190942 research outputs found
Sort by
Ternära metalloxid-nanokatalysatorer för syreutvecklingsreaktionen
No full textAs the demand for sustainable energy sources is continuously growing, green hydrogen produced by water splitting is considered one of the key energy sources of the future. In this thesis, catalysts with low noble metal content for the acidic Oxygen Evolution Reaction (OER) have been synthesized, and their electrochemical performance have been investigated. OER is one of the half-cell reactions in water-splitting, the other being the Hydrogen Evolution Reaction (HER). The OER is the more energy demanding and harder to optimize half-cell reaction, because of its more complex kinetics. The currently well-studied catalysts for the acidic OER, namely IrO2 and RuO2, are expensive, scarce and not feasible to use on a large scale. For these reasons, in this thesis, catalysts mainly consisting of WOx with low content of Ru and Ir have been developed. Five different catalysts of the general form RuxIryW1-x-yOz have been synthesized starting from RuCl3, IrCl3, WCl6 and EtOH in a three-step synthesis. These catalysts were characterized by UV-Vis spectroscopy, XRD, Raman spectroscopy, SEM and TEM, and their electrocatalytic performance was evaluated using Linear Sweep Voltammetry (LSV), Electrochemical Impedance Spectroscopy (EIS) and Cyclic Voltammetry (CV). SEM and TEM showed that the catalysts had a sphere-like morphology with some crystalline features, while Raman and XRD confirmed that the catalysts contained RuO2, IrO2 and WOx. The key findings of the electrochemical analysis were that the catalysts with increased Ir content compared to Ru and W performed better than commercial IrO2 in terms of current density and overpotential, while increased Ru content compared to Ir and W showed negligible improvement of the catalyst performance. The catalysts with increased Ru content also performed worse than commercial IrO2. The best performing catalyst, Ru0.1Ir0.3W0.6Ox, was also stability tested. The stability test showed that the catalyst was only stable for around 15 minutes. In addition to the poor stability, the electrochemical performance in terms of current density and overpotential of the catalysts are worse than the best performing materials already reported in literature.Eftersom behovet av hållbara energikällor konstant ökar, så anses grönt väte framställt från vattenspjälkning vara en av framtidens viktigaste energikällor. I den här avhandlingen har katalysatorer med låga halter av ädelmetaller för syreutvecklingsreaktionen (OER) i sura förhållanden syntetiserats, och deras elektrokemiska prestanda har utvärderats. OER är den ena halvcellsreaktionen i vattenspjälkning, den andra halvcellsreaktionen är väteutvecklingsreaktionen (HER). OER är den mer energikrävande och utmanande halvcellsreaktionen att optimera, på grund av mer komplex kinetik. De katalysatorer som för tillfället har studerats mest för OER i sura förhållanden, dvs. IrO2 och RuO2, är dyra och sällsynta, vilket gör att de inte är realistiska att använda i stor skala. På grund av dessa orsaker har katalysatorer som i huvudsak består av WOx med låga halter av Ru och Ir utvecklats i den här avhandlingen. Fem olika katalysatorer, med den generiska formeln RuxIryW1-x-yOz har syntetiserats utgående från RuCl3, IrCl3, WCl6 och EtOH i en trestegs-syntes. Dessa katalysatorer har karaktäriserats med UV-Vis spektroskopi, XRD, Ramanspektroskopi, SEM och TEM, medan deras elektrokatalytiska prestanda utvärderades med hjälp av voltammetri med linjära potentialsvep (LSV), elektrokemisk impedansspektroskopi (EIS) och cyklisk voltammetri (CV). SEM och TEM visade att katalysatorerna hade en sfärisk morfologi, med vissa kristallina kännetecken, medan Raman och XRD bekräftade att katalysatorerna innehöll RuO2, IrO2 och WOx. De viktigaste upptäckterna från den elektrokemiska analysen var att katalysatorerna med förhöjd Ir-halt jämfört med Ru och W hade bättre prestanda än kommersiell IrO2 när det kommer till strömdensitet och överspänning, medan förhöjd Ru-halt jämfört med Ir och W uppvisade försumbar förbättring av katalysatorernas prestanda. Katalysatorerna med förhöjd Ru-halt presterade också sämre än kommersiell IrO2. För den bäst presterande katalysatorn, nämligen Ru0.1Ir0.3W0.6Ox, utfördes också stabilitetstest. Stabilitetstestet visade att katalysatorn endast var stabil i ungefär 15 minuter. Utöver den låga stabiliteten så är också de elektrokemiska resultaten i fråga om strömdensitet och överspänning sämre än de bästa publicerade katalysatorerna
Peroksisomien Dynamiikka Lihasten Kantasoluissa In Vitro Myogeneesin Aikana
No full textBackground: Skeletal muscle is highly organized postmitotic tissue with robust regenerative capacity, which depends on muscle stem cells (MuSCs). MuSCs are quiescent under homeostatic conditions and activate after muscle injury to repair the myofiber or to self-renew and replenish the quiescent MuSC pool. It has been demonstrated that altering MuSC metabolism, such as mitochondrial maturation and the activity of oxidative phosphorylation guides MuSC fate. Other organelles, such as peroxisomes, exhibit similar effects on stem cell dynamics in human mammary epithelial cells. Furthermore, peroxisome numbers increase during the myogenic progression in the C2C12 cell line, and the activity of catalase increases during the differentiation of primary human myoblasts and is altered in aging. In addition, disturbing peroxisomal biogenesis or FAO in the C2C12 cell line or primary cells, respectively, affects MuSC dynamics suggesting a key role for peroxisomes in muscle regeneration. However, the connections between an increase in peroxisomes, the myogenic progression, and the effects of altering peroxisomal functions in primary MuSCs haven’t been examined in detail. Thus, this thesis aims to characterize changes in MuSC peroxisome quantity and changes in peroxisomal catalase at various time points during the in vitro myogenic progression and to characterize how inhibition of catalase affects myogenesis and stem cell fate in primary MuSCs.
Methods: Primary MuSCs from wild-type C57BL/6J mice were isolated with fluorescence or magnetic-activated cell sorting and cultured for one to six days, in order to characterize peroxisome numbers and catalase localization and content through time points with immunofluorescent microscopy and western blotting. Pharmacological irreversible inhibition of catalase was used to evaluate the effects of losing peroxisomal enzyme function at selected time points.
Results/Discussion: A rise in peroxisomal numbers throughout the myogenic progression that seemed to scale with cell size was observed. However, a distinct jump in peroxisome biogenesis and catalase abundance from 24h to 48h in culture, and large heterogeneity in the peroxisome density and catalase expression of freshly isolated MuSCs, was revealed. Catalase expression declined between 48h and 72h of culture. Catalase inhibition with 3-AT affected MuSC proliferation and differentiation capacity, dose, and timepoint dependently. Low-dose catalase inhibition indicated to increase MuSC proliferation and myotube formation in preliminary experiments while a higher dose decreased both outcomes. These findings suggest that MuSCs show great metabolic heterogeneity and invest in peroxisomal biogenesis and catalase upregulation in the proliferative state and that the myogenic progression and proliferation can be altered by affecting catalase function. Uncovering the role of peroxisomal metabolism in muscle stem cell dynamics and muscle regeneration in detail could yield new strategies for treating muscle wasting disorders and the functional decline associated with aging.Taustaa: Luustolihas on hyvin organisoitunut postmitoottinen kudos, jonka vankka uusiutumiskyky on riippuvainen lihaskantasoluista. Lihaskantasolut ovat lepotilassa homeostaattisissa olosuhteissa, ja aktivoituvat lihasvamman jälkeen korjaamaan lihassyitä tai uusiutumaan täydentääkseen lepotilassa olevia kantasoluja. On osoitettu, että mitokondrioiden kypsymistä ja oksidatiivisen fosforylaation aktiivisuutta muuttamalla voidaan ohjata lihaskantasolujen kohtaloa. Muilla soluelimillä, kuten peroksisomeilla, on samanlainen vaikutus ihmisen maitorauhasen epiteelin kantasolutoimintaan. Lisäksi peroksisomien määrä lisääntyy myogeneesin aikana C2C12-solulinjassa, ja katalaasin aktiivisuus lisääntyy ihmisen primaaristen myoblastien erilaistumisen aikana ja heikkenee ikääntyessä. Lisäksi peroksisomaalisen biogeneesin tai metabolian häiritseminen C2C12-solulinjassa tai primaarisoluissa vaikuttaa lihaskantasolujen dynamiikkaan, mikä viittaa peroksisomien keskeiseen rooliin lihaksen uudistumisessa. Peroksisomien lisääntymisen, myogeneesin, ja peroksisomaalisten toimintojen muuttamisen vaikutusten välistä yhteyttä primaarisissa MuSC-soluissa ei kuitenkaan ole tutkittu yksityiskohtaisesti. Niinpä tämän tutkielman tavoitteena on luonnehtia lihaskantasolujen peroksisomien määrän muutoksia ja katalaasin muutoksia eri aikapisteissä koko In Vitro myogeneesin aikana sekä luonnehtia, miten katalaasin inhiboiminen vaikuttaa myogeneesiin ja primaaristen kantasolujen kohtaloon. Menetelmät: Villiä tyyppiä edustavista C57BL/6J-hiiristä peräisin olevat primaariset lihaskantasolut eristettiin fluoresenssi- tai magneettisesti aktivoidulla solulajittelulla ja niitä kasvatettiin yhdestä kuuteen vuorokautta, jotta peroksisomien lukumäärää ja katalaasin lokalisaatiota ja pitoisuutta voitiin karakterisoida eri aikapisteissä immunofluoresenssimikroskopialla ja western blottaamalla. Katalaasin farmakologista irreversiibeliä estoa 3-AT:lla käytettiin arvioimaan peroksisomaalisen entsyymitoiminnan menettämisen vaikutuksia valituissa aikapisteissä. Tulokset ja keskustelu: Havaittiin peroksisomaalisten lukumäärän kasvua koko myogeneesin ajan, joka näytti skaalautuvan solujen koon mukaan. Kuitenkin havaittiin selvä hyppäys peroksisomien biogeneesissä ja katalaasin runsaudessa 24 tunnista 48 tuntiin viljelyssä sekä suurta heterogeenisuutta peroksisomien tiheydessä ja katalaasin ilmentymisessä juuri eristetyissä lihaskantasoluissa. Katalaasin ilmentyminen väheni 48 tunnin ja 72 tunnin viljelyn välillä. Katalaasin estäminen 3-AT:lla vaikutti lihaskantasolujen proliferaatioon ja erilaistumiskykyyn annoksesta ja ajankohdasta riippuvaisella tavalla. Katalaasin esto pienellä 3-AT-annoksella (5μm) lisäsi alustavissa kokeissa lihaskantasolujen proliferaatiota ja myotuubien muodostumista, kun taas suurempi annos (5mM) 3-AT:ta vähensi molempia lopputuloksia. Nämä havainnot viittaavat siihen, että lihaskantasolut osoittavat suurta metabolista heterogeenisuutta ja panostavat peroksisomaaliseen biogeneesiin proliferatiivisen tilan aikana ja että myogeneesia ja proliferaatiota voidaan muuttaa vaikuttamalla katalaasin toimintaan. Peroksisomaalisen aineenvaihdunnan roolin paljastaminen yksityiskohtaisesti lihaskantasolujen dynamiikassa ja lihaksen uudistumisessa voisi tuottaa uusia strategioita lihaksenkadon ja ikääntymiseen liittyvän toiminnallisen heikkenemisen hoitoon
Ohjelmoitavan kiraaliplasmonisen nanoswitch-teknologian kehittäminen sepsiksen vieritestidiagnostiikkaan
No full textBacterial sepsis, a life-threatening and urgent condition resulting from the body's dysregulated response to infection, poses a significant global health challenge, underscoring the urgent need for rapid and reliable diagnostic tools. However, current point-of-care tests face limitations in accuracy and robustness, particularly in complex biological samples. Advances in DNA nanotechnology and chiral plasmonics offer promising solutions by enabling programmable molecular recognition and highly sensitive biosensing.
This thesis explores an aptamer-based chiral plasmonic nanoswitch system designed for the detection of procalcitonin, a prominent biomarker for sepsis diagnostics. The nanoswitch system was developed through the bottom-up assembly of DNA origamis, functionalization with gold nanoparticles, and incorporation of aptamers. Circular dichroism spectroscopy was utilized to monitor changes in chirality upon target binding, facilitating the assessment of the nanoswitch's sensitivity and specificity. Optimization and validation efforts were concentrated on determining whether the challenges encountered were due to the approach itself or to defective materials.
The nanoswitch platform was successfully validated using thrombin, a well-characterized endogenous protein, as a benchmark, confirming the fundamental functionality of the approach. However, the sensitivity and robustness for procalcitonin detection were found to be inadequate, likely due to suboptimal aptamer binding affinity or target protein degradation. This study highlights critical challenges in aptamer-based diagnostics, including the necessity for high-affinity aptamers and the complexities involved in working with biological samples.
Future research should prioritize enhancing aptamer selection—potentially through artificial intelligence and machine learning-assisted in silico screening—and optimizing nanoswitch performance in real-world sample matrices. Beyond diagnostics, the nanoswitch concept may also have applications in quality control for biomolecules within the pharmaceutical and medical industries. Although the system did not achieve diagnostic-grade detection, this work provides valuable insights into DNA nanotechnology and aptamer-based sensing, paving the way for future advancements in point-of-care diagnostics.Bakteerisepsis on hengenvaarallinen ja kiireellistä hoitoa vaativa tila, joka johtuu elimistön häiriintyneestä immuunivasteesta infektioon. Sepsis on merkittävä maailmanlaajuinen terveyshaaste, mikä korostaa kriittistä tarvetta nopeille ja luotettaville diagnostiikkamenetelmille. Nykyisillä vieritestiratkaisuilla on kuitenkin rajoitteita tarkkuuden ja luotettavuuden suhteen, erityisesti koostumukseltaan monimutkaisissa ihmisperäisissä näytteissä. Toisaalta, DNA-nanoteknologian ja kiraalisen plasmoniikan edistysaskeleet tarjoavat lupaavia ratkaisuja mahdollistamalla ohjelmoitavan ja erittäin herkän molekyylitunnistuksen.
Tässä maisterintutkielmassa kehitettiin ja tutkittiin aptameeripohjaista kiraalista plasmonista nanoswitch-järjestelmää prokalsitoniinin – sepsiksen keskeisen biomarkkerin – havaitsemiseksi. Nanoswitch-järjestelmä rakennettiin kokoamalla DNA-origameja niin kutsutulla "bottom-up" -menetelmällä, funktionalisoimalla ne kultananopartikkeleilla ja liittämällä niihin spesifisiä aptameereja. Kohdemolekyylin sitoutumisesta aiheutuvia kiraalisia muutoksia analysoitiin ympyrädikroismispektroskopian avulla, minkä perusteella arvioitiin nanoswitch-järjestelmän herkkyyttä ja täsmällisyyttä. Optimointi- ja validointikokeet keskittyivät selvittämään, olivatko havaitut haasteet seurausta itse menetelmästä vai käytettyjen materiaalien puutteellisuudesta.
Nanoswitch-alusta validoitiin onnistuneesti trombiinilla – hyvin karakterisoidulla endogeenisella proteiinilla – mikä vahvisti järjestelmän perustoiminnallisuuden. Prokalsitoniinin havaitsemisen herkkyys ja luotettavuus eivät kuitenkaan olleet riittäviä, mikä todennäköisesti johtui aptameerien heikosta sitoutumisaffiniteetista tai kohdeproteiinin rakenteen hajoamisesta. Tämä tutkielma tuo esiin keskeisiä haasteita aptameeripohjaisessa diagnostiikassa, kuten tarpeen korkean affiniteetin aptameereille ja ihmisperäisten näytteiden analysoinnin haasteellisuuden.
Tulevan tutkimuksen tulisi keskittyä aptameerien kehitysprosessin tehostamiseen, esimerkiksi hyödyntämällä tekoälyyn ja koneoppimiseen perustuvaa in silico -seulontaa, sekä nanoswitch-järjestelmän suorituskyvyn optimointiin aidoissa potilasnäytteissä. Diagnostiikan lisäksi nanoswitch-konseptilla voisi mahdollisesti olla sovelluksia myös biomolekyylien laadunvalvonnassa lääke- ja terveysteknologian aloilla. Vaikka kehitetty järjestelmä ei saavuttanut tässä tutkielmassa diagnostista herkkyystasoa, tämä työ tarjoaa hyödyllisiä näkemyksiä DNA-nanoteknologiasta ja aptameereistä tunnistuselementteinä, luoden pohjaa tuleville edistysaskeleille vieritestidiagnostiikassa
Driver fatigue: crashes, the law, and traffic police officers’ experiences and views
No full textPeer reviewe
Recent advances in modern extracellular vesicle isolation and separation techniques
No full textSince slightly sluggish beginning some twenty years ago, heterogeneous nanosized extracellular vesicles (EVs), secreted by almost all mammalian cells, all Gram-negative and Gram-positive bacteria and several plant tissues, have grabbed an enormous attention especially during the last years due to their functional role in intercellular and interorgan communications, offering possibilities for great innovations in diagnostics and therapeutic monitoring and prognosis. The availability of their reliable isolation and separation techniques that can conquer most of the challenges related to purity, yield, scalability, integrity, nanoscale contaminants, operation time, and complex sample matrices is a crucial prerequisite for the exploitation of EVs and their subpopulations. The utmost goal of this review, that is a continuation of our overview published in 2021, is to introduce the most recent modern and emerging isolation and separation approaches and trends, with their specific properties and applications. The focus is again on size-, charge-, and affinity-based techniques and ultracentrifugation and precipitation-based techniques are included only for comparison purposes. The isolation techniques will be briefly compared with each other in terms of different important parameters. Furthermore, a few industrial promising applications are introduced. Although in the newly developed isolation and separation techniques and methods especially the presence of interfering nanocomponents, causing lower purity, has been much better minimized or even eliminated, and although even in vivo studies have more frequently been carried out, it is obvious that only combined and hyphenated techniques can overcome at least most of the challenges.Peer reviewe
Distribution and characteristics of Campylobacter spp. in turkeys at slaughter
No full textCampylobacter is the leading cause of bacterial foodborne gastrointestinal infection in humans, and poultry is among the most important reservoirs. Turkeys have been shown to frequently carry Campylobacter, and the isolates recovered from turkeys show high antimicrobial resistance. We studied the prevalence of Campylobacter in Finnish slaughter turkeys during 2013–2023 from the monitoring data of a Finnish turkey slaughterhouse, covering 1856 turkey flocks originating from 41 farms over the monitoring period. Additionally, we used whole-genome sequencing to identify bacterial species, resistance profiles, sequence types, and core genome allelic profiles for 103 Campylobacter isolates from turkey faecal samples collected at the slaughterhouse between 2013 and 2021. In total, 9.5 % of the flocks and 82.9 % of the farms were Campylobacter positive. The prevalence varied significantly between years, months, and farms. Campylobacter jejuni (87.6 %) was the most common species followed by Campylobacter coli (4.0 %) and Campylobacter lari (1.1 %). We obtained 34 sequence types, with ST45 being the most common (20.2 % of the isolates). Genetically closely related isolates originated mostly from the same farm with a tight temporal connection. Five antimicrobial genotypic resistance profiles were identified. Most of the isolates carried only the blaOXA gene, and only one Campylobacter isolate carried several resistance genes. We showed that Campylobacter prevalence and the occurrence of antimicrobial resistance genes in the isolates is low in Finnish slaughter turkeys. Further, we demonstrated that Campylobacter spread between the flocks on the same farm, but the same STs did not persist.Peer reviewe
Eu and HTO sorption and diffusion in crystalline rock in the presence of MX-80 bentonite colloids
No full textThe diffusion of 152Eu and HTO through two crystalline rock cores (metatexitic gneiss, granitic pegmatoid) was studied in low saline, pH 8 groundwater conditions. Diffusion was examined both in the presence and absence of MX-80 bentonite colloids. Determination of the penetration depth by autoradiography showed that 152Eu did not penetrate the studied rocks during the four-month experimental period but was mostly retained on the rock surface through sorption. Furthermore, the presence of bentonite colloids was found to decrease the sorption of 152Eu on the rock surface. The size of bentonite colloids at pH 8 groundwater conditions prevented the migration of 152Eu through the connected pore network of low-porous rock cores. This finding was investigated by determining the size of the rock pore and micro fissure apertures from 14C-PMMA autoradiograms, which were determined to be mostly smaller than the size of the colloids. The effective diffusion coefficient De of HTO was (4.34 f 2.88) center dot 10- 13 m2/s and (7.70 f 2.19) center dot 10- 13 m2/s for metatexitic gneiss and granitic pegmatoid, respectively. The distribution coefficient Kd of 152Eu in Allard water was determined to be (7.26 f 0.64) m3/kg for metatexitic gneiss and (3.50 f 1.44) m3/kg for granitic pegmatoid. In bentonite colloid solution, distribution coefficient values of (0.22 f 0.07) m3/kg and (0.20 f 0.05) m3/kg were determined for metatexitic gneiss and granitic pegmatoid, respectively.Peer reviewe
Mobile-CRISPRi menetelmällä muodostettu uropatogeeninen Escherichia coli, jolla on hiljennetty quorum sensing reitti
No full textThe antimicrobial resistance problem is worsening rapidly, and there is an urgent need to discover alternative treatment options. One potential alternative is blocking quorum sensing (QS), as it controls among others the expression of virulence genes. The QS autoinducer 3 (AI-3) pathway has been found so far from 25 pathogens, potentially being a broad-spectrum antivirulence drug target.
Urinary tract infections (UTIs) are among the most common bacterial infections and uropathogenic Escherichia coli (UPEC) is the most common causing agent in these infections. Due to the recurrent nature of these infections, antibiotics are prescribed both as treatment and as preventives, causing increased selective pressure on bacteria to develop resistance. Therefore, finding alternative treatments for UTIs is of most importance.
Quorum sensing systems are complex circuitries, and studying the function of their genes isolated from the system is difficult. Therefore, the CRISPRi method was chosen to silence the key gene of the AI-3 pathway, qseC, to study the effect of this pathway on UPEC virulence. This method allows silencing the desired gene inducibly, without removing it from its regulon.
This study aimed to establish the Mobile-CRISPRi method for UPEC CFT073 and to construct a QS pathway AI-3 silenced mutant so that it can be used to study the effects of this pathway on UPEC virulence and screen for potential inhibitors of this pathway.
Initially, the CRISPRi method was tested with pre-made fluorescent plasmids verifying the suitability of this method for silencing genes in UPEC CFT073 strain. In the next stage, a qseC-specific sgRNA was designed and the CRISPRi silencing machinery was incorporated into the chromosome of UPEC CFT073 at the attTn7 site via triparental mating. The obtained clones were tested for qseC-gene silencing with real-time quantitative polymerase chain reaction (RT-qPCR).
The CRISPRi method was compatible with the tested strains as the difference between unsilenced and silenced GFP fluorescent signals was explicit. Moreover, the GFP silencing intensity increased with incubation time. Gradual silencing with inducer gradient was, however, not accomplished. The silencing of the qseC gene in UPEC CFT073, verified by RT-qPCR, was established with continuous silencing even without induction. The fold reduction of mutant qseC-gene expression compared to the wild type was over 30 in some of the clones obtained.
The CRISPRi method provides a powerful tool to study important genes with inducibility and tunable silencing efficacy. The GFP silencing was inducible with an on-off mechanism. The lack of silencing gradient might be due to the different transport mechanisms of the inducer, IPTG, in low and high concentrations. The qseC-gene was silenced in UPEC CFT073 but with continuous silencing. One reason for continuous silencing could be a leaky lac promoter. Despite the lack of inducibility, this mutant can be used to study the effect of qseC silencing on UPEC virulence in the future.Mikrobilääkeresistenssin ongelma pahenee nopeasti ja vaihtoehtoisia hoitomuotoja tulisi löytää kiireellisesti. Yksi mahdollinen vaihtoehto on bakteerien quorum sensingin (QS) esto, koska se säätelee muiden muassa virulenssigeenien ilmentämistä. QS autoindusori 3 (AI-3) reitti on löydetty tähän mennessä 25 patogeenisestä bakteerista, mikä tekee siitä varteenotettavan laajakirjoisen virulenssin vastaisen lääkekohteen.
Virtsatieinfektiot ovat yleisimpiä bakteeri-infektioita ja uropatogeeninen Escherichia coli (UPEC) on yleisin näiden infektioiden aiheuttajista. Koska nämä infektiot ovat usein toistuvia, antibiootteja määrätään niin hoidoksi kuin estohoidoksikin, mikä lisää valintapainetta bakteereissa kehittää resistenssiä. Näin ollen vaihtoehtoisten hoitomuotojen löytäminen virtsatieinfektioiden hoitoon on erityisen tärkeää.
QS järjestelmät ovat monimutkaisia ja niiden geenien toiminnan tutkiminen irrallaan järjestelmästä on vaikeaa. Tästä syystä CRISPRi -menetelmä valittiin hiljentämään AI-3 reitin keskeinen geeni, qseC, jotta tämän reitin vaikutusta UPEC:in virulenssiin voitaisiin tutkia. Tämä menetelmä mahdollistaa halutun geenin hiljentämisen indusoitavasti geenin säilyessä säätely-yksikössään.
Tämän tutkimuksen tarkoitus oli saada Mobile-CRISPRi menetelmä toimimaan UPEC CFT073 kannassa ja muodostaa QS AI-3 reitin hiljennetty mutantti, jolla voitaisiin tutkia tämän reitin hiljennyksen vaikutusta UPEC:in virulenssiin sekä seuloa mahdollisia inhibiittoreita tälle reitille.
Aluksi CRISPRi menetelmän soveltuvuutta UPEC CFT073 kannan geenien hiljentämiseksi testattiin valmiilla fluoresoivilla plasmideilla. Seuraavassa vaiheessa suunniteltiin qseC-geenille komplementaarinen sgRNA ja CRISPRi hiljentämisjärjestelmä liitettiin UPEC CFT073 kromosomiin attTn7 kohtaan triparental mating -menetelmällä. qseC-geenin hiljennys todennettiin saaduista klooneista RT-qPCR -menetelmällä.
CRISPRi-menetelmä soveltui molemmille testatuille kannoille, sillä hiljentämättömän ja hiljennetyn kannan GFP fluoresoivien signaalien ero oli selkeä. Lisäksi GFP hiljennyksen voimakkuus kasvoi inkubaatioajan kasvaessa. Osittaista hiljennystä ei kuitenkaan onnistuttu saamaan aikaan indusorin määrää säätelemällä. Muodostetulla mutantilla oli jatkuva qseC-geenin hiljennys, joka oli riippumaton indusoinnista. Mutantin qseC-geenin ilmentymisen kertoimen muutos villityyppiin verrattuna oli joillakin klooneilla jopa yli 30.
CRISPRi menetelmä on tehokas työkalu tärkeiden geenien tutkimiseen indusoitavasti ja säädettävällä hiljennystehokkuudella. GFP hiljennys oli indusoitava, mutta toimi "päällä-pois päältä" -mekanismilla. Hiljennysgradientin puutos saattaa johtua indusorin, IPTG, erilaisista kuljetustavoista soluun alhaisissa ja korkeissa pitoisuuksissa. qseC-geeni hiljennettiin kannassa UPEC CFT073, mutta jatkuvalla hiljentämisellä. Yksi syy jatkuvaan hiljentämiseen voi olla vuotava lac promoottori. Vaikka muodostettu kanta ei olekaan indusoitava, sillä voidaan tutkia qseC-geenin hiljentämisen vaikutusta UPEC:in virulenssiin tulevaisuudessa
Finnish translation, validation, and reproducibility of BREAST-Q modules relevant to breast cancer treatment
No full textBackground and objective: Breast cancer and its treatments can have a marked impact on the patient health-related quality of life. The aim of this study was to produce and validate Finnish versions of the breast-conserving treatment, mastectomy and breast reconstruction modules of the BREAST-Q, a patient-reported outcome tool designed specifically for women undergoing treatment for breast cancer. Methods: The relevant BREAST-Q modules were translated to Finnish according to established guidelines. Altogether 777 preoperative women were recruited at the Breast Surgery Unit and the Plastic Surgery Unit of Helsinki University Hospital between December 2019 and March 2021. This included 541 patients scheduled for breast-conserving surgery, 86 for mastectomy, and 150 for breast reconstruction. Postoperative patients were recruited through a postal survey, approaching 500 women operated for breast cancer in 2017, including 250 women who had undergone breast-conserving surgery and 250 women who had undergone mastectomy, as well as 339 women who had undergone breast reconstruction between August 2017 and July 2019. The patients were invited to fill the BREAST-Q modules relevant to their treatment and the general health-related quality-of-life instrument SF-36. A repeat administration of the BREAST-Q was done 2 weeks later. Results: A total of 665 (41%) women participated in the study, 339 (44%) preoperatively and 326 (39%) postoperatively. The BREAST-Q subscales showed high internal consistency with most Cronbach’s alphas > 0.8. The repeatability of the subscales was excellent with most intra-class correlation coefficients > 0.75. Low or negligible correlation was observed between BREAST-Q subscales and SF-36 domains. Conclusions: The Finnish version of the BREAST-Q modules breast-conserving treatment, mastectomy, and breast reconstruction performs well in assessing the health-related quality of life of women undergoing surgery for breast cancer or breast reconstruction.Peer reviewe
War exposure and changes in eating behaviours in Ukrainian school-aged children : A cross-sectional online survey
No full textThe war in Ukraine has exposed children to extremely high levels of acute and chronic stressors, which can impact their eating behaviour (EB). We aimed to determine the prevalence of war-induced, stress-related disruptions in EB of Ukrainian children. We conducted a cross-sectional online survey among parents of 5- to 17-year-old children, who had experienced the war in Ukraine in February–May 2023. Guardians reported their child's various war exposure changes in EBs using a modified version of the Child Eating Behaviour Questionnaire. We assessed associations between total and medium-term EB changes and age, sex, and war exposure using bivariate correlations and χ2 tests. Logistic regression models were fitted to explore the associations between socio-demographic factors, war exposure and frequency of EB changes. Of the 4854 children, 63% had changes in EBs during the war. The most common EB changes included food cravings (38%), food fussiness (37%), and aversion to certain foods (29%). Of the reported EB changes, 40% were medium-term, lasting over a month, and related to altered attitudes towards food. Food insecurity (adjusted OR 2.35, 95% CI: 1.76–3.14), and displacement (internally 2.01, 1.19–3.42) emerged as the most influential determinants of medium-term EB changes. The findings underscore a significant and robust association between war-related exposures and an increased risk of frequent EB changes. As healthy EBs are learned during childhood and have been shown to track into adulthood, the identified disruptions in EB may have medium-term consequences for the physical and mental health of Ukrainian children.Peer reviewe