Rakkude paljunemist soodustav AKT signaalirada kui potentsiaalne kasvajavastase ravi sihtmärk

Väitekirja elektrooniline versioon ei sisalda publikatsioone.AKT kinaas on seriin/treoniinkinaas, mis kuulub AGC valkude perekonda. Sihtmärkvalke fosforüleerides reguleerib AKT rakkudes mitmeid olulisi füsioloogilisi protsesse nagu rakkude elulemus, rakutsükli edenemine, metabolism, transkriptsioon, valkude süntees, rakkude liikuvus jne. Kontrollimatult aktiivset PI3K/AKT signaalirada on kirjeldatud inimese maksa-, aju-, rinna-, soole- ja eesnäärmekasvajates ning seda loetakse mõnede kasvajate puhul halva prognoosi markeriks. Käesoleva töö raames leidsime, et aktiveeritud AKT signaalirada osaleb ka healoomulise hüperproliferatiivse patoloogia – Dupuytreni kontraktuuri - arengus. AKT kinaasi olulisuse ja sagedase seotuse tõttu kasvajate tekkimisega, peetakse teda heaks märklauaks kasvajavastaste terapeutikumide väljatöötamisel. Praeguseks on avastatud ja kliinilistesse katsetustesse võetud juba mitmeid AKT signaaliraja inhibiitoreid, kuid paraku on tugevad mittespetsiifilised kõrvaltoimed ja toksilisus takistanud nende edasise kasutamise raviskeemides. Käesoleva doktoritöö põhieesmärk oli leida ja iseloomustada AKT signaaliraja aktiivsust pärssivaid väikesemolekulaarseid aineid, mida võiks kasutada potentsiaalsete kasvajavastaste ravimitena. Otsitavate inhibiitorite täpsem sihtmärk oli AKT1 ja PDPK1 valkude omavaheline interaktsioon, mis on vajalik AKT1 esmaseks aktiveerimiseks. Väikesemolekulaarsete ainete skriinimine valgu komplementaarsusel põhinevat lähenemisviisi kasutades ning sellele järgnenud erinevatest meetoditest koosnenud valideerimisprotsess tuvastas ühe kemikaali – NSC156529 - mis pidurdas erinevat päritolu kasvajarakkude paljunemist nii koekultuuri tingimustes kui in vivo hiire kasvajamudelis. Tuumorirakkude töötlemine NSC156529 kemikaaliga mõjutas lisaks AKT kinaasi enda aktiivsusele ka AKT-i märklaudvalkude aktiivsust, viidates sellele, et antud kemikaal mõjutab AKT signaaliraja poolt reguleeritud raku biokeemilisi protsesse. Kuna in vivo kasvajamudelitest eraldatud tuumorite analüüsimisel leiti NSC156529 kemikaaliga töödeldud kasvajates rakkude diferentseerumisele viitavate markerite suurenenud ekspressioon, järeldati, et lisaks suudab NSC156529 tuumorirakkude paljunemist pärssida neid diferentseeruma suunates. Käesoleva töö kokkuvõtteks võib öelda, et AKT kinaasiga seotud signaalirada on sobiv märklaud kasvajavastaste terapeutikumide väljatöötamiseks ning testitud ühend NSC156529 võiks olla potentsiaalne kandidaat AKT signaaliraja aktiivsuse pärssimiseks kasvajarakkudes.AKT protein is a serine/threonine kinase, which belongs to AGC group of protein kinases. By substrate phosphorylation AKT regulates several physiological processes in the cells, such as cell survival, cell cycle promotion, metabolism, transcription, translation, cell migration etc. Aberrantly activated PI3K/AKT signaling has been frequently found in several human tumors, like liver, brain, breast, colorectal and prostate cancers, and it is a poor prognostic marker for a number of cancer types. In this work we found that the activated AKT signaling might play a role in the progression of benign hyperproliferative pathology – the Dupuytren’s contracture. Due to AKT involvement in critical steps of human tumor pathogenesis, targeting AKT pathway has become a promising strategy in anti-cancer therapy. Although a number of small molecule AKT kinase inhibitors have been developed and tested in clinical trials, severe side effects have prevented their use in current treatment schemes. The main purpose of the present thesis was to identify and characterize the inhibitors of AKT signaling pathway that could be used as potential anti-cancer drugs. The exact target of the inhibitors was the AKT1-PDPK1 interaction, which is the first step in AKT activation cascade. Protein complementation-based screening and the following experiments revealed one chemical – NSC156529 – which inhibited the growth of tumor cells from different origin in cell culture as well as in a mouse tumor xenograft model. In addition to AKT activity inhibition, cancer cell incubation with NSC156529 chemical also reduced the phosphorylation of AKT downstream target proteins, which confirmed that NSC156529 treatment inhibited the key biochemical activities of the AKT signaling pathway. The analysis of NSC156529-treated xenografts revealed increased expression level of differentiation markers, suggesting that NSC156529 could limit tumor growth at least in part by directing cancer cells to differentiate. Conclusively, inhibiting activated AKT-regulated signaling pathway in tumor cells is a promising anti-cancer drug target and the small molecular compound NSC156529 could be a potent suppressor of AKT1 pathway in tumor cells harboring active AKT signaling

Proteases—From Basic Structure to Function to Drug Design as Targeted Therapy

In the two last decades, proteases have constituted one of the primary and important targets in drug discovery. The U.S. FDA has approved more than 12 protease therapies in the last 10 years, and a number of next-generation or completely new proteases are under clinical development. Protease inhibition strategies are one of the fastest expanding areas in the field of of drugs that show considerable promise. This Special Issue will focus on the recent advances in the discovery and development of protease inhibitors, covering the synthesis of protease inhibitors, the design of new chemical entities acting as inhibitors of special/particular types of proteases, and their mode of actions (Frolova et al. 2020; Slapak et al. 2020; Künnapuu et al. 2021). In addition, the new applications of these interesting compounds/biomolecules and their limitations have been discussed and described (Wang et al. 2020; Bartošová-Sojková et al. 2021)

tmRNA kasutamine markermolekulina bakterite tuvastamisel mikrokiibi ja biosensor tehnoloogia kaudu

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Bakterite uurimiseks on traditsiooniliselt kasutatud erinevaid kultuuris kasvatamise meetodeid. Kuigi mainitud meetodid on töökindlad ja täpsed, on nad samas ka küllaltki töö- ja ajamahukad ning ei võimalda kõikide bakterite uurimist. Seetõttu kasutatakse tänapäeval tihti nende uurimiseks erinevaid molekulaarbioloogilisi meetodeid, mis põhinevad spetsiifiliste nukleiinhappe järjestuste tuvastamisel ja kirjeldamisel. Käesolevas töös tutvustatakse erinevaid tehnoloogiaid, mida kasutatakse nukleiinahappe põhises bakteriaalses diagnostikas. Põhirõhk on erinevatel nukleiinhapete paljundamise meetoditel ning hübridisatsiooni-põhistel detektsiooni tehnoloogiatel. Käsitletud on erinevate mikrokiibi ja biosensor tehnoloogiate põhimõtteid ning nende võimalikke kasutusviise bakterite tuvastamisel. Lisaks antakse ülevaade DNA ja RNA järjestustest, mida saab kasutada markerjärjestusena erinevate bakterite tuvastamisel ja üksteisest eristamisel. Pikemalt tutvustatakse tmRNA molekule, mida kasutatakse markerjärjestusena käesoleva doktoritöö raames välja töötatud diagnostiliste meetodite puhul. tmRNA on kõikides bakterites leiduv, keskmiselt 300-400 nukleotiidi pikkune spetsiifiline RNA molekul, mis abistab rakus valgusünteesi mehhanismi, ning mille molekuli järjestuse põhjal on võimalik tuvastada ning eristada erinevaid bakteriliike ja ka kõrgemaid taksonoomilisi üksusi. Töö praktilises osas kirjeldatakse kahte erinevat meetodit, kus tmRNA detektsiooni kaudu tuvastatakse erinevaid baktereid. Nendest esimene põhineb tmRNA molekulide spetsiifilisel paljundamisel NASBA tehnoloogia abil, millele järgneb märgistatud tmRNA molekulide tuvastatamine ja täpne identifitseerimine mikrokiibi tehnoloogia abil. Teise puhul toimub tmRNA-de detektsioon märkevaba reaal-ajas toimiva biosensor süsteemi abil, mis põhineb optilisel mikroring resonaator tehnoloogial. Kuigi mõlema meetodi puhul kasutati testsüsteemina erinevaid hingamisteede haigusi põhjustavaid baktereid ning nende vastavaid liigispetsiifilisi tmRNA molekule, on kirjeldatud tehnoloogiad lihtsasti rakendatavad ka teiste RNA järjestuste ning erinevate bakteri-liikide korral.There is a growing need for faster and more reliable approaches for microorganism detection and identification that could complement or replace conventional rather time- and labor-consuming culture-based technologies. A common tactics nowadays is to analyze the nucleic acid component of analyte solution and determine the bacterial composition according to specific nucleic acid profiles that are detected and identified. Theoretically every bacterial species and strain contain unique characteristic target regions that can be used for their specific identification. In the first part of current thesis a literature overview is given about the different technologies that are used for nucleic acid-based bacterial detection. Main focus is on nucleic acid amplification and hybridization-based detection methods with emphasis on microarray and biosensor technologies, and their practical application in bacterial diagnostics. In second part of the literature overview, a description of different DNA and RNA molecules that have been targeted for bacterial detection and identification is reviewed. Longer explanation is given about the trans-translation mediating RNA molecule called tmRNA that is used as a target marker molecule in the current thesis. The research section describes two different methods that apply tmRNA for bacterial detection and identification. Firstly, a microarray-based technology is described where target tmRNA molecules are amplified using Nucleic Acid Sequence Based Amplification (NASBA) and labeled fluorescently prior the hybridization experiment. The developed method was applied for tmRNA detection from bacterial total RNA samples. In second part of the research tmRNA molecules are specifically targeted using real-time label-free biosensing platform that is based on the optical microring resonator technology. Potential quantitative nature and sensitivity of the biosensor is demonstrated using in vitro synthesized tmRNA molecules

TAXONOMY, MORPHOLOGY, AND RNA-SEQ TRANSCRIPTOMICS OF THE CUBOZOAN ALATINA ALATA, AN EMERGING MODEL CNIDARIAN

Cnidarians are often considered simple animals, but the more than 13,000 estimated species (e.g., corals, hydroids and jellyfish) of the early diverging phylum exhibit a broad diversity of forms, functions and behaviors, some of which are demonstrably complex. In particular, cubozoans (box jellyfish) are cnidarians that have evolved a number of distinguishing features. Some cubozoan species possess complex mating behaviors or particularly potent stings, and all possess well-developed light sensation involving image-forming eyes. Like all cnidarians, cubozoans have specialized subcellular structures called nematocysts that are used in prey capture and defense. The objective of this study is to contribute to the development of the box jellyfish Alatina alata as a model cnidarian. This cubozoan species offers numerous advantages for investigating morphological and molecular traits underlying complex processes and coordinated behavior in free-living medusozoans (i.e., jellyfish), and more broadly throughout Metazoa. First, I provide an overview of Cnidaria with an emphasis on the current understanding of genes and proteins implicated in complex biological processes in a few select cnidarians. Second, to further develop resources for A. alata, I provide a formal redescription of this cubozoan and establish a neotype specimen voucher, which serve to stabilize the taxonomy of the species. Third, I generate the first functionally annotated transcriptome of adult and larval A. alata tissue and apply preliminary differential expression analyses to identify candidate genes implicated broadly in biological processes related to prey capture and defense, vision and the phototransduction pathway and sexual reproduction and gametogenesis. Fourth, to better understand venom diversity and mechanisms controlling venom synthesis in A. alata, I use bioinformatics to investigate gene candidates with dual roles in venom and digestion, and review the biology of prey capture and digestion in cubozoans. The morphological and molecular resources presented herein contribute to understanding the evolution of cubozoan characteristics and serve to facilitate further research on this emerging cubozoan model

COVID19 Disease Map, a computational knowledge repository of virus-host interaction mechanisms.

Funder: Bundesministerium für Bildung und ForschungFunder: Bundesministerium für Bildung und Forschung (BMBF)We need to effectively combine the knowledge from surging literature with complex datasets to propose mechanistic models of SARS-CoV-2 infection, improving data interpretation and predicting key targets of intervention. Here, we describe a large-scale community effort to build an open access, interoperable and computable repository of COVID-19 molecular mechanisms. The COVID-19 Disease Map (C19DMap) is a graphical, interactive representation of disease-relevant molecular mechanisms linking many knowledge sources. Notably, it is a computational resource for graph-based analyses and disease modelling. To this end, we established a framework of tools, platforms and guidelines necessary for a multifaceted community of biocurators, domain experts, bioinformaticians and computational biologists. The diagrams of the C19DMap, curated from the literature, are integrated with relevant interaction and text mining databases. We demonstrate the application of network analysis and modelling approaches by concrete examples to highlight new testable hypotheses. This framework helps to find signatures of SARS-CoV-2 predisposition, treatment response or prioritisation of drug candidates. Such an approach may help deal with new waves of COVID-19 or similar pandemics in the long-term perspective

Healing Spaces

The field of design and health, formerly known as the domain of healthcare design professionals, has now reached a turning point with the proliferation of a plethora of non-invasive wearable technologies, to provide the objective and near-real-time measurement of the impact of many features of the built environment on aspects of health, wellbeing and performance. In turn, new materials and the Internet of Things are allowing the development of smart buildings, which can interact with occupants to optimize their health, wellbeing, performance and overall experience. Companies that have previously focused on positioning themselves as “green” are now turning to positioning themselves in the marketplace as both green and healthy. This Special Issue will include articles that address new cutting edge technologies and materials at the interface between design and health, and review some of the latest findings related to studies which use these technologies. This SI will also suggest exciting future directions for the field. It will include articles which focus on the objective data gathered to document the effects of the built environment on health. Importantly, it will focus on the use of innovative methods of measurement, such as state-of-the-art wearable and environmental sensors, quantifying some aspects of health, such as stress and relaxation responses, activity, posture, sleep quality, cognitive performance and wellbeing outcomes. It will also examine the impacts of different elements of the built environment on these health and wellbeing outcomes. The published articles will focus on the design interventions informed by these measurements, along with innovative integrated building materials that can shape the design of built environments for better health, productivity, and performance. It will also address the return on investment (ROI) of such design interventions. This Special Issue will provide both the foundational knowledge and fundamentals for characterizing human health and wellbeing in the built environment, as well as the emerging trends and design methods for innovations in this field

Plant Virus Emergence

This compilation of articles elaborates on plant virus diseases that are among the most recent epidemiological concerns. The chapters explore several paradigms in plant virus epidemiology, outbreaks, epidemics, and pandemics paralleling zoonotic viruses and that can be consequential to global food security. There is evidence that the local, regional, national, and global trade of agricultural products has aided the global dispersal of plant virus diseases. Expanding farmlands into pristine natural areas has created opportunities for viruses in native landscapes to invade crops, while the movement of food and food products disseminates viruses, creating epidemics or pandemics. Moreover, plant virus outbreaks not only directly impact food supply, but also incidentally affect human health