Identification of critical enzymes in the salmon louse chitin synthesis pathway as revealed by RNA interference-mediated abrogation of infectivity

Treatment of infestation by the ectoparasite Lepeophtheirus salmonis relies on a small number of chemotherapeutant treatments that currently meet with limited success. Drugs targeting chitin synthesis have been largely successful against terrestrial parasites where the pathway is well characterised. However, a comparable approach against salmon lice has been, until recently, less successful, likely due to a poor understanding of the chitin synthesis pathway. Post-transcriptional silencing of genes by RNA interference (RNAi) is a powerful method for evaluation of protein function in non-model organisms and has been successfully applied to the salmon louse. In the present study, putative genes coding for enzymes involved in L. salmonis chitin synthesis were characterised after knockdown by RNAi. Nauplii I stage L. salmonis were exposed to double-stranded (ds) RNA specific for several putative non-redundant points in the pathway: glutamine: fructose-6-phosphate aminotransferase (LsGFAT), UDP-N-acetylglucosamine pyrophosphorylase (LsUAP), N-acetylglucosamine phosphate mutase (LsAGM), chitin synthase 1 (LsCHS1), and chitin synthase 2 (LsCHS2). Additionally, we targeted three putative chitin deacetylases (LsCDA4557, 5169 and 5956) by knockdown. Successful knockdown was determined after moulting to the copepodite stage by real-time quantitative PCR (RT-qPCR), while infectivity potential (the number of attached chalimus II compared with the initial number of larvae in the system) was measured after exposure to Atlantic salmon and subsequent development on their host. Compared with controls, infectivity potential was not compromised in dsAGM, dsCHS2, dsCDA4557, or dsCDA5169 groups. In contrast, there was a significant effect in the dsUAP-treated group. However, of most interest was the treatment with dsGFAT, dsCHS1, dsCHS1+2, and dsCDA5956, which resulted in complete abrogation of infectivity, despite apparent compensatory mechanisms in the chitin synthesis pathway as detected by qPCR. There appeared to be a common phenotypic effect in these groups, characterised by significant aberrations in appendage morphology and an inability to swim. Ultrastructurally, dsGFAT showed a significantly distorted procuticle without distinct exo/endocuticle and intermittent electron dense (i.e. chitin) inclusions, and together with dsUAP and dsCHS1, indicated delayed entry to the pre-moult phase.publishedVersio

FAST INdiCATE Trial protocol. Clinical efficacy of functional strength training for upper limb motor recovery early after stroke : neural correlates and prognostic indicators

© 2013 The Authors. International Journal of Stroke published by John Wiley & Sons Ltd on behalf of World Stroke Organization. Funded by: Efficacy and Mechanism Evaluation programme. Grant Number: 10/60/30 Medical Research Council (MRC)Peer reviewedPublisher PD

HUMAN VOICE AND THE ABILITY OF ATTUNEMENT. THE SIGNIFICANCE OF NON-VERBAL VOCAL ENCOUNTERING IN THE EXPERIENCES OF PEOPLE IN NEED OF PSYCHIATRIC HELP

Defence is held on 6.5.2022 12:00 – 14:00 Zoom https://aalto.zoom.us/j/65419798839MY DOCTORAL RESEARCH uses the process of artistic research to develop a method fornon-verbal vocal encountering for situations when verbal communication breaks down. The research has been conducted in co-operation with Helsinki University Central Hospital of Psychiatry, with participants who were receiving psychiatric treatment in the hospital. Eight participants and a psychiatric nurse took part in the participatory vocalizing that consisted of 15 meetings. In this vocal work, the participants activated and joined their voices together. The shared non-verbal vocalizing created a phenomenon of vocal encountering. In its sphere of influence the interaction unfolded through silence, vulnerability, interoceptive sensing, one’s own voice, rhythm, space, the listener’s share, spectrum of vitality and shared resonance sphere. I recorded, composed, and published the shared voices of the sessions as a series of research artworks called Hospital Symphonies in co-operation with artistic working groups.The site- and situation sensitive works where then shared in the psychiatric hospital space (Laulunpesä/Vocal Nest, 2015), in a concert hall (Hospital Symphonies, 2018) and via radio (Sairaalasinfonia 3.0). I have investigated the effects of shared vocalizing through artistic and qualitative means, and by collecting questionnaires from the participants and listeners. Based on the ethically sensitive research material and three artistic components, my research shows that shared vocal and sensorial attunement can foster affective connection between people, in situations in which verbal communication breaks down. Non-verbal encountering developed strong interoceptive experiences for participants and listeners that enabled them to experience vitalization of the connection between self, others, and the surrounding world. The non-verbal encountering affirmed the participants’ involvement and agency and widened the spectrum of becoming heard and understood. The resonance sphere, experienced bodily, decreased the participants’ sense of petrification and enabled existential and mutual experiences of being alive, which are difficult to achieve otherwise. Working as an artist-researcher in the challenging environment increased my skill of sensorial attunement. The ethics of sensorial attunement become crucial for enabling the vulnerable encounter-events between participants. The shared attunement regulates the connections between people by enlivening the participants own abilities for attunement which has transformational potentiality for the recovery of the participants. It can also be helpful for the psychiatric healthcare to foster situations in which language-based connection is not feasible. In the shared resonance sphere social cohesion can be experienced without the institutional and functional roles between people which can enhance social recognition and human dignity. By creating connections for affective interaction, the skill of vocal attunement destabilizes the structures of ordinary interaction and shows the psychiatric environment in a new light.VÄITÖSTUTKIMUKSESSANI olen kehittänyt taiteellisen tutkimusprosessin avulla sanattoman äänellisen kohtaamisen menetelmän tilanteisiin, joissa sanallinen vuorovaikutus on murtunut. Olen toteuttanut tutkimuksen yhteistyössä Helsingin yliopistollisen keskussairaalan HUS Psykiatriakeskuksen sekä osallistujien kanssa, jotka ovat saaneet sairaalan piirissä psykiatrista apua. 15 tapaamisen osallistavan äänityöskentelyn jaksoon osallistui kahdeksan osallistujaa ja psykiatrinen sairaanhoitaja. Äänityössä osallistujat käyttivät aktiivisesti omaa ääntään yhdessä. Sanaton yhdessä äänteleminen synnytti äänellisen kohtaamisen ilmiön, jonka vaikutuspiirissä inhimillinen vuorovaikutus tapahtui hiljaisuuden, haavoittuvuuden, kehoaistisuuden, oman äänen, rytmin, tilan, kuulijan osuuden, vitaalisuuden kirjon sekä yhteisen resonanssitilan muodoissa. Äänityön prosessissa talteen saadun aineiston pohjalta muodostin yhdessä taiteellisten työryhmien kanssa kolmiosaisen Hospital Symphonies -teossarjan, joka koostuu sairaalatilaan toteutetusta Laulunpesästä (2015), konserttisaliin toteutetusta Hospital Symphonies-teoksesta (2018) sekä Sairaalasinfonia 3.0 (2019) -radioesseestä. Olen tutkinut yhdessä ääntelemisen vaikutuksia taiteellisin, laadullisin ja kyselytutkimuksen keinoin. Väitöskirjani sekä siihen sisältyvät kolme eettisesti herkkävireistä taiteellista osiota osoittavat, että tilanteissa, joissa sanallinen vuorovaikutus ei ole mahdollista, voidaan ihmisten välille luoda äänellisen ja aistimellisen virittäytymisen avulla mahdollisuus affektiivisesti latautuvaan sanattomaan kohtaamiseen. Äänellinen kohtaaminen synnytti voimakkaita interoseptiivisesti tuntuvia kokemuksia osallistujille ja kuulijoille, joiden varassa itsen, toisen ja ympäröivän maailman välinen kytkös elävöityi. Sanaton kohtaaminen vahvisti osallistujien toimijuutta ja osallisuutta, sekä laajensi kuulluksi ja ymmärretyksi tulemisen spektriä. Osallistujien ja kuulijoiden kehollisesti kokema resonanssitila vähensi jähmettyneisyyttä ja mahdollisti sellaisten eksistentiaalisten ja vuorovaikutuksellisten elossaolon tunteiden kokemisen, joita on vaikea saavuttaa muilla keinoilla. Haastavassa ympäristössä taiteilija-tutkijana toimiminen muodosti aistimellisen virittäytymisen taidon, jonka mahdollisuus avautui osallistujille ja kuulijoille. Aistimellisen virittäytymisen etiikka on keskeistä haavoittuvuutta huomioivan kohtaamisen mahdollistamisessa. Yhdessä äänteleminen säätelee yhteyttä itseen ja toiseen elävöittämällä osallistujien ja kuulijoiden omaa virittäytymisen taitoa, joka voi edistää osallistujien toipumista. Se saattaa olla hyödyllinen psykiatrisen hoidon kannalta tukemaan tilanteita, joissa kieleen pohjaava vuorovaikutus murtuu. Yhteisessä resonanssitilassa voidaan aistimellisella tasolla kokea yhteenkuuluvuutta ja läheisyyttä ilman ihmisten välisiä funktionaalisia rooleja, joka voi lisätä kokemusta sosiaalisesta tunnustetuksi tulemisesta ja ihmisarvosta. Luomalla olosuhteita affektiiviselle kanssakäymiselle, aistimellinen kokemus horjuttaa tavanomaisen vuorovaikutuksen rakenteitaja näyttää psykiatrisen toimintaympäristön uudessa valossa

Non-verbal voice as embodied encountering

Peer reviewe

Identification of critical enzymes in the salmon louse chitin synthesis pathway as revealed by RNA interference-mediated abrogation of infectivity

Treatment of infestation by the ectoparasite Lepeophtheirus salmonis relies on a small number of chemotherapeutant treatments that currently meet with limited success. Drugs targeting chitin synthesis have been largely successful against terrestrial parasites where the pathway is well characterised. However, a comparable approach against salmon lice has been, until recently, less successful, likely due to a poor understanding of the chitin synthesis pathway. Post-transcriptional silencing of genes by RNA interference (RNAi) is a powerful method for evaluation of protein function in non-model organisms and has been successfully applied to the salmon louse. In the present study, putative genes coding for enzymes involved in L. salmonis chitin synthesis were characterised after knockdown by RNAi. Nauplii I stage L. salmonis were exposed to double-stranded (ds) RNA specific for several putative non-redundant points in the pathway: glutamine: fructose-6-phosphate aminotransferase (LsGFAT), UDP-N-acetylglucosamine pyrophosphorylase (LsUAP), N-acetylglucosamine phosphate mutase (LsAGM), chitin synthase 1 (LsCHS1), and chitin synthase 2 (LsCHS2). Additionally, we targeted three putative chitin deacetylases (LsCDA4557, 5169 and 5956) by knockdown. Successful knockdown was determined after moulting to the copepodite stage by real-time quantitative PCR (RT-qPCR), while infectivity potential (the number of attached chalimus II compared with the initial number of larvae in the system) was measured after exposure to Atlantic salmon and subsequent development on their host. Compared with controls, infectivity potential was not compromised in dsAGM, dsCHS2, dsCDA4557, or dsCDA5169 groups. In contrast, there was a significant effect in the dsUAP-treated group. However, of most interest was the treatment with dsGFAT, dsCHS1, dsCHS1+2, and dsCDA5956, which resulted in complete abrogation of infectivity, despite apparent compensatory mechanisms in the chitin synthesis pathway as detected by qPCR. There appeared to be a common phenotypic effect in these groups, characterised by significant aberrations in appendage morphology and an inability to swim. Ultrastructurally, dsGFAT showed a significantly distorted procuticle without distinct exo/endocuticle and intermittent electron dense (i.e. chitin) inclusions, and together with dsUAP and dsCHS1, indicated delayed entry to the pre-moult phase