24 research outputs found
The Heroes We Mistake for Villains: the Truth Behind Self-Sacrifice and Transformation
Throughout the centuries of human civilization, the notion of the self-sacrificing hero has evolved in parallel to the evolution of societies. Heroes are shaped by their creators the populace of a society in regards to the ideals that society wishes to uphold. A hero is a person who represents the greatest aspects of human beings, epitomizing the characteristics that society holds most dear. These characteristics have transitioned in their levels of importance over time, shifting the ideal self-sacrificing hero from one figure to another. However, as these transitions occur, inevitable discrepancies will evolve in the varying minds of the participants in society. Those who wish to value one attribute above another will disagree with another\u27s archetype of the ideal self-sacrificing hero. Ultimately, one view will prevail in the mind of society itself, while the other will fall by the wayside. This thesis has been written to contend that the contemporary view of the self-sacrificing hero is incomplete, and that another type of self-sacrifice should be recognized for its contribution to society\u27s greater good. The introduction portion of this thesis will explain the evolution of the heroic figure over the course of time. In this process, we will examine the attributes of the hero that are most valued by society as the hero morphs from one form into another. Next, we will examine the relationship between ethics and heroism and utilize this connection to describe the contemporary view of heroism through modern ethicists. Finally, a brief overview of the rest of the thesis will be laid out in order to provide an understanding of the structure of the argument that some self-sacrificing heroes sacrifice more than their lives to save their societies, and that they merit recognition for their contributions as well as other heroes
Post-Transcriptional Shaping of Neurons: The Role of miRNAs and FMRP-Interacting P-Body Components in Regulating Neuronal Structure
Changes in synaptic structure in response to neuronal stimulation are believed to underlie the processes of learning and long-term memory. However, the mechanisms for these structural modifications are poorly understood. It is well-known that activity-dependent synaptic modifications rely upon new protein synthesis, and rapid new protein synthesis, at that. Therefore, it is widely believed that pools of messenger RNAs held in a state of translational repression are transcribed in a neuronal cell body prior to stimulation, and transported to the synapse, where they reside until stimulation occurs.
This study investigates the roles and interactions of translational repression mechanisms to better understand how new synaptic growth is repressed or enhanced for the purposes of long-term memory and learning. We found that miRNAs -315, -275, -11 and the miR-9 family are of particular interest for neuronal growth in Drosophila larvae because they are extremely enriched in the larval CNS compared to the adult brain, and are predicted to regulate mRNA targets that significantly contribute to neuronal development. Furthermore, miR-315 and the miR-9 family bind and regulate a Futsch (Drosophila homolog of mammalian MAP1B known to affect synaptic growth) reporter in vitro, and the miR-9 family exhibits an increase in bouton numbers at muscles 6/7 of the NMJ characteristic of an increase in Futsch levels when under-expressed. Curiously, this same effect with seen with miR-9 family overexpression.
While miRNAs are translational repressors and can clearly affect synaptic structure on their own, components of the miRNA pathway further interact with other translational repressors, including the Fragile-X Mental Retardation Protein (FMRP). Although FMRP has been shown to interact with the miRNA pathway, and to regulate Futsch, we could not discern down-regulation of a Futsch reporter from FMRP overexpression in S2 cells, nor an interaction between FMRP and these miRNAs that regulated a Futsch reporter in vitro. However, FMRP did interact with several P-body components, including co-localization with HPat, Twin, and Me31B, as well as coimmunoprecipitation with HPat, Me31B and Dcp1. Genetic interactions between FMRP and HPat and FMRP and Twin produced discernible phenotypes at the Drosophila NMJ, suggesting this interaction is important for synaptic growt
Use of Rapid-Scan EPR to Improve Detection Sensitivity for Spin-Trapped Radicals
AbstractThe short lifetime of superoxide and the low rates of formation expected in vivo make detection by standard continuous wave (CW) electron paramagnetic resonance (EPR) challenging. The new rapid-scan EPR method offers improved sensitivity for these types of samples. In rapid-scan EPR, the magnetic field is scanned through resonance in a time that is short relative to electron spin relaxation times, and data are processed to obtain the absorption spectrum. To validate the application of rapid-scan EPR to spin trapping, superoxide was generated by the reaction of xanthine oxidase and hypoxanthine with rates of 0.1–6.0 μM/min and trapped with 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO). Spin trapping with BMPO to form the BMPO-OOH adduct converts the very short-lived superoxide radical into a more stable spin adduct. There is good agreement between the hyperfine splitting parameters obtained for BMPO-OOH by CW and rapid-scan EPR. For the same signal acquisition time, the signal/noise ratio is >40 times higher for rapid-scan than for CW EPR. Rapid-scan EPR can detect superoxide produced by Enterococcus faecalis at rates that are too low for detection by CW EPR
Re-Cloning the N27 Dopamine Cell Line to Improve a Cell Culture Model of Parkinson's Disease.
Parkinson's disease is characterized by the death of dopaminergic neurons in the substantia nigra. To understand the molecular mechanisms of the disease, an in vitro model is important. In the 1990s, we used the SV40 large T antigen to immortalize dopaminergic neurons derived from Embryonic Day 14 rat mesencephalon. We selected a clone for its high expression of dopaminergic neuron markers such as tyrosine hydroxylase (TH), and we named it 1RB3AN27 (N27). Because the original N27 cell line has been passaged many times, the line has become a mixture of cell types with highly variable expression of TH. In the current study, we have performed multiple rounds of clonal cultures and have identified a dopaminergic cell clone expressing high levels of TH and the dopamine transporter (DAT). We have named this new clone N27-A. Nearly 100% of N27-A cells express TH, DAT and Tuj1. Western blots have confirmed that N27-A cells have three to four times the levels of TH and DAT compared to the previous mixed population in N27. Further analysis has shown that the new clone expresses the dopamine neuron transcription factors Nurr1, En1, FoxA2 and Pitx3. The N27-A cells express the vesicular monoamine transporter (VMAT2), but do not express dopamine-beta-hydroxylase (DβH), the enzyme responsible for converting dopamine to norepinephrine. Functional analysis has shown that N27-A cells are more sensitive than N27 cells to neurotoxins taken up by the dopamine transporter such as 6-hydroxydopamine and 1-methyl-4-phenylpyridine (MPP+). The DAT inhibitor nomifensine can block MPP+ induced toxicity. The non-selective toxic effects of hydrogen peroxide were similar in both cell lines. The N27-A cells show dopamine release under basal and depolarization conditions. We conclude that the new N27-A clone of the immortalized rat dopaminergic cell line N27 should provide an improved in vitro model for Parkinson's disease research
Immunocytochemistry of purified N27-A and unpurified N27 cells for dopamine neuron markers TH and DAT.
<p>The N27 cells were cultured on 8-well chamber slides and immunostained for the dopamine neuron markers TH <b>(A-D)</b> and DAT <b>(E-H).</b> Other wells were double-stained for TH and the neuronal marker Tuj1 <b>(I-L).</b> To image every cell in each well, the nuclear marker DAPI was added to all wells. <b>(A-B):</b> The purified N27-A clone showed strong TH staining in all cells as demonstrated by dual-staining with TH and DAPI. <b>(C-D):</b> The unpurified N27 cell mixture revealed that only a small fraction of the DAPI-labeled cells were TH positive. <b>(E-F):</b> In the purified N27-A clone, all cells had moderate DAT staining as shown with DAT and DAPI double staining. <b>(G-H):</b> In the unpurified N27 cell mixture, very few cells were positive for DAT immunostaining. <b>(I-J)</b>: In the purified N27-A clone, all cells were double-positive for Tuj1 and TH. <b>(K-L)</b>: While there were few TH-positive cells in the unpurified N27 cell mixture, most cells were Tuj1 positive, demonstrating that the mixed cell population was neuronal. Bar, 50 μm for <b>A-L</b>.</p
Purified N27-A cells are more sensitive to 6-OHDA toxicity than unpurified N27 cells.
<p>Purified N27-A and unpurified N27 cells were cultured in 24-well plates (for trypan blue staining) and 96-well plates (for MTT assay). Two days after plating, cells were treated with 0–150 μM of 6-OHDA for 24 hr. The cell viability was measured by trypan blue staining <b>(A)</b> and MTT assay <b>(B)</b>. <b>(A):</b> Cell viability data from trypan blue staining showed that there were significantly fewer viable cells in purified N27-A cultures compared to unpurified N27 cultures. <b>(B)</b>: Cell viability results from the MTT assay also showed that purified N27-A cells had greater cell death than unpurified N27 cells after exposure to 6-OHDA. Reduced cell survival in both assays indicate that purified N27-A cells are more sensitive to 6-OHDA than unpurified N27 cells (n = 12 for <b>A</b>, n = 15 for <b>B</b>, **<i>p</i><0.01).</p
Growth properties of purified N27-A and unpurified N27 cells.
<p><b>(A-F):</b> Both cell types were plated at 20,000 cells in each well of 6-well plates. Representative images were taken at Day 1, 3, and 5 for each cell type. Purified N27-A cells (<b>Images A-C</b>) grew more slowly than unpurified N27 cells (<b>Images D-F</b>). <b>(G)</b>: Growth charts of purified N27-A (red line) and unpurified N27 cells (green line) from Day 0 to Day 7. Data present the average cell number from two wells of purified and unpurified cells in three experiments (n = 6, each cell type). Bar, 20 μm for <b>A-F</b>.</p
Process of N27 cells clonal purification.
<p><b>(A):</b> Phase contrast image of unpurified N27 cells grown at low density. <b>(B):</b> Immunostaining for TH with green fluorescence in unpurified N27 cells. Shown is a cluster of cells exhibiting bright TH-positive staining. Most cells shown in phase contrast have no TH-immunoreactivity. <b>(C):</b> Schematic drawing of the clonal culture procedures for purifying N27-A cells. Cells were plated at low density to form individual colonies which were picked up and screened in 48- and 96-well plates. The positive clones were expanded in 6-well plates and 10-cm dishes. Bar, 20 μm for both <b>A</b> and <b>B</b>.</p
Western blots for TH and DAT in purified N27-A and unpurified N27 cells.
<p><b>(A)</b>: Representative images show TH, DAT and β-actin Western blots from purified and unpurified N27 cells. Strong TH bands were seen in purified N27-A cells, while much reduced TH protein levels were seen in unpurified N27 cells. There were moderate DAT protein levels in purified N27-A cells but only faint DAT bands in unpurified N27 cells. <b>(B):</b> Quantification of TH and DAT Western blots relative to β-actin bands. The TH and DAT levels in unpurified N27 cells were set at 100%. Results show that purified N27-A cells have four-fold higher TH and three-fold higher DAT protein levels compared to unpurified N27 cells. (n = 6, **<i>p</i><0.01)</p
N27-A cells are more sensitive to MPP+ toxicity than unpurified N27 cells, and nomifensine can block MPP+ induced toxicity.
<p>Purified N27-A and unpurified N27 cells were cultured in 96-well plates at equal starting density. Two days after plating, cells were treated with 0–1000 μM of MPP+ for 24 hr. <b>(A):</b> Cell viability data from MTT assays showed that there was significantly greater cell death in N27-A cultures compared to that in N27 cultures after 100 μM to 1000 μM of MPP+ treatment (n = 12, *<i>p</i><0.05). <b>(B)</b>: After nomifensine pre-treatment, exposure to MPP+ did not significantly reduce cell viability in either N27-A or N27 cells, even when MPP+ reached 1000 μM (n = 12, <i>p</i>>0.3).</p