Browsing by Subject "Genetics, Cell Biology and Development"

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Correction of Mucopolysaccharidosis Type I (MPS I) with Multipotent Adult Progenitor Cells (MAPCs) in an Immunodeficient Mouse Model
(2010-04-21) Evavold, Carrie
Hurler Syndrome or Mucopolysaccharidosis I (MPS I) is an autosomal recessive lysosomal storage disease characterized by skeletal abnormalities, hepatosplenomegaly, and neurological degeneration. Children who exhibit MPS I lack α-L-iduronidase (IDUA), a crucial enzyme in the degradation pathway of glycosaminoglycans (GAGs), specifically heparan and dermatan sulfate. GAG accumulation in lysosomes interferes with normal cell function creating multi-systemic problems. Current treatments including enzyme replacement therapy and hematopoietic stem cell transplantation are not widely available and fail to correct the majority of symptoms, particularly mental retardation and skeletal anomalies. Using a NOD/SCID immunodeficient mouse model of MPS I, we attempted to correct the enzyme deficiency by transplanting human multipotent adult progenitor cells (MAPCs) directly into the striatum within five days of birth. Through the process of cross-correction, the IDUA enzyme released from the transplanted cells is taken up by the defective cells. The efficacy of the MAPCs was investigated through measurement of IDUA levels in different tissues, immunohistochemical staining, and sensorimotor testing. The transplanted MAPCs were detected throughout the central nervous system along with decreased levels of GAGs indicating sufficient delivery of IDUA to the cells. Sensorimotor coordination on a rotarod test improved in the MAPC-treated MPS I mice compared to untreated MPS I mice. These results denote that transplantation of MAPCs into the striatum greatly reduces GAG tissue levels in the brain and ameliorates sensorimotor function in MPS I mice.
Cyclophilin A – Antizyme Fusion as a Potential HIV-1 Restriction Factor
(2013-08-12) Williams, Brady;
During the HIV-1 retroviral life cycle, endogenous cellular proteins serve to both promote and inhibit the retroviral reproductive cycle. Cyclophilin A (CypA) is one such protein that binds the viral capsid. The interaction between CypA and the capsid, especially in the target cell, has been demonstrated to be essential for efficient infection and progression through the HIV-1 reproductive cycle. However, other native proteins inhibit HIV-1 at various stages of the reproductive cycle. For example, TRIM5α catalyzes premature and accelerated capsid uncoating thereby blocking reverse transcription in certain species. Tetherin inhibits budding by tethering the virion to the cell and preventing release. Other proteins exploit a combination of interactions to assist in degradation. TRIMCyp fusions in certain primate species contain a TRIM5α domain fused to a Cyclophilin A domain. The combination results in capsid binding, premature uncoating, and prevention of reverse transcription. Here we attempt to exploit a similar interaction in the creation of a novel HIV-1 restriction factor by fusing CypA to the protein Antizyme-1. Endogenously, Antizyme plays a role in regulating polyamine levels by regulating levels of ornithine decarboxylase (ODC) by mediating ODC degradation via the 26S proteasome. We hypothesized that the combination of the capsid binding CypA domain and the proteasome associated Antizyme domain would result in premature degradation of the viral capsid resulting in reduced viral titer and infectivity. In contrast to our hypothesis, we found that expression of the CypA-Antizyme fusion did not affect viral titer or viral infectivity when expressed in both target and producer cell lines due in part to proteasome mediated degradation of our fusion construct. Despite the negative result, our data suggests future modifications that could be made to our construct to help evade cellular degradation mechanisms and eventually test the full therapeutic potential of the fusion.
Deregulation of microRNA Processing in Idiopathic Pulmonary Fibrosis
(2015) Guenther, Kacey;
Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease with an incidence in the United States of 42.7 to 63 per 100,000 and an incidence in Europe of 16.3 to 17.4 per 100,000 (Nalysnyk et al. 2012). Incidence and severity of IPF increases with age, but on average projected survival is just 3-4 years post-diagnosis (Raghu et al. 2014). Until recently, treatment options have been limited and mostly palliative, with little impact on survival. For many years, the only established treatment shown to extend life was lung transplantation. However, lungs are the most difficult organs to transplant, with patients surviving an average of just 4.6 years post-transplantation (NIH 2014). Just recently, two drugs (pirfenidone and nintedanib) have been approved for treatment of IPF. However, even with such treatments the unyielding progression of IPF is only slowed, and a more effective treatment for IPF remains both necessary and illusive (as reviewed in Jenkins and Goodwin 2014).
Effects of Rapamycin on Dia2 Localization
(2012-02-15) Peter, Newhouse IX
Expression of Surfactant Protein-A (SP-A) in the Developing MurineIntestinal Tract
(2011-04-13) Theisen, Erin
Surfactant protein-A (SP-A) plays a critical role in the innate immune system and has well characterized effects in the lung where it attenuates inflammatory responses and controls invasion of bacteria. Extra-pulmonary sources of SP-A have also been indentified : SP-A mRNA has been detected in the murine neonatal and adult gastrointestinal (GI) tract, while significant levels of SP-A protein have been detected in amniotic fluid. A novel finding by the George research lab showed that SP-A knockout (-/-) newborn mice raised in a bacterial laden corn dust environment exhibited intestinal inflammation-- instead of pulmonary inflammation--and higher rates of death. Further studies have shown that SP-A -/- mice exhibit abnormal bacterial colonization patterns in the GI tract compared to their wild-type counterparts, indicating a role for SP-A in the newborn intestinal tract. To date it is not clear if newborn intestinal exposure to SP-A comes from ingested amniotic fluid or from production in the newborn intestinal tract. RT-qPCR showed low levels of SP-A gene expression in the newborn murine GI tract; yet, we and others have had mixed results regarding the detection of SP-A protein via immunohistochemistry. To address the question of intestinal exposure of SP-A in the newborn, I will perform RNA in situ hybridization to identify gene expression in specific cells of the GI tract. I have designed a 438 bp digoxigenin-labeled antisense RNA probe specific for the SP-A gene. This probe will be used on flash-frozen GI and lung tissue sections of mice at post-natal days of life 5 and 6 with an in situ hybridization protocol designed by the Panoskaltsis-Mortari laboratory.
Functions of NOM1 required for Cell Growth and Proliferation
(2011-04-13) Meyer, Melissa
Our lab has identified a protein called NOM1 that is highly conserved from yeast to humans and that we have shown is required for ribosome biogenesis. We have also demonstrated that decreased levels of NOM1 result in cells that are smaller than normal and that fail to proliferate. We hypthosize that NOM1 depletion activates a p38 MAPK-dependent pathway that establishes both a G1 and a G2/M cell cycle arrest. I am investigating the functions of NOM1 required for normal cell growth and proliferation and am focusing on (1) NOM1’s ability to localize to and to target proteins to the nucleolus, the sight of ribosome biogenesis, and (2) NOM1’s ability to bind Protein Phosphatase I (PP1), a protein required for cell signaling, cell division, and metabolism. This experiment is accomplished by first decreasing levels of endogenous NOM1 in the cell using lentiviral vectors that encode short hairpin RNAs that bind NOM1 mRNA and target it for degradation. Next, these cells are either left untreated or transfected with one of three different NOM1 expression constructs. One encodes the full length NOM1 protein that should rescue growth and proliferation of the NOM1 depleted cells. The other two constructs contain either a deletion of the nucleolar targeting domain of NOM1 or a mutation within the NOM1 PPI binding site. The ability of these mutant constructs to rescue NOM1 depleted cells will reveal whether either nucleolar targeting and/or PP1 binding is essential for NOM1 function. These studies are important to define pathways and downstream events that respond to deficiencies in ribosome production, a process disrupted in several human diseases including several bone marrow failure syndromes.
Genetic Approach to Generating a Novel Mouse Model of Mammary Tumorigenesis
(2011-04-13) Miller, Matthew
Fibroblast growth factor receptors (FGFRs) and their ligands contribute to cellular functions including proliferation, survival, differentiation, migration, and angiogenesis. The growth factor receptor, FGFR1, chromosomal locus is amplified in 10% of breast cancer patients. Patients with this amplification do not respond well to current therapies and have been shown to develop a resistance to endocrine therapies, thus the inducible fibroblast growth factor receptor-1 (iFGFR1) was engineered. When activated, iFGFR1 promotes increased lateral budding of epithelial structures which develop into hyperplasias that progress to multicellular invasive lesions, characteristic of breast cancer. One pro-inflammatory protein upregulated by iFGFR1 activation is osteopontin. Osteopontin is a secreted glycophosphoprotein that is involved in a variety of different cancer types, including breast cancer. Data suggests that osteopontin is synthesized by breast carcinomas and acts to promote traits associated with increased aggressiveness. To study iFGFR1-induced osteopontin expression and the role osteopontin plays in cancer development and progression in mouse mammary glands in vivo, I developed a novel mouse model where mice are heterozygous for our FGFR transgene and osteopontin null (FGFR1 +/-; osteopontin -/-). In order to do this I backcrossed osteopontin -/- mice on a C57BL/6 genetic background to the FVB genetic background of the FGFR1 mice. The goal of this project was to master techniques in mouse husbandry and PCR-based genotyping as well as tissue staining. With the new transgenic mouse model we can better study the correlation between osteopontin levels and breast cancer in hopes of making osteopontin a targetable factor for therapeutic intervention.
Identification of Genes Required for Minisatellite Instability
(2009-04-08) Swanlund, Seth
Rare alleles of minisatellites, repetitive DNA tracts whose repeat units range from 15 to 100 base pairs in length, have been correlated with human diseases including breast, colon, and urinary cancer. Little is known about the regulation of minisatellite stability. The Kirkpatrick lab conducted a color-based screen that identified factors involved in minisatellite stability during stationary phase using the yeast Saccharomyces cerevisiae. Factors were identified by screening for mutants that destabilized a minisatellite repeat construct in the coding region of the ADE2 gene. One of the factors identified in this screen was the zinc transporter gene ZRT1. I followed up these findings by identifying and sequencing mutations that suppressed the minisatellite instability phenotype in the ZRT1 null strains. To identify a suppressor gene, I transformed a yeast genomic plasmid library into a suppressor mutant stain, selected transformants that reverted to the parental phenotype, then rescued the candidate plasmids and sequenced them. I also identified specific mutations in the suppressor genes PET112 and IFM1 by amplification and sequencing. Both of these genes are involved in mitochondrial function. This suppressor screen has shown that many processes are involved in minisatellite stability during stationary phase. Further investigation is needed to identify other genes involved in the ZRT1 pathway, and to determine how these genes affect minisatellite stability. Understanding the regulation of minisatellite stability in yeast could lead to effective treatment and prevention for many human diseases.
Maternal Folic Acid Supplementation and Risk of Medulloblastoma in Offspring
(2012-04-18) Marek, Courtney
Folate deficiency is reportedly the most common vitamin deficiency in the United States. Various clinical studies have researched the implications of such a deficiency on the health of individuals, particularly regarding developing embryos in expectant mothers. Such previous research has found a convincing correlation with a number of developmental abnormalities, including neural tube defects as well as a form of brain cancer known as medulloblastoma. Medulloblastoma is classified as a primitive neuralectodermal tumor (PNET) that affects the cerebellum, and it is the leading type of brain cancer occurring in children. Despite the existing theories and clinical studies that have pointed to an association between folate deficiency and medulloblastoma, it has yet to be shown that folate in fact plays a causal role in the formation of these tumors. To test our hypothesis that folate deficiency directly causes an increased incidence of medulloblastoma, we created an experiment in which three cohorts of mice of the heterozygous Ptch+/- background were fed different diets that included high, control, and low folate supplementation. After allowing these mice to breed, the offspring were observed for the development of medulloblastoma. Unexpectedly, we found a significantly lower incidence of medulloblastoma in the low folate cohort, while the differences in the high and control cohorts were not statistically significant. Our findings indicate that folate does appear to play a role in medulloblastoma formation, although whether folate actually induces the tumors or progresses preexisting ones remains unclear. Thus, future directions aiming to clarify the role of folate in cancer cells are necessary in order to fully understand the effect of folate deficiency on medulloblastoma tumorigenesis.
Mechanisms of Androgen-Mediated Repression of the Maspin Tumor Suppressor Gene in Prostate Cancer
(2010-04-21) Bader, David
Prostate Cancer (PCa) is the second leading cause of cancer death in American men; one in six will be diagnosed with the disease during his lifetime. The androgen receptor (AR) is a transcription factor necessary for normal prostate cell growth as well as for growth of PCa. When AR is activated by androgens, it translocates to the nucleus and exerts transcriptional activation and repression of target genes. Significant efforts have focused on characterizing genes that are activated by AR such as prostate specific antigen (PSA), a marker for PCa screening. Genes that are transcriptionally repressed by AR are also likely to play a role in the progression of prostate cancer, yet the mechanisms behind their repression have received less attention. One such gene is maspin, a tumor suppressor gene that is repressed by androgens. Maspin expression is associated with increased cellular adhesion, increased sensitivity to cellular apoptosis, and decreased angiogenesis in the tumor microenvironment. In this study, we demonstrated that (1) Maspin is repressed following androgen treatment, (2) the repression is mediated in a direct manner, and (3) Androgen antagonists such as bicalutamide do not affect the ability of AR to repress maspin. Ongoing research will continue to investigate AR’s role in the repression of maspin. Understanding the underlying mechanisms by which AR represses maspin and other target genes may reveal novel opportunities for developing new prostate cancer therapies.
Observing Bacterial Diversity in Glacial Till using 16S rRNA Sequencing
(2012-04-18) Burnes, Andrew
Recent surveys of domestic wells in west central Minnesota have shown that the levels of arsenic in 50% of wells are higher than standards set by the EPA, creating a serious health risk for the surrounding populations. Investigations show that the arsenic contamination originates from naturally occurring arsenic in the surrounding glacial sediments that are part of the Des Moines Lobe; However, wells present in the Des Moines lobe are not universally contaminated with high levels of arsenic, suggesting that the process of contamination is controlled by other factors such as the presence of bacteria. This experiment aimed to observe if there are bacteria present in these soil samples that could play a role in arsenic contamination. To do this, DNA was extracted from core soil samples and the 16S rRNA gene was amplified using PCR. The PCR product was then cleaned and sent to a private lab to be sequenced using 454 pyrosequencing. Results are expected to indicate the presence of bacterial genera that have the ability to trigger arsenic contamination. These findings could further show the importance and scope of microbial populations on a geological level and could lead to avenues of bioremediation.
The Role of sgd-8 in the Control of Oocyte Meiotic Maturation in Caenorhabditis elegans
(2009-04-08) Lee, Carrie K.
Oocyte meiotic maturation is a highly conserved biological process required for sexual reproduction. In human females, the frequency of meiotic errors increases with maternal age, and these mistakes can lead to miscarriage, infertility, or Down Syndrome. At present, C. elegans is the only genetic model organism in which we can observe oocyte development, meiotic maturation, and ovulation in the intact living animal. In C. elegans, major sperm signal (MSP) is a hormone used to promote oocyte meiotic maturation. A receptor of this signal, VAB-1 (variable abnormal morphology)/Eph receptor, functions as part of a sperm-sensing control mechanism that regulates oocyte meiotic maturation. The gsa-1 gene encodes the stimulatory G protein alpha subunit, is necessary and sufficient to promote oocyte meiotic maturation. A large-scale forward genetic screen from prior work identified mutations that suppress the requirement of gsa-1 for oocyte meiotic maturation effect. One of the mutations identified, sgd-8 (suppressor of gsa-1 maturation defect) is represented by five alleles. Preliminary studies show that sgd-8 mutants are sensitive to somatic RNAi, but resistant to RNAi in the germline. To better understand how sgd-8 functions as a regulator of oocyte meiotic maturation, we are conducting genetic and phenotypic analysis of sgd-8 mutants. Using snip-SNP mapping, sgd-8 was mapped to LG III. Currently, we are fine mapping sgd-8 to an interval for positional cloning, and have successfully narrowed the interval.
ShRNA Knockdown of ID genes in Human Embryonic Stem Cells as a Possible Path Towards B and T Cell Development
(2009-04-08) Taylor, Russ
Human Embryonic Stem Cells (hESCs) are pluripotent, self-renewing cells capable of becoming any cell in the human body. Previously, our lab has derived multiple cell types from hESCs, with a particular interest in hematopoietic (blood cell) development. We have been able to successfully derive natural killer (NK) cells, a type of lymphocyte with potent anti-tumor activity. However, to date we have been unable to derive other lymphocytes (T and B cells) from hESCs. Using Umbilical Cord Blood (UCB) as a comparison for early hematopoietic development, we deduced that one possible factor for this difference could be a relatively high expression of the Inhibitor of Differentiation (IDs) transcription factors in hESCs compared to UCB. ID proteins bind to and negatively regulate basic Helix-Loop-Helix (bHLH) proteins responsible for many differentiation programs within the cell. In particular, ID2 and ID3 are known to promote NK cell development and inhibit B and T cell development. My project has been to introduce shRNA constructs into hESCs to inhibit expression of ID2 and ID3 as a means to better promote T and B cell development from hESCs. Two shRNA systems have been designed. The first uses a lentiviral vector in which the shRNA construct is constitutively expressed. The second uses a lentiviral vector containing a CRE-conditional shRNA expression system. We have introduced both of these vectors into Ntera2 embryonal carcinoma cells and hESCs. For the constitutive shRNAs, we demonstrate partial knockdown of ID2 and ID3 via qRTPCR. These constitutive knockdown ID hESCs become more difficult to culture in an undifferentiated state, and over time may have lost some of their potency. This potential problem with constitutive knockdown cells highlights the need for the inducible system, which is in progress. We anticipate that the ability to inhibit ID2/3 expression at specific timepoints of hESCs differentiation into hematopoietic cells could solve this problem and facilitate development of B and T cells from hESCs.
Solving the “Cocktail Party Problem:” Fluctuating Background Noise Effects on Signal Recognition in Cope’s Grey Tree Frog
(2011-04-13) Linehan-Skillings, Betsy
If you have ever had trouble understanding what a person was saying to you in a noisy group setting, then you have experienced a condition known as “the cocktail-party problem.” The cocktail party problem is a condition in which an individual has trouble hearing and understanding an isolated target signal in a noisy group setting. When natural increases and decreases in the levels of background noise occur, a phenomenon known as “modulation masking release” may allow the listener to decipher the target signal during the times when background noise is lowest. Frogs breed in noisy groups known as choruses. Within these choruses, females must isolate the most attractive male call possible from the cacophony around them—thus, they are also subject to the cocktail party problem. Using simulated mating calls and modulated chorus noise, my colleagues and I attempted to study how grey tree frogs may use masking release to resolve the cocktail party problem within mating groups. I hypothesized that exposure to low-frequency modulated chorus-like noise would allow frogs to experience modulation masking release, resulting in an overall improved phonotaxis (sound response) compared to unmodulated (constant) levels of background noise. Additionally, I expected the effect of masking release to be more prominent when the mating call was longest. However, experimental results revealed that the presence of modulated chorus noise confers no significant benefit to females when they are isolating and choosing male calls with respect to flat, unmodulated noise. Future successful studies such as this one could ultimately benefit the hearing-impaired by helping audiologists to create better hearing technology.

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