Pantagruel

Conjunt de particel·les dels següents instruments: 1r violí, 2n violí, viola, contrabaix, flauta, 1r clarinet, 2n clarinet, trompes, 1r cornetí, 2n cornetí, 1r trombó, 2n trombó, 3r trombó, fiscorn, caixa, bomboHi figura escrita a mà la data 1886Rigodo

Electron micrographs showing unusual aspect of inner membrane complex development of the ΔNΔRep mutant.

<p><b>A.</b> Low power of a mid-stage oocyst showing the retracted plasmalemma and areas of IMC invagination into the cytoplasmic mass (arrows). Bar is 1 µm. <b>B.</b> Detail of the surface of an early oocyst showing extensive growth of the IMC (arrows) but no evidence of budding. Bar is 100 nm. <b>C.</b> Detail of a more advanced stage in development showing areas of abnormal IMC/plasmalemma formation and invagination into the cytoplasmic mass of the sporoblasts (arrows). Bar is 100 nm. <b>D.</b> Cross section through two sporozoites showing loss of shape, adhesion, and folding of the plasmalemma of the sporozoites (arrows). R – rhoptry; Mt - microtubule. Bar is 100 nm. <b>E, F.</b> Enlargement of cross sections through ΔNΔRep (E) and WT (F) parasites, showing the relative distance between the plasmalemma of adjacent sporozoites. Note in the ΔNΔRep mutant the plasma membranes appeared tightly adhered (similar to that between the IMC membranes) (<b>E</b>) compared to the significantly wider space observed in the WT (<b>F</b>). I – IMC; Mt - sub-pellicular microtubules; P – plasmalemma. Bar is 100 nm.</p

Phenotypic analyses of ΔRep and ΔNΔRep mutants in the mosquito.

<p><b>A.</b> Oocyst numbers. On day 14 post-infection, midguts from 20–30 mosquitoes were scored for number of oocysts by phase or fluorescence microscopy. Shown is the mean ± SEM for each line. This analysis was performed 3 times with different batches of mosquitoes and a representative experiment is shown. <b>B.</b> Number of sporozoites per oocyst. On the indicated day post-infective blood meal, equal numbers of 10–20 mosquito midguts were collected and used either to count oocysts or were homogenized and sporozoites were counted. The number of sporozoites was then divided by the number of oocysts. Each point represents the mean ± SEM of 4 independent experiments. ΔNΔRep parasites did not produce sporozoites. <b>C.</b> Midgut sporozoite numbers. At each of the indicated days post-infective blood meal, midguts were dissected from 10–20 mosquitoes per parasite line, sporozoites were counted and the number of sporozoites per mosquito was calculated. Shown is the mean ± SEM of pooled data from 4 independent experiments. No sporozoites could be detected by light microscopy in the ΔNΔRep line. <b>D.</b> Hemolymph sporozoite numbers. On days 16 and 19 post-infective blood meal, hemolymph was collected from 15 mosquitoes and sporozoites were counted. Shown is the mean ± SEM of three independent experiments. No hemolymph sporozoites were observed in ΔRep infected mosquitoes. <b>E.</b> Salivary gland sporozoite numbers. On day 21 post-infective blood meal, salivary glands from 20 mosquitoes were dissected and sporozoites were counted. Shown is the mean ± SEM of 3 independent experiments. No salivary gland sporozoites were ever observed in ΔRep and ΔNΔRep infected mosquitoes. <b>F.</b> Representative differential interference contrast (DIC) microscopy images of oocysts from wild type, ΔRep and ΔNΔRep infected mosquitoes at the indicated days post infection. Bars represent 10 µm.</p

Light microscopy analysis of cell death in ΔRep oocysts.

<p><b>A.</b> Photographs of representative mosquito midguts on days 14 and 21 post infection. Control and ΔRep parasites express GFP in most oocysts at day 14 post infection whereas by day 21 most of the ΔRep oocysts have lost the GFP fluorescence. Top panel shows a mosquito midgut and lower panel shows representative oocysts at higher magnification with degenerated features and absence of GFP fluorescence due to loss of plasmalemma integrity at day 21 post infection in ΔRep oocysts. <b>B.</b> Quantification of GFP positive and GFP negative oocysts at 14, 16, 18, and 21 days post infection for control and ΔRep oocysts.</p

Generation of CSP repeatless mutants.

<p><b>A.</b> Schematic representation of CSP structure in wild type and mutant parasites ΔRep and ΔNΔRep. Region I is shown as hatched, repeat region as light grey and the TSR domain as dark grey. <b>B.</b> Western blot analysis of wild type (WT), WT-GFP and RCon as control parasites and the two repeat mutants: ΔRep and ΔNΔRep. Lysates from midgut sporozoites or infected midguts were probed using antisera specific for each of the three CSP domains: polyclonal antisera specific for the CSP NH₂-terminus, anti-repeat region (mAb 3D11) and polyclonal antisera specific for the CSP COOH-terminus. Molecular weight markers (kDa) shown on the left of each gel photograph.</p

Electron micrographs of sporogony in WT, ΔRep and ΔNΔRep mutants.

<p>A series of electron micrographs of oocysts illustrating the progressive stages in the sporogonic process undergone by WT, ΔRep, and ΔNΔRep oocysts in the mosquito midgut. The structure of the oocysts at the end of the growth phase was similar for WT (<b>A</b>) and both mutants. (<b>B, C</b>) The initiation of sporozoite formation with retraction of the plasmalemma was also similar (<b>D-F</b>). However, while sporozoite formation continued by a budding process in both WT (<b>G</b>) and the ΔRep mutant (<b>H</b>) there was no budding seen in the ΔNΔRep mutant (<b>I</b>). This budding process continued until the sporozoites were fully formed in the WT (<b>J</b>) and ΔRep (<b>K</b>). In contrast the mature oocyst of the ΔNΔRep mutant contained a tightly adhered mass of sporozoites (<b>L</b>). Bars represent 10 µm.</p

Electron micrographs illustrating the process of cell death observed in oocysts of the ΔRep and ΔNΔRep mutants.

<p><b>A.</b> Low power of a ΔNΔRep oocyst with a degenerating, undifferentiated central cytoplasmic mass. Bar is 10 µm. <b>B.</b> Detail from the degeneration of a ΔNΔRep oocyst similar to that in <b>A</b> showing a dilated nuclear membrane containing a number of nuclei (N) exhibiting peripheral chromatin condensation. Bar is 100 nm. <b>C.</b> Low power of a ΔRep oocyst in which sporozoite formation had occurred but now exhibited features of cell degeneration. Bar is 10 µm. <b>D.</b> Detail of the nuclei observed in an intact ΔNΔRep oocyst showing the absence of any peripheral nuclear condensation. Bar is 100 nm. <b>E.</b> Longitudinal section through a sporozoite showing the nucleus with peripheral chromatin condensation and dilatation of the nuclear membranes. N – nucleus. Bar is 500 nm. <b>F.</b> Low power of a ΔRep oocyst in which there is a cross section of a central mass of degenerating sporozoites (S). Bar is 10 µm. <b>G.</b> Histogram comparing the relative number of immature mature and degenerate oocysts at two time points (12–14 days and 18–21 days) for WT, ΔRep and ΔNΔRep oocysts. (Based on EM examination of multiple midguts from multiple experiments – number of oocysts evaluated: 405 wild type; 236 ΔRep mutant; 165 ΔNΔRep mutant).</p

Characterisation of the cyclin repertoire of the <i>Apicomplexa</i>.

<p>(A) Maximum likelihood phylogeny based on an alignment of cyclins from <i>Plasmodium falciparum</i>, <i>Toxoplasma gondii</i>, <i>Cryptosporidium parvum</i> and <i>Homo sapiens</i>. CYCP1 and CYCH1 from <i>Arabidopsis thaliana</i> have been included for clarity. Topology support from bootstrapping is shown at nodes. (B) Distribution of cyclin families across <i>Apicomplexa</i>. Presence (filled dot) or absence (empty circle) of specific families of cyclin in each predicted proteome is shown. Dot area is proportional to number of putative proteins. The Group I cyclins in <i>Cryptosporidium</i> cannot be placed reliably into any specific families within the group (“Orphaned”). *The <i>Plasmodium berghei</i> predicted proteome (release 9.3; plasmodb.org/) contains no apparent orthologue of CYC1, as the likely gene encoding the protein on Chromosome 13 (downstream of PBANKA_132730, syntenic with <i>cyc1</i> in other <i>Plasmodium</i> species) is interrupted by a sequence gap.</p

CYC3-GFP protein expression throughout most stages of the life cycle.

<p>(A) Transcription of <i>cyc1</i>, <i>cyc3</i> and <i>cyc4</i> as analysed by qRT-PCR, normalised against two endogenous control genes, <i>arginine-tRNA synthetase</i> and <i>hsp70</i>. Each bar is the mean of three biological replicates ± SEM. All asexual blood stages: AS; schizonts: Sch; non-activated gametocytes: NAG; activated gametocytes: AG; ookinete: Ook; 14 dpi oocysts/sporozoites: Spor. (B) Expression of CYC3-GFP in trophozoites, schizonts, gametocytes, zygotes, ookinetes, oocysts and sporozoites. 13.1, a cy3-conjugated antibody which recognises P28 on the surface of activated females, zygotes, and ookinetes was used with the sexual stages. Scale bar = 5 μm. (C) Deconvolved 2D projections of live trophozoite, gametocyte, and ookinete expressing CYC3-GFP (green), co-stained with Hoechst 33342 (blue) and cy3-conjugated anti-P28 antibody, 13.1 as a marker for the ookinete surface (red). Scale bar = 5 μm. Line profiles (red) in the black and white images indicate pixel intensity for that channel.</p

Expression of CYC3-GFP during sporogony in mosquitoes.

<p>Fluorescence microscopy of CYC3-GFP at different time points: 5, 7, 10, 14 and 21 dpi during development in the mosquito. Scale bar = 20 μm. Representative percentage of oocysts that either: do not express GFP (black number), have a low expression of GFP (red number) or have a high expression of GFP (green number).</p