A three-dimensional digital brain atlas and stereotaxic coordinates for the Anna’s Hummingbird, Calypte anna

This thesis presents a new three-dimensional atlas for analyzing the neuroanatomical structures of the avian brain. Using magnetic resonance imaging (MRI) and X-ray computed tomography (CT), the brain of an Anna’s Hummingbird (Calypte anna) was scanned. These datasets were co-registered using three-dimensional visualisation software and overlaid with thionin stained slices in the sagittal and coronal view. Results show that previously developed techniques for creating three-dimensional atlases in other birds can be successfully used for representing the anatomical structures of hummingbirds (Güntürkün et al., 2013; Poirier et al., 2008; Vellema et al., 2011). There is no previous atlas, neither two nor three-dimensional, available for hummingbirds or any other wild-caught bird. Studies requiring avian neural recordings have not included hummingbirds, but this combination of imaging protocols along with descriptions from the literature provides the necessary and sufficient information for outlining many of the major functional structures in the Anna’s hummingbird brain. The co-registered datasets are also sufficient for constructing histological data into a three-dimensional representation that is functional in guiding classical tract tracing and electrophysiology experiments. By creating brain area delineations in three dimensions and anchoring these data to a skull, researchers will now be able to reach target structures from outside of the brain. For two regions of the brain, the lentiformis mesencephali and nucleus of the basal optic route, suggested stereotaxic coordinates and angles are provided. Through expanding the collection of atlases to include hummingbirds we introduce a new animal that will be especially useful for future studies of avian motor control.Science, Faculty ofZoology, Department ofGraduat

A three-dimensional digital brain atlas and stereotaxic coordinates for the Anna’s hummingbird, Calypte anna - June 2013

3D imaging of transposed neuroanatomical images of a hummingbird

Combined RNAi-mediated suppression of Rictor and EGFR resulted in complete tumor regression in an orthotopic glioblastoma tumor model.

The PI3K/AKT/mTOR pathway is commonly over activated in glioblastoma (GBM), and Rictor was shown to be an important regulator downstream of this pathway. EGFR overexpression is also frequently found in GBM tumors, and both EGFR and Rictor are associated with increased proliferation, invasion, metastasis and poor prognosis. This research evaluated in vitro and in vivo whether the combined silencing of EGFR and Rictor would result in therapeutic benefits. The therapeutic potential of targeting these proteins in combination with conventional agents with proven activity in GBM patients was also assessed. In vitro validation studies were carried out using siRNA-based gene silencing methods in a panel of three commercially available human GBM cell lines, including two PTEN mutant lines (U251MG and U118MG) and one PTEN-wild type line (LN229). The impact of EGFR and/or Rictor silencing on cell migration and sensitivity to chemotherapeutic drugs in vitro was determined. In vivo validation of these studies was focused on EGFR and/or Rictor silencing achieved using doxycycline-inducible shRNA-expressing U251MG cells implanted orthotopically in Rag2M mice brains. Target silencing, tumor size and tumor cell proliferation were assessed by quantification of immunohistofluorescence-stained markers. siRNA-mediated silencing of EGFR and Rictor reduced U251MG cell migration and increased sensitivity of the cells to irinotecan, temozolomide and vincristine. In LN229, co-silencing of EGFR and Rictor resulted in reduced cell migration, and increased sensitivity to vincristine and temozolomide. In U118MG, silencing of Rictor alone was sufficient to increase this line's sensitivity to vincristine and temozolomide. In vivo, while the silencing of EGFR or Rictor alone had no significant effect on U251MG tumor growth, silencing of EGFR and Rictor together resulted in a complete eradication of tumors. These data suggest that the combined silencing of EGFR and Rictor should be an effective means of treating GBM

Transfection of siRNA sequences specific to Rictor and EGFR results in downregulation of their respective proteins in U251MG cell line.

<p>Optical density values shown are expressed relative to values obtained from untreated cells, which correspond to a value of 1. <b>a</b>) Representative immunoblots showing ILK, Rictor, p(473)AKT, AKT and β-actin from U251MG cells 96hrs after transfection of siRNA against ILK or Rictor or the negative control sequence (Ng ctrl). <b>b</b>) Representative immunoblots showing EGFR, p(473)AKT, AKT and β-actin from U251MG cells 96hrs after transfection of siRNA against EGFR or the negative control sequence (Neg ctrl). <b>c</b>) Representative immunoblots showing Rictor, EGFR, p(473)AKT, AKT and β-actin from U251MG cells 96 hrs after transfection of the combination of Rictor and EGFR siRNAs or the combination of two negative control sequences (Ng2x). Optical density values shown under each band represent the average obtained from three independent experiments (±SEM) normalized to β-actin, and AKT in the case of p(473)AKT. <b>d</b>) Representative fluorescence photomicrograph (n = 3) of U251MG cells showing nuclei (Draq5; red), F-actin (Texas red phalloidin; Yellow), and p(473)-AKT (Alexa 488; blue) 96 hrs after transfection of siRNA against Rictor, EGFR, the combination of Rictor and EGFR, or the combination of two negative sequences (Ng2x).</p

Fluorescence micrographs showing EGFR (Alexa 488; green), Rictor (Alexa 488; yellow) and cell nuclei (Hoechst 33342; blue) in GBM4 GBM-derived cancer stem-like cell line, and Gli36, U251MG, U118MG and LN229 GBM cell lines.

<p>Fluorescence micrographs showing EGFR (Alexa 488; green), Rictor (Alexa 488; yellow) and cell nuclei (Hoechst 33342; blue) in GBM4 GBM-derived cancer stem-like cell line, and Gli36, U251MG, U118MG and LN229 GBM cell lines.</p

The combination of EGFR and Rictor silencing results in a reduction in cell migration.

<p><b>a</b>) Example of scoring chart for the scratch-wound healing assay. <b>b</b>) Scratch width scoring of U251MG, U118MG and LN229 cells 96 hrs after transfection of siRNA against Rictor, EGFR, the combination of Rictor and EGFR or the combination of two negative control sequences (Ng2x), obtained from three independent experiments. ***p-value ≤0.001 compared to untreated cells.</p

Induction of lentiviral shRNA-transduced cells results in downregulation of corresponding proteins <i>in vitro</i> and downstream effectors, and reduction in cell migration.

<p><b>a</b>) Representative immunoblots showing Rictor, EGFR and β-actin from parental U251MG cells, U251^Ng2x, U251^Rictor, U251^EGFR and U251^EGFR/Rictor in the absence (-) or presence (+) of doxycycline. Average of band optical density normalized to β-actin from three independent experiments (+/−SEM), and expressed as relative to values obtained from parental cells, is shown under each band. *p-value ≤0.05; **p-value ≤0.01; ***p-value ≤0.001 compared to parental cells. <b>b</b>) Representative immunoblots showing Rictor, EGFR, p(473)-AKT, p(346)-NDRG1, p(422)-SGK, p(657)-PKCα and β-actin from U251^Ng2x, U251^Rictor, U251^EGFR and U251^EGFR/Rictor exposed to doxycycline for 72hrs. Average of band optical density normalized to β-actin and expressed as relative to values obtained from U251^Ng2x is shown under each band. <b>c</b>) Representative immunoblots showing Rictor, p(473)-AKT and β-actin from U251^Rictor in the absence (-) of doxycycline or exposed to doxycycline for 24–120 hrs. Average of band optical density normalized to β-actin and expressed as relative to values obtained from U251^Ng2x in the absence of doxycycline is shown under each band. <b>d</b>) Scratch width scoring of U251^Ng2x, U251^Rictor, U251^EGFR and U251^EGFR/Rictor 18hrs after scratching in presence of doxycycline and after pre-incubation with doxycycline for 72 hrs.**p-value ≤0.01 compared to U251^Ng2x cells.</p

Transfection of siRNA sequences specific to Rictor and EGFR results in downregulation of their respective proteins in U118MG and LN229 GBM lines.

<p>Histograms showing Rictor, p(473)AKT, AKT and β-actin protein levels relative to untreated cells. Optical density values were normalized to the β-actin value, and the AKT value in the case of p(473)AKT, and represent the average obtained from three independent experiments from <b>a</b>) U118MG and <b>b</b>) LN229 cells 96 hrs after transfection of siRNA against Rictor, EGFR, the combination of Rictor and EGFR siRNAs or the combination of two negative control sequences (Ng2x). *p-value ≤0.05; **p-value ≤0.01; ***p-value ≤0.001.</p

The combination of EGFR and Rictor silencing results in an increase in cell sensitivity to chemotherapeutic drugs.

<p>Changes in fraction affected measured by MTT assay of <b>a</b>) U251MG, <b>b</b>) U118MG and <b>c</b>) LN229 cells 96 hrs after transfection of siRNA against Rictor, EGFR or the combination of Rictor and EGFR, and treated with irinotecan, vincristine or temozolomide at EC50 concentrations determined for control non-transfected cells at 72 hrs. Transfection of the two negative control sequences did induce an increase in Fa of LN229 exposed to vincristine of 0.13 compared to non-transfected cells and in this case only, changes in Fa are expressed relative to Ng2x-transfected cells. For all other cases, changes in Fa are expressed relative to control non-transfected cells. *p-value ≤0.05; **p-value ≤0.01; ***p-value ≤0.001.</p

The combined silencing of Rictor and EGFR <i>in vivo</i> results in a complete inhibition of tumor growth.

<p>U251^Ng2x, U251^Rictor, U251^EGFR and U251^EGFR/Rictor cells were implanted into the right caudate nucleus-putamen of Rag2M mice (n = 6−8). Induction of shRNA expression in mice was initiated on day 21 by dissolving 2 mg/mL doxycyline and 5% sucrose in drinking water. <b>a</b>) On day 49, animals were imaged by Maestro™ fluorescence imaging unit for the expression of tRFP co-expressed with the shRNA sequences upon doxycycline-induced expression. Mice were then terminated and brains were harvested, sectioned and stained for nuclei, Rictor, EGFR and p(473)-AKT and imaged for all markers in addition to tRFP by robotic fluorescence microscopy. No tumor was detected in the U251^EGFR/Rictor group. <b>b</b>) A representative brain section from U251^Ng2x, U251^Rictor, U251^EGFR and U251^EGFR/Rictor tumor groups is shown: tRFP (red) and Hoechst (blue). <b>c</b>) A representative tumor section from U251^Ng2x, U251^Rictor and U251^EGFR tumor groups is shown: nuclei (blue), rRFP (red), Rictor (yellow), EGFR (green) and p(473)-AKT (orange). <b>d</b>) The expression of EGFR (left axis), Rictor (right axis) and p(473)-AKT (right axis) in U251^Ng2x, U251^Rictor, U251^EGFR tumor sections were quantified (positive staining normalized to Hoechst nuclei staining). <b>e</b>) Tumor sizes were estimated by quantification of tumor areas in brain sections from all groups (left axis). The expression of the proliferation marker Ki67 in the tumor (proliferating fraction) was also quantified (right axis). *p-value ≤0.05; **p-value ≤0.01; ***p-value ≤0.001 compared to control untreated cells. ‡: No tumor was detected in the U251^EGFR/Rictor group.</p