<html><body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space; "><!--StartFragment--><p class="MsoNormal" align="center" style="margin-bottom:0in;margin-bottom:.0001pt;
text-align:center;line-height:normal"><span style="font-size:22.0pt;font-family:
ComicSansMS;color:#DA211F"><b>Seminario de Química Orgánica 2009 <o:p></o:p></b></span></p><p class="MsoNormal" align="center" style="margin-bottom:0in;margin-bottom:.0001pt;
text-align:center;line-height:normal"><span style="font-size:16.0pt;font-family:
"Comic Sans MS Bold""><b>Viernes 27/11/09, 11 hs, aula de seminario del Departamento de Química Orgánica, FCEyN</b></span></p><p class="MsoNormal" align="center" style="margin-bottom:0in;margin-bottom:.0001pt;
text-align:center;line-height:normal"><span style="font-size:14.0pt;font-family:
"Comic Sans MS Bold""><b> <o:p></o:p></b></span></p><p class="MsoNormal" align="center" style="margin-bottom:0in;margin-bottom:.0001pt;
text-align:center;line-height:normal"><span style="font-size:14.0pt;font-family:
"Comic Sans MS Bold""><b>Expositor: </b></span><span style="font-size:18.0pt;
font-family:"Comic Sans MS Bold""><b>Enrico Gratton</b></span><span style="font-size:14.0pt;font-family:"Comic Sans MS Bold""><b><o:p></o:p></b></span></p><p class="MsoNormal" align="center" style="margin-bottom:0in;margin-bottom:.0001pt;
text-align:center;line-height:normal"><span style="font-size:12.0pt;font-family:
"Comic Sans MS""><b>Laboratory for Fluorescence Dynamics; University of
California, Irvine, USA<o:p></o:p></b></span></p><p class="MsoNormal" align="center" style="margin-bottom:0in;margin-bottom:.0001pt;
text-align:center;line-height:normal"><span style="font-size:14.0pt;font-family:
"Comic Sans MS Bold""><b> <o:p></o:p></b></span></p><p class="MsoNormal" align="center" style="margin-bottom:0in;margin-bottom:.0001pt;
text-align:center;line-height:normal"><span style="font-size:14.0pt;font-family:
"Comic Sans MS Bold""><b>Título: Aggregation of Proteins in cells:<span style="mso-spacerun: yes"> </span>the N&B method</b></span><span style="font-size:12.0pt;font-family:"Comic Sans MS""><b> <o:p></o:p></b></span></p><p class="MsoNormal" align="center" style="margin-bottom:0in;margin-bottom:.0001pt;
text-align:center;line-height:normal"><span style="font-size:12.0pt;font-family:
"Comic Sans MS""> <o:p></o:p></span></p>
<span style="font-size:11.0pt;font-family:"Comic Sans MS";mso-ansi-language:
EN-US;mso-fareast-language:EN-US">Aggregation of misfolded proteins is a
hallmark of several neurodegenerative diseases such as Huntington's
disease.<span style="mso-spacerun: yes"> </span>Here we describe the
utility of the recently developed Number and molecular Brightness method
(N&B) to monitor the aggregation process of Hungtintin exon 1
(Httex1).<span style="mso-spacerun: yes"> </span>N&B measures the
molecular brightness of the protein aggregates in the entire cell
non-invasively based on the fluctuation dynamics at each pixel of an image.<span style="mso-spacerun: yes"> </span>Dynamic imaging by the N&B method
allowed detection of monomers, oligomers and inclusions in different regions of
the cell simultaneously at different time points. We observed the behavior of
Httex1-EGFP, with polyglutamine (polyQ) repeats of different lengths
(Httex1-97QP-EGFP, Httex1-46QP-EGFP, Httex1-25QP-EGFP in transfected COS-7
cells. We found that Httex1 is present throughout the cell and that the
non-pathogenic protein (Httex1-25QP-EGFP) does not aggregate while the
pathogenic proteins (Httex1-46Q-EGFP and Httex1-97QP-EGFP) form inclusions. We
establish that the process of nucleation leading to inclusion formation has
four phases: i) Initially only monomers are present; ii) Following an increase
in protein concentration, due to protein accumulation, small oligomers (8-15
proteins) form throughout the cell; iii) At higher protein concentrations, an
inclusion is formed in the cytoplasm; iv) The inclusion recruits most of the
Httex1 protein in the cell, including those in the nucleus, leaving only
monomers at very low concentration.<span style="mso-spacerun: yes">
</span>The protein with 46 glutamine repeats (Httex1-46QP-EGFP) aggregated
after a longer lag phase (50 hours) compared to Httex1-97QP-EGFP (24 hours). In
a different cell type, ST14A cells, the aggregation process followed similar
dynamics.</span><!--EndFragment--></body></html>