Light-sheet fluorescent microscopy in tardigrade anoxybiosis

Tardigrades are invertebrates that are known for their tolerance to extreme conditions. This research focuses on anoxia, the total lack of oxygen, and the adaptation and survival of anoxiainduced experiments. Cryptobiosis is a form of dormancy that enables the survival of the animals, however, many forms of cryptobiosis are still poorly understood and the mechanisms and physiological responses are not entirely explained. This study aimed to create a protocol that utilizes fluorescent dyes to visualize the transition into anoxybiosis and morphometric changes at the cellular level involved in the phenomenon. Lightsheet fluorescent microscopy enabled fast 3D volumetric scanning of the transition and revealed what happened to the animal when anoxia was chemically applied. Results were aligned with the expectations: during anoxybiosis, tardigrades became immobilized and swollen leading to the relocation and reorganization of cells. Importantly it was observed that variation throughout the experiments was quite significant and in further studies, this should be outlined. In this research, two specimens of M. ripperi were used and obtained data were compared including cell number, volume, and displacement over time. The most fundamental issue is how to gain stable and reproducible results. Tardigrade cuticle and overall variation of body state of the animals create sources of error, particularly dyeing by soaking. By soaking, there are very few possibilities to control, how the dye is distributed and attached to the cellular compartments, therefore in this study we did not pay so close attention to the statistical significance of the results. Animals chosen for the experiments were random and therefore variables such as sex, age, and fasting were not included. Fluorescent microscopy is a widely used method in biological studies and this study showed that it can be used in live imaging of tardigrades. However, as a pilot experiment, this led to many open questions and features to improve, especially in the image analysis part. Large datasets need a lighter pipeline to gain a higher throughput method. In the future, the (light sheet) fluorescent imaging can be a beneficial tool for similar cellular studies; however, the individual sources of variation need to be minimized. Tardigrades can be a promising model organism for studies including cell survival, cancer research, and storage and storing solutions for various drug components when understanding the mechanisms that enable the animal stress tolerance and survival

Distinctive effects of SGLT2 inhibitors on angiogenesis in zebrafish embryos

Sodium glucose cotransporter 2 (SGLT2) inhibitor canagliflozin has been found to increase the risk for lowerlimb amputations in type 2 diabetics about two-fold. Conversely, empagliflozin and dapagliflozin do not display a similar effect. A question arises whether the increased risk for minor amputations is associated only with canagliflozin or whether it is a class effect of SGLT2 inhibitors. Defective angiogenesis has a role in amputations. We compared the effects of empagliflozin, dapagliflozin and canagliflozin on angiogenesis in vivo using zebrafish model, and in vitro using human umbilical vein endothelial cells (HUVECs). The effects of SGLT2 inhibitors on the formation of intersegmental blood vessels (ISVs) of the zebrafish embryos were clarified. Additionally, transcriptome analysis was performed to explore whether putative angiogenesis-associated genes are differentially regulated by SGLT2 inhibitors. The effects of SGLT2 inhibitors on the viability of HUVECs were examined. We noticed that especially empagliflozin and also dapagliflozin significantly accelerated the formation of ISVs of zebrafish embryos. In contrast, canagliflozin was not able to stimulate ISV formation, and at high concentration, it was lethal to the embryos. Transcriptome analysis demonstrated that in empagliflozin-treated embryos compared to canagliflozin-treated embryos seven genes previously shown to contribute to angiogenesis were upregulated, and four downregulated. Canagliflozin at high concentrations, but not empagliflozin or dapagliflozin, decreased the viability of HUVECs and disrupted their capability to sprout. SGLT2 inhibitors differed in their effects on angiogenic processes in zebrafish embryos and on the viability of HUVECs suggesting that the risk of SGLT2 inhibitors for peripheral amputations likely differs

Indiumtinaoksidin etsauskemia ja sen vaikutus reunakarheuksista aiheutuviin defekteihin

Opinnäytetyö tehtiin Beneq Oy:lle, joka tarjoaa atomikerroskasvatukseen (ALD) pohjautuvia pinnoitusratkaisuja, valmistaa laitteita ALD-prosesseihin sekä soveltaa ALD-tekniikkaa näyttöihin. Opinnäytetyö keskittyi näyttöpuolen tuotannon litografiaprosessin syvempää ymmärtämiseen liittyen etsauskemiaan ja sen suhdetta indiumtinaoksidin (ITO) sekä alumiinititaanioksidin (ATO) käyttäytymiseen tässä prosessin vaiheessa. Etsauskemian optimoinnilla pyrittiin vaikuttamaan reunakarheuden suuruuteen, sillä reunakarheuksien on todettu vaikuttavan tuotteen kestävyyteen. Indiumtinaoksidi on mielenkiintoinen metallioksidi, keraami. Se on puolijohtava materiaali, jolla on sekä johtava että eristävä ominaisuus. Indiumtinaoksidi on näkyvän valon aallonpituudella läpinäkyvä. Tämä on hyvin haluttu ominaisuus monissa teknologian sovelluksissa. Laajalti levinneestä käytöstä huolimatta indiumtinaoksidin ominaisuuksia ei täysin tunneta. Prosesseissa on herkästi hajontaa sekä optimien hakeminen on työlästä. Jotta voidaan saavuttaa riittävä läpinäkyvyys ja sähköinen johtavuus on yhdistettä ymmärretävä erityisen tarkasti. Kaikkien haluttujen ominaisuuksien valjastaminen vaatii syvää prosessin tuntemusta. Opinnäytetyön tavoite oli löytää märkäkemialliseen prosessiin parannuksia hakemalla etsausreagenssien kautta parempi etsausreunan laatu ja selvittää tämän muutoksen merkitys lopputuotteeseen. Kokeellisessa osuudessa tuotetta kuormitettiin lämpötilalla ja korkealla ajotaajuudella. Paranneltua etsausliuosta verrattiin käytössä olevaan liuokseen ja tuloksissa vertailtiin rakenteellisten muutosten syntymistä ajan suhteen. Näitä defektejä kuvattiin valomikroskoopilla sekä elektronipyyhkäisymikroskoopilla. Testieriä ehdittiin vanhentaa reilu 1 000 tuntia. Testit osoittivat, ettei tuotteen laatu ja kestävyys dramaattisesti muutu etsauskemian myötä, mutta jatkotutkimuksille on syytä varata lisää työtunteja