Pattern of aphid tissues infected with <i>D. dadantii</i>.

<p>Immunostaining with anti KdgM antibodies of aphids infected with the wild type <i>D. dadantii</i> strain. Anti KdgM antibodies were detected with anti IgG labeled with Alexa fluor 488 (green labeling) and DNA was stained with DAPI (blue labeling). <b>2A to 2D</b>: early infection stage: day 1 of infection (aphids collected on infected diet). <i>D. dadantii</i> is located mainly in the fat body (fb, 2A), within the gut lumen (gl, 2B), and can also be detected in some gut cells (gc, 2C) as well as in the embryonic fat body (efb, 2D). <b>2E to 1K</b>: early infection stage: day 2 post infection. <i>D. dadantii</i> is located as a general infection of the fat body (2E), as dense aggregates in the gut lumen (2F), in gut cells (2G) and, occasionnally, in all of the following tissues: brain (2H), cornicles (co, 2I) and in many embryos showing either embryonic fat body (efb, 1J) or embryonic gut infections (egc egl, 2K). <b>2L to 2O</b>: late infection stage: day 4 post-infection. A heavy infection of <i>D. dadantii</i> is seen in all the maternal and embryonic fat bodies (2L), the gut tissue and lumen (2M), and large parts of the embryonic fat body (2N) and embryo gut cells, but not in the embryonic bacteriocytes (egc, eba, 2O). Abbreviations - ba: bacteriocytes, brc: brain cells, co: cornicles, emb: embryo, eba: embryonic bacteriocyte; efb: embryonic fat body, egc: embryonic gut cells, egl: embryonic gut lumen, fb: fat body, gc: gut cells, gl: gut lumen, oe:eonocytes.</p

Bacterial population growth in aphids.

<p>Numeration of <i>D. dadantii</i> populations within infected pea aphids. <i>wt</i>: infection with wild-type <i>D. dadantii</i>; <i>Δcyt</i>: infection with strain A4977, with the four <i>cyt</i> genes deleted. Data are means ± SE of bacterial numerations (n from 6 to 28, depending on day and survival rates). Ingestion dose was 10⁶ cell/mL. Only aphids still infected with bacteria were included in the counts.</p

Expression of CytC toxin in aphid tissues.

<p>Immunostaining with anti CytC antibodies (except 3C, left) of aphids infected with the wild type <i>D. dadantii</i> strain. Anti CytC antibodies were detected with anti IgG labeled with Alexa fluor 488 (green labeling) and DNA was stained with DAPI (blue labeling). <b>3A to 3B</b>: early infection stage: day 1 of infection. In most samples, no expression of the toxin was detected (3A) although bacterial presence was readily observed in the same sample, for example in the gut lumen (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030702#pone-0030702-g002" target="_blank">Fig. 2</a>); in some individual insects, toxin expression was detected in the gut lumen (3B). <b>3C to 3E</b>: Primary infection stage: day 2 post infection; 3C, left and right: typical staining difference (in the same aphid) between bacterial detection at many locations, including the fat body (anti-KdgM antibody, 3C left) and toxin expression detection, restricted to the digestive tract (anti-CytC antibody, 3C right); In some instances, toxin expression was also detected in clustered areas of the fat body, in addition to typical gut lumen localization (gl, fb, 3D–E); <b>2F</b>: late infection stage, day 4 post-infection. Toxin detected as a marked stain only in the gut lumen (2F). Abbreviations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030702#pone-0030702-g002" target="_blank">Figure 2</a>, plus: oe: oenocytes.</p

Infection of pea aphids by the toxin defective mutant strain.

<p>Immunostaining with anti KdgM or anti CytC antibodies of aphids infected with <i>D. dadantii</i> strain A4977. Anti KdgM and anti CytC antibodies were detected with anti IgG labelled with Alexa fluor 488 (green labelling) and DNA was stained with DAPI (blue labelling). <b>4A</b>: Staining with anti-CytC antibody of an aphid at late infection stage (day 4 post infection). Stain was never detected on such mutants. All the following images are with anti-KdgM stain to detect <i>D. dadantii</i> cell localization. <b>4B to 4D</b>: early infection stage (day 1). When detected, bacterial cells were localized at standard sites, as compared to the wild-type strain, such as in the fat body (4B), the gut lumen and gut cells (4C), and even in the embryonic fat body (4D); <b>4E to 4F</b>: late infection stage, day 4 post infection. <i>D. dadantii</i> are detected in the maternal fat body (4E), and in the gut lumen and cells (4F). Abbreviations are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030702#pone-0030702-g002" target="_blank">Figure 2</a>.</p

Factores condicionantes de la variabilidad fenotípica de los síndromes arritmogénicos hereditarios: análisis funcional de mutaciones asociadas a los síndromes de Brugada y de QT largo

El síndrome de Brugada (SBr) y el de QT largo (SQTL) son los dos síndromes arritmogénicos hereditarios (SAH) más prevalentes. En ambos casos, la expresividad y la penetrancia entre los portadores de las mutaciones causales son muy variables y dependen, entre otros, de factores demográficos. Cada vez existen más evidencias que sugieren que variantes en genes que no son los que hasta la fecha se han relacionado con dichos síndromes y que producen “gran o pequeño impacto” funcional, modulan la penetrancia y la expresividad de los SAH.El OBJETIVO GENERAL de la presente TESIS DOCTORAL es analizar funcionalmente las consecuencias electrofisiológicas y moleculares de las variantes identificadas en tres familias españolas con SAH para discernir su posible papel como responsables o modificadoras de la expresividad y penetrancia de los SAH de los pacientes portadores de dichas mutaciones..

Survival of various mutants to polymyxin.

<p>Wild type and various mutants in genes involved in resistance to AMP (<i>phoP</i>, <i>pmrA</i>, <i>dltB</i>, <i>arnB</i>) were incubated in the presence of 1 µg ml⁻¹ polymyxin for 1 h. Samples were diluted and plated on LB agar plates to assess bacterial viability. Survival values are relative to the original inoculum. Data correspond to mean values of three independent experiments.</p

Regulation of <i>sstE</i> and <i>outC</i>.

<p>The <i>sttE-uidA</i> and <i>outC-uidA</i> fusions of strain A4206 and A1919, respectively, were assayed in the presence of 5 µg/ml of polymyxin or 10 mg/ml Mg²⁺ in the wt, <i>phoP</i> and <i>pmrA</i> backgrounds. Activities are the mean value from at least four separate experiments and are expressed in µmoles of <i>p</i>-nitrophenol produced per minute and per milligram of bacterial dry weight ± standard deviation.</p

A subset of <i>D. dadantii</i> genes whose expression varies in aphid.

a<p>Positive values represent genes upregulated in aphids, whereas negative values represent genes downregulated.</p

Survival of pea aphids after oral infection by wt and mutants of <i>D. dadantii</i> 3937.

<p>Survival is shown for aphids treated with wt bacteria (red), <i>arnB</i> (green), <i>dltB</i> (blue), and <i>dltB arnB</i> (brown) mutants. Results were obtained with 2×30 third instar aphid nymphs per treatment, including a diet-treated control (no mortality, not shown). The experiment was repeated twice with very similar results (p<0.06 in all comparisons of wt with <i>arnB</i> mutants). Median survival times (LT50s) were calculated with a Weibull fit (inlet), and give the following series [95% confidence intervals]: wt, 2.92 [2.18–3.92]; <i>arnB</i>, 4.74 [3.42–6.55]; <i>dltB</i> 3.58 [2.62–4.88] and <i>arnB</i>-<i>dltB</i> double mutant, 4.46 [3.23–6.18].</p

Regulation of <i>arnB</i>.

<p>The <i>arnB-uidA</i> fusion of strain A5256 was assayed in the presence of increasing concentrations of polymyxin (A), protamine (B), and Mg²⁺ (C). Effect of <i>phoP</i> and <i>pmrA</i> mutations on <i>arnB-uidA</i> regulation by Mg²⁺ (D) and protamine (E) was assayed. Cultures were performed in LB medium for A, C and D and in M63 medium for B and E since protamine precipitates in LB medium. Activities are the mean value from at least four separate experiments and are expressed in µmoles of <i>p</i>-nitrophenol produced per minute and per milligram of bacterial dry weight ± standard deviation.</p