosis
    (2017-09) Munshi, Manit Nikhil
    Multiple Sclerosis (MS) is a neurodegenerative autoimmune disease that affects millions of people worldwide. Although the exact cause of MS is unknown, it is clear that CD4+ T helper cells play a significant role, namely T helper 1 (Th1) and T helper 17 (Th17) cells. The Notch family of proteins plays a role in the development and differentiation of T helper cells. Previous data has shown that inhibition of Notch impairs the ability of T helper cell differentiation. Additionally specific inhibition of certain Notch members inhibits specific T helper cell differentiation, for example the inhibition of Notch 1 inhibits Th1 and iTreg polarization [Samon et al., 2008]. However, the effects of the other Notch family members on CD4+ T cells are not fully studied. We propose that Notch 3 plays an extensive role in the regulation of Th1, Th2, Th17, and iTreg polarizations. In addition, we propose that Notch 3 regulates function of T helper cell function in the mouse model of MS, experimental autoimmune encephalomyelitis (EAE). Data in this thesis show that Notch 3 plays a significant role in the polarization of Th1, Th17 and iTreg polarization [Karlsson et al., 2011]. We present evidence that the heterozygous and homozygous Notch 3 knockout exhibits a significant decrease in polarization toward Th1, Th17 and iTreg cell fates. Exopolysaccharide (EPS) is a compound that has been previously shown to play a protective role in other inflammatory diseases. EPS has been shown to produce anti-inflammatory macrophages. We propose that a similar anti-inflammatory effect might be possible in EAE. We found that EPS had a significant effect on EAE induction, decreasing the onset and peak disease score. EPS also reduced the concentration of IFN-γ, IL17A, and GM-CSF in the supernatants of the splenocytes after restimulation with MOG. Further experimental data is needed to prove the effects of EPS on EAE and the method by which EPS function. These data indicate that Notch 3 could be crucial in regards to EAE due to the effects on Th1 and Th17 which are instrumental in EAE induction [Raphael et al., 2015].
  • Publication
    UNDERSTANDING THE FUNCTIONAL IMPACT OF DISEASE-ASSOCIATED PHOSPHORYLATION SITES ON THE NEURODEGENERATIVE PROTEIN TAU
    (2024-02) Sebastian, Navya T
    Microtubule Associated Protein Tau (Tau) is found intracellularly along the axons of neurons in the central nervous system where they stabilize the microtubules by binding along its lattice. In disease, Tau becomes hyperphosphorylated, dissociates from the microtubule, oligomerizes, and eventually forms neurofibrillary tangles (NFT). These NFTs then spread in a prion-like manner starting from the entorhinal cortex to the frontal lobe, as it does in Alzheimer’s Disease (AD). AD is the most common form of dementia, characterized by the decline of cognition and function. Since the 1980’s the field has shown that post-mortem brains of AD patients have ‘hyperphosphorylated’ Tau tangles and the working model suggests that hyperphosphorylation is the cause for dissociation and thus aggregation. However, the definition of hyperphosphorylated Tau remains ambiguous. Previous findings show specific sites of Tau modification being significant in disease. Mass spectrometric data further corroborates these findings by indicating specific sites of phosphorylation with high frequency and extent modification, including epitopes AT100 (T212, S214, T217), 12E8 (T262, S263), and PHF1 (S396, S400, T403, S404). In this study, I aim to show that the term ‘hyperphosphorylation’ is misleading as it is truly the aberrant phosphorylation of Tau at specific sites that increase aggregation propensity in disease. I will do this by studying co-factor free aggregation kinetics using a Thioflavin T probe with different Tau constructs modified at specific sites of interest. These results will provide a new perspective into the Tau hypothesis of AD and serve to update AD therapeutics.
  • Publication
    Chromatin Accessibility Impacts Knockout of Mt-Bell4 Transcription Factor
    (2024-02) Redden, Thomas
    Medicago truncatula is a model legume plant that is used extensively in research to study nitrogen fixation in plants. Among the techniques used to study this interaction, CRISPR-Cas9 mutagenesis is a recent advancement that has allowed researchers to experiment with the genome more easily and efficiently. Even with this new technology, issues still arise where the designed gRNAs often seem to suffer from low mutagenesis rate. In this study, I aim to uncover whether chromatin accessibility plays a role in gRNA cleavage success. To this end, I tested this hypothesis on a novel gene, Mt-Bell4, which is an evolutionarily conserved transcription factor proposed to function during nodulation across many species. The findings in this study showed that there appears a higher likelihood of successful large genomic deletions if regions of open chromatin are utilized as gRNA sites. Additionally, I showed that the Mt-Bell4 gene is highly expressed in nodule tissue, with its function possibly linked to many other transcription factors. The findings of this study highlight a new metric to consider when choosing gRNA sites, which can be generally applicable to engineering genomes whose chromatin accessibility is known. Additionally, understanding the function of Mt-Bell4 can show a new potential target to understand legume nitrogen fixation.
  • Publication
    Investigating the Role of Got2 in Murine Organogenesis and Placenta Development
    (2024-02) Macrorie, Olivia
    According to the Knockout Mouse Project (KOMP), loss of glutamatic oxaloacetic transaminase 2 (Got2) results in lethality between embryonic day (E) 9.5 and E12.5, which is a critical period for organ and placenta development. The first aim of this project is to characterize the KOMP-generated Got2 null allele. In situ hybridization reveals that while Got2 is ubiquitous throughout much of the embryo, it is highly expressed in the liver and throughout the developing labyrinth layer of the placenta by E9.5. While Got2-/- embryos are lethal at E10.5, at E9.5 Got2-deficient embryos have pharyngeal arch defects, do not turn properly, and have oedemic hearts. Because of the widespread and high levels of Got2 in the placenta and its putative role in energy production, we hypothesized that the embryonic lethality observed was caused by placenta dysfunction. The second aim of this project is to test the hypothesis, using a conditional Got2 allele, which phenocopies the original null alleles when induced to make a full null allele, to create an epiblast-only conditional knockout (cKO). Compared to heterozygous littermates cKO Got-/- embryos are slightly delayed, but otherwise grossly normal at E9.5, are pale and smaller than littermates at E11.5 and are smaller and markedly reduced or absent liver at E12.5. Histological and molecular analysis demonstrates that the cKO embryos, like the null embryos, have a normal liver bud at E9.5. By E10.5 the cKO livers are highly reduced. Mass spectrometry of single intact E10.5 cKO embryos demonstrated that their metabolism exhibits significant changes relative to controls, with alterations in malate aspartate shuttle metabolites, as well as pyruvate and pyrimidine and purine precursor metabolites. These results suggest that placental expression of Got2 is critical for embryo development between E8.5 to E10.5 and that embryonic expression of Got2 is conspicuously required for embryonic liver development by E10.5.
  • Publication
    The Impact of a Non-ionic Adjuvant to the Persistence of Pesticides on Produce Surfaces
    (2024-02) Barnes, Daniel
    Adjuvants can enhance the performance of the pesticide active ingredients in many ways including decreasing surface tension and reducing evaporation. Understanding how adjuvants effect pesticide behavior (e.g., surface persistence) is crucial for developing effective pesticide formulations, as well as facilitating the development of effective approaches to reduce pesticide residues from the surface of fresh produce post-harvest. The objective of this study is to investigate the effect of a non-ionic surfactant, Surf-Ac 910, on the persistence of two model pesticides, thiabendazole and phosmet on apple surfaces. The result shows that the addition of Surf-Ac 910 increased both the maximum wetted area and evaporation rate of thiabendazole, a systemic pesticide, and phosmet, a non-systemic pesticide. Utilizing surface-enhanced Raman spectroscopy to explore the surface and penetrative behaviors of thiabendazole and phosmet revealed that the addition of Surf-Ac 910 influenced the Raman signal of pesticides as well. The addition of Surf-Ac 910 decreased the Raman signal intensity when added to phosmet but did not affect the Raman signal intensity when added to thiabendazole. In terms of penetration, the addition of Surf-Ac 910 did not affect the penetration depth of phosmet but slightly increased the penetration depth of thiabendazole. These findings were true for both short-term, 40 minutes, and long-term, 3 days, exposure. Next, the effects of adjuvants on the removal of pesticide residues were investigated. Common household materials, such as baking soda, were effective at removing surface pesticide residues. After testing a variety of baking soda concentrations and starch granules, 2% baking soda and 2% corn starch were found to be the most effective baking soda concentration and starch granule respectively. 2% corn starch was the most effective removal method overall, with 99% of pesticide with/without adjuvant removed in just 5 minutes of wash time. Overall, this study demonstrated that although adjuvant Surf-Ac 910 could affect the surface persistence of pesticides, washing with common household materials such as 2% corn starch can be used as an effective, safe, and economic way to reduce pesticide exposure through fresh produce.
  • Publication
    Investigating Diterpene Biosynthesis in Medicago Truncatula
    (2023-09) Hwang, Sungwoo
    Terpenes are secondary metabolites produced by plants and they have promising roles in plant defense and pharmaceuticals. They are synthesized by terpene synthases and these enzymes are part of a complex plant metabolic pathway. Diterpene biosynthesis requires co-expression of class II and class I diterpene synthases (diTPSs) to convert geranylgeranyl diphosphate (GGPP), the common precursor, into a C20 intermediate substrate. These substrates then use cytochrome p450s (CYPs) as their final steps to form diterpene scaffolds. CYPs are monooxygenases that change the redox status of their substrates into final diterpene products. Medicago truncatula was used as my model organism to investigate how legumes synthesize these secondary metabolites to contribute to crop defense improvement in the future. Seven diTPSs - MtTPS17, MtTPS18, MtTPS19, MtTPS37, MtTPS38, MtTPS39, and MtTPS40 - in M. truncatula have been identified. MtTPS38 was found to produce ent-CPP and MtTPS37 used ent-CPP to yield ent-kaurene. Combinatorial expression showed that MtTPS38 and MtTPS37 react together to produce ent-kaurene, a precursor for an important plant hormone gibberellin (GA). CYPs have also been discovered to be clustered around MtTPS19, suggesting the possibility of MtTPS19 utilizing these CYPs for downstream reactions.
  • Publication
    The Discovery of a Novel Bacteria from a Large Co-assembly of Metagenomes
    (2023-09) Finkelberg, Matthew
    In the summer of 2022, a co-assembly of metagenome was created using the microbes found at Barres Woods in Harvard Forest. 14 samples were taken, and sample was split into the organic and mineral layer, which totals 28 Bulk MAGs. Within this Co-assembly, 4 different genomes were found which were designated with the phylum of FCPU426. Three of which were considered medium quality and one being assigned high quality. The novel phyla first appeared in NCBI and GTDB databases in June 2018. The name FCPU426 dates to 2010 and was named based on the 16s amplicon sequencing. The novel phylum is shown to have common ancestors with the phyla of Goldbacteria and Firestonebacteria. Goldbacteria was determined to have the ability to break down cellulose to further enhance carbon cycling. In this analysis of FCPU426, the taxonomy of the phyla will be further investigated. FCPU426 was then annotated to determine if any of the cellulase genes are shared between itself and Goldbacteria. Further annotations were done to spot any genes of interest that create any distinctions between any of its counterparts.