アラビドプシス ノ ヒダリマキ ネジレ セイチョウ ニ オケル ビショウカン ノ カンヨ

https://library.naist.jp/mylimedio/dllimedio/show.cgi?bookid=100034195&oldid=61725博士 (Doctor)バイオサイエンス (Bioscience)博第216号甲第216号博士（バイオサイエンス）奈良先端科学技術大学院大

Experience with the implementation of peer consultants model to the organization providing services to people with mental illness

Peer support is gaining increasing interest of professional public abroad and at home in recent years. Peer support consists in the active participation of people with personal experience with mental illness to mental health care system. This thesis presents this model as one of the forms of participation of people with mental illness in the services provided to them. Using a concrete example of an organization it describes the experience of its implementation in practice. The theoretical part shows how peer support is related to the transformation of attitudes of the society in which there is a remarkable shift to the recognition and enforcement of fundamental human rights and freedoms of people with mental illness. The practical part focus on a specific organization providing social services to people with mental illness. A new position of peer specialist has been introduced into practice the organization in recent years. Using methods of case studies there is for more than a year monitored, as whether and how the implementation of this position has affected the organization, staff, service users and the actual peer consultant. Subsequently, we discussed what the findings say on the topic of participation of people with mental illness and whether the introduction of peer specialist position..

Canopy1, a positive feedback regulator of FGF signaling, controls progenitor cell clustering during Kupffer's vesicle organogenesis

The assembly of progenitor cells is a crucial step for organ formation during vertebrate development. Kupffer’s vesicle (KV) is a key organ required for the left-right asymmetric body plan in zebrafish, and is generated from a cluster of approximately 20 dorsal forerunner cells (DFCs). Although several genes are known to be involved in KV formation, how DFC clustering is regulated and how cluster formation then contributes to KV formation remain unclear. Here we show that positive feedback regulation of FGF signaling by Canopy1 (Cnpy1) controls DFC clustering without affecting DFC specification and DFC number. Cnpy1 positively regulates FGF signals within DFCs, which in turn promotes Cadherin1-mediated cell adhesion between adjacent DFCs to sustain cell cluster formation. When this FGF positive feedback loop is disrupted, the DFC cluster fails to form, eventually leading to KV malformation and defects in the establishment of laterality. Our results therefore uncover both a previously unidentified role of FGF signaling during vertebrate organogenesis and a regulatory mechanism underlying cell cluster formation, which is an indispensable step for formation of a functional KV and establishment of the left-right asymmetric body plan

AP4 method for two-tube nested PCR detection of AHPND isolates of Vibrio parahaemolyticus

Our previous work on the mechanism of virulence for the unique isolates of Vibrio parahaemolyticus that cause acute hepatopancreatic necrosis disease (VPAHPND) revealed that it was mediated by a binary Pir-like toxin pair ToxA and ToxB. These toxins are located on the pVA plasmid, a plasmid carried by AHPND-causing strain of V. parahaemolyticus with a size of approximately 69 kbp. Using the coding sequences of ToxA, a one-step PCR detection method for VPAHPND was introduced in June 2014 but had the limitation that attempts to adapt it into a nested PCR protocol were unsuccessful. As a result, low levels of VPAHPND in shrimp or other samples could not be detected without first preparing an enrichment broth culture to allow bacterial growth before extraction of template DNA. Here, we describe the AP4 (abbreviation of AHPND detection version 4) method, a two-tube nested PCR method that targets the tandem genes ToxA and ToxB, including the 12 bp spacer that separates them on pVA plasmid. Testing of the method revealed that it gave 100% positive and negative predictive values for VPAHPND using a panel of 104 bacterial isolates including 51 VPAHPND isolates and 53 non-AHPND isolates, the latter including 34 isolates of V. parahaemolyticus and 19 isolates of other bacteria found in shrimp ponds, including other Vibrio species. The AP4 nested PCR method was 100 times more sensitive (100 fg total DNA template) than the one-step AP3 (10 pg total DNA template) method, and it could detect VPAHPND in experimentally challenged shrimp by 6 h post immersion (n = 2/3), while AP3 could not detect is until 12 h post immersion (n = 1/3). Thus, the AP4 method may be useful in detecting VPAHPND isolates in samples where target material is limited (e.g., small tissue quantity or archived DNA) and enrichment cannot be employed (i.e., frozen samples or samples preserved in alcohol)

Characterization and PCR Detection Of Binary, Pir-Like Toxins from Vibrio parahaemolyticus Isolates that Cause Acute Hepatopancreatic Necrosis Disease (AHPND) in Shrimp.

Unique isolates of Vibrio parahaemolyticus (VPAHPND) have previously been identified as the causative agent of acute hepatopancreatic necrosis disease (AHPND) in shrimp. AHPND is characterized by massive sloughing of tubule epithelial cells of the hepatopancreas (HP), proposed to be induced by soluble toxins released from VPAHPND that colonize the shrimp stomach. Since these toxins (produced in broth culture) have been reported to cause AHPND pathology in reverse gavage bioassays with shrimp, we used ammonium sulfate precipitation to prepare protein fractions from broth cultures of VPAHPND isolates for screening by reverse gavage assays. The dialyzed 60% ammonium sulfate fraction caused high mortality within 24-48 hours post-administration, and histological analysis of the moribund shrimp showed typical massive sloughing of hepatopancreatic tubule epithelial cells characteristic of AHPND. Analysis of the active fraction by SDS-PAGE revealed two major bands at marker levels of approximately 16 kDa (ToxA) and 50 kDa (ToxB). Mass spectrometry analysis followed by MASCOT analysis revealed that both proteins had similarity to hypothetical proteins of V. parahaemolyticus M0605 (contig034 GenBank accession no. JALL01000066.1) and similarity to known binary insecticidal toxins called 'Photorhabdus insect related' proteins A and B (Pir-A and Pir-B), respectively, produced by the symbiotic, nematode bacterium Photorhabdus luminescens. In in vivo tests, it was shown that recombinant ToxA and ToxB were both required in a dose dependent manner to cause AHPND pathology, indicating further similarity to Pir-A and -B. A single-step PCR method was designed for detection of the ToxA gene and was validated using 104 bacterial isolates consisting of 51 VPAHPND isolates, 34 non-AHPND VP isolates and 19 other isolates of bacteria commonly found in shrimp ponds (including other species of Vibrio and Photobacterium). The results showed 100% specificity and sensitivity for detection of VPAHPND isolates in the test set

Examples of HP histology of moribund shrimp administered single or combined doses of ToxA and ToxB by reverse gavage.

<p>(A) Tissue sections from a BSA (10/20 μg/g) negative control shrimp (tubule longitudinal sections) and (B) a ToxA (5 μg/g) treated shrimp (tubule cross sections) showing only normal histology with morphology and cell types as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126987#pone.0126987.g001" target="_blank">Fig 1A to 1C</a>. (C) ToxB only (5 μg/g) and (D) ToxA+ToxB (2 μg/g each) showing mostly normal histology, but with some thin HP tubule epithelia (black arrows) when compared to epithelia of normal thickness (grey arrows). (E) ToxA+ToxB (5 g/g each) showing enlarged HP tubules (compared to A-C) with collapsed epithelia (black arrows) but no cell sloughing. (F) ToxA+ToxB (10 μg/g each) showing massive sloughing (black arrows) and dissolution of HP tubule epithelial cells (i.e., severe AHPND histopathology).</p

Bacterial expression of ToxA and ToxB.

<p>(A) ToxA expressed with a 6-His tag and purified by Ni-NTA affinity chromatography. Lane 1: Bacterial cell lysate from a non-induced bacterial culture; Lane 2: Bacterial cell lysate from an IPTG-induced culture; Lane 3: Eluted protein from the Ni-NTA column. The deduced molecular weight for ToxA-His was 12.7 kDa. (B) ToxB was expressed as a GST-fusion protein. Lane 1: Bacterial cell lysate from a non-induced culture; Lane 2: Bacterial cell lysate from an IPTG-induced culture; Lane 3: Eluted fraction from Sepharose 4B beads; Lanes 4&5: Fraction eluted from Sepahrose 4B after thrombin-cut. The estimated molecular weights for GST-ToxB and ToxB were approximately 76 and 50 kDa, respectively.</p