Exploring the Potential of Cryo-Electron Tomography on Protein Nanocrystals for Molecular Structure Determination

The three-dimensional structure of a protein molecule, challenging to determine and close to impossible to predict, plays a key role in understanding protein function and has implications in drug design. When it comes to structure determination, there exist many complementary methods, each with their specific advantages and disadvantages. Most of those methods rely on a combined signal from thousands of individuals and cannot be used for directly reconstructing an actual 3d volume as it appears inside the sample. This thesis focuses on developing the methodology and providing proof of concept for a novel approach in structure determination by reconstructing small protein nanocrystals via cryo-electron tomography. Real-space imaging gets past the phase problem that is a challenging companion of conventional diffraction-based methods. With electron tomography we can reconstruct and visualize a 3d nanocrystal in its entirety and study the properties of small biological crystal from a new perspective. Being a relatively unexplored territory, nanocrystal tomography sets several challenges, such as creating nanocrystals small enough for imaging with transmission electron microscope and developing algorithms for going from a tilt-series to a 3d structure. For a proof of concept we create, image and reconstruct nanocrystals of hen egg white lysozyme that, having molecular weight of only 15 kDa, is generally considered unfeasible for electron tomography. Nanocrystals make finding and determining the relative orientations of the individual molecules possible, symmetry relations help reduce the effects of missing information, and by averaging we are able to reconstruct a molecular structure at a medium resolution of around 13 Å. Using Fourier Transform (FFT) we get a direct objective measure of the resolution of details within the reconstruction in the form of a diffraction pattern and show that in specific directions the resolution reaches as high as 7 Å in a single tomogram. Additionally, this work explores two other tightly related ideas. First, we study the concept of extended field and show with extensive simulations that extending the reconstruction space in various regularized iterative reconstruction procedures helps reduce the overall error and prevent over-smoothing. Second, calculating FFT of an image comes at a computational cost, and when the image is not periodic, the discontinuity of the opposing edges causes undesirable strong artifacts in the FFT that could obstruct important details. In this project we implemented a simultaneous 2d FFT and edge artifact removal for real-time applications on a Field Programmable Gate Array (FPGA) reconfigurable computing system.Okinawa Institute of Science and Technology Graduate Universit

Farmakoloogiline väljakutse diabeedi fenotüübi arengu pidurdamiseks Wolframi sündroomi näriliste mudelites

Väitekirja elektrooniline versioon ei sisalda publikatsiooneWolframi sündroom (WS) on autosomaalne retsessiivne haigus, mida põhjustavad mutatsioonid Wolframin1 (WFS1) geenis. Sündroomi põhisümptomiks on varajane suhkrudiabeet, millele järgneb optilise närvi atroofia, kurtus, psühhiaatrilised häired ja neurodegeneratsioon. Patsientid surevad 30-40 aastaselt piklikaju degeneratsioonist tingitud hingamise seiskumise tagajärjel. WS sagedus populatsioonis on 0.001% ja heterosügootsette WFS1 mutatsiooni kandjate sagedus 0.3-1%. On leitud, et heterosügootsetel WFS1 mutatsiooni kandjatel on suurenenud risk teist tüüpi diabeedi tekkeks. Täna puudub WS ravi, mis aitaks haiguse progressiooni efektiivselt pidurdada. Sellest tulenevalt üritasime leida uusi ravimikandidaate, mis võiksid aidata WS patsiente. Uurimistööks kasutasime transgeenseid Wfs1 puudulikke hiiri ja rotte. Esmalt leidsime, et muskariini 3 ja glükagooni-sarnase peptiidi-1 (GLP1) retseptorite akuutne stimulatsioon võimendas insuliini sekretsiooni ja normaliseeris veresuhkru taseme glükoositolerantsi testis Wfs1 puudulikel närilistel. Edasi uurisime GLP-1 retseptor agonisti liraglutiidi võimalikku WS kulgu pidurdavat toimet geeni puudulikus rotis. Erinevalt Wfs1 puudulikust hiirest arenes antud rottidel välja insuliinist sõltuv diabeet. Kahe kuu vanustel transgeensetel rottidel, kellel puudub Wfs1 geeni 5. kodeeriv ekson, on normaalne glükoositaluvus ja insuliini sekretsioon. Vanemaks saades hävinevad neil järk-järgult pankrease Langerhansi saarte beeta-rakud, süveneb glükoositalumatus ja 13 kuu vanuselt ilmneb neil insuliinist sõltuv diabeet. Alustades Wfs1 puudulike rottide ravi liraglutiidiga 2 kuu vanuselt (enne esimesi haiguse sümptomeid) leidsime 4.5 kuu möödudes, et ravimit saanud transgeensete rottide glükoositaluvus ei erinenud metsiktüüpi rottide omast. Näitasime, et liraglutiidi manustamine parandas insuliini ja glükagooni sekretsiooni ning vähendas Langerhansi saartes endoplasmaatilise retiikulumi stressi ja põletikumarkerite geeniekspressioone. Ravi tulemusena ei vähenenud rottide pankrease Langerhansi saarte mass, mis oli täheldatav ravi mittesaanud loomadel. Käesolevas töös leidsime esmakordselt, et ennetaval ravil GLP-1 agonistiga võib olla tugev WS vastane toime.Wolfram syndrome (WS) is an autosomal recessive disorder, caused by mutations in Wolframin1 (WFS1) gene. Main initial symptom is non-autoimmune diabetes mellitus, followed by optic nerve atrophy, deafness, psychiatric disorders, and neural degeneration. Patients usually die in their 30- and 40-s due to central apnea caused by brain stem atrophy. WS prevalence is estimated up to 0.001%, the carrier frequency is approximately 0.3-1%. Carriers of heterozygous WFS1 mutation have been shown to have increased risk for development of type 2 diabetes. Currently there is no cure to delay the progression of WS. Therefore, in current thesis, we aimed to identify new drug candidates for the treatment of WS. For this purpose, transgenic Wfs1 deficient mice and rats were used. Our first findings showed that stimulating muscarinic 3 and glucagon-like peptide-1 (GLP-1) receptors on Langerhans islets beta cells was able to stimulate insulin secretion in Wfs1 deficiency and to normalize blood glucose levels in glucose tolerance test. Next we aimed to study whether repeated treatment with GLP-1 receptor agonist liraglutide was able to delay disease progression in the rat model of WS. Unlike Wfs1 deficient mice these transgenic rats developed insulin dependent diabetes. Rats, missing 5th exon in Wfs1 gene, had normal glucose tolerance and insulin secretion at 2 months of age. After that they started to lose their pancreatic Langerhans islets beta cells and develop progressive glucose intolerance, resulting in insulin dependent diabetes at 13 months of age. Starting the chronic treatment of Wfs1 deficient rats at 2 months of age, before onset of disease symptoms, we established no changes in glucose tolerance of rats receiving medication for 4.5 months. We showed that liraglutide improved insulin and glucagon secretion and decreased endoplasmic reticulum stress and inflammatory markers gene expression in Langerhans islets. Due to these improvements liraglutide receiving rats did not lose Langerhans islet mass, as did untreated animals. In current study we established for the first time the potential therapeutic effect of preventive treatment with GLP-1 analogue against the progression of WS

Krooniline neeruhaigus lapseeas

Kroonilise neeruhaiguse (KNH) esinemissagedus on viimastel aastatel kogu maailmas suurenenud ja see on muutunud tõsiseks rahvatervise probleemiks. Võrreldes täiskasvanutega esineb lapseeas kroonilist neeruhaigust märkimisväärselt harvem. Lapsed moodustavad kõikidest KNH-patsientidest erinevatele kirjanduse allikatele tuginedes 5– 6%. Kõige enam on kirjanduses andmeid lõppstaadiumi neerupuudulikkuse kohta, kui muutub vajalikuks neeruasendusravi (NAR) alustamine. KNH varasemate staadiumite esinemissageduse kohta on infot vähem, sest haigus kulgeb sel perioodil enamasti asümptoomselt. KNH diagnostilises ja terapeutilises käsitluses tuleb pöörata tähelepanu neeruhaiguse primaarsele preventsioonile, varajasele avastamisele ja agressiivsele ravile. Artiklis on esitatud andmed lapseea KNH definitsiooni, klassifikatsiooni, esinemissageduse, sagedasemate põhjuste ja ravivalikute kohta. Toodud on ka andmed Tallinna Lastehaiglas jälgimisel olevate laste kohta, kellel on diagnoositud KNH

Reforms of Social Insurance Governance in a Quasi-Bismarckian Welfare State: How Estonia Moved from Tripartism to Direct State Management

Continental welfare regimes in Western Europe have in last decades gone through a set of paradigmatic reforms that significantly altered the initial Bismarckian principles of social insurance and welfare governance. Existing research overwhelmingly concludes that social partners have rendered up their power to the governments that inter alia facilitated expansion of non-contributory social security instruments. This article asks to what extent this reform trajectory can be found in quasi-Bismarkian welfare states, which already initially lacked solid social dialogue and favoured market-oriented approach to the social security. Tracing the process of main social policy governance reforms in Estonia 2009-2019 we test the assumption that fiscal prudence is in quasi-Bismarkian regimes important similarly to the typical Bismarckian countries, yet – it is achieved by using different means. The main empirical finding is that the governments in Estonia acted much more autocratically and used social insurance funds to execute various priorities of government programs. Differently from full Bismarckian regimes, insurance contributions were neither lowered nor complemented by tax instruments. We discuss these findings in the light of the institutional theory and welfare state change in times of increasing EU pressure

Microwave integrated circuit for Josephson voltage standards

A microwave integrated circuit comprised of one or more Josephson junctions and short sections of microstrip or stripline transmission line is fabricated from thin layers of superconducting metal on a dielectric substrate. The short sections of transmission are combined to form the elements of the circuit and particularly, two microwave resonators. The Josephson junctions are located between the resonators and the impedance of the Josephson junctions forms part of the circuitry that couples the two resonators. The microwave integrated circuit has an application in Josephson voltage standards. In this application, the device is asymmetrically driven at a selected frequency (approximately equal to the resonance frequency of the resonators), and a d.c. bias is applied to the junction. By observing the current voltage characteristic of the junction, a precise voltage, proportional to the frequency of the microwave drive signal, is obtained

Tool Support for Privacy-Enhanced Business Process Model and Notation

Käesolev töö käsitleb tugitööriista äriprotsessimudeli ja -notatsiooni privaatsuslaiendusele, mis täiendab äriprotsesside modelleerimiskeelt võimalustega lisada äriprotsessi mudelitele privaatsustehnoloogiate kirjeldusi. Äriprotsessimudeli ja -notatsiooni privaatsuslaiendus võimaldab visualiseerida privaatse informatsiooni liikumist ja avalikustamist äriprotsessides erinevate osapoolte vahel.Töö tulemusena valminud tööriist nimega PE-BPMN editor võimaldab luua privaatsuslaiendusega äriprotsessimudeli ja -notatsiooni mudeleid. Sealjuures pakub tööriist võimalust kontrollida nende mudelite süntaktilist korrektsust, mis on aluseks nende mudelite edasisteks analüüsideks. Praeguseks on võimalik kasutada kahte analüüsimeetodit, mille kombineeritud tulemus annab ülevaate äriprotsessis kasutatavast privaatsest informatsioonist, millel on oht lekkida. Saadud tulemus võimaldab täiustada olemasolevaid ja planeerida uusi turvalisemaid äriprotsesse.This paper presents an implementation tool for a Privacy-Enhanced Business Process Model And Notation language (PE-BPMN) that extends Business Process Modal And Notation (BPMN) by adding constructs to specify privacy enhancing technologies to be used in process models. PE-BPMN language allows to visualize the movement and disclosure of private information between participants of business processes. The language is used as a basis for detecting privacy leakages in business processes.The result of this work, the PE-BPMN editor provides a modelling tool for PE-BPMN. In addition, the tool supports the user by providing analyzers to check the syntactical correctness of these extended models. Syntactical correctness is a prerequisite of further analysis on PE-BPMN models. Currently, there are two analysis implemented. Combined results of these analysis give an overview of whether some information used in the business process is at risk of being leaked. Also, these results give an insight how to improve already existing processes or how to plan more secure new processes

Grid-scale Fluctuations and Forecast Error in Wind Power

The fluctuations in wind power entering an electrical grid (Irish grid) were analyzed and found to exhibit correlated fluctuations with a self-similar structure, a signature of large-scale correlations in atmospheric turbulence. The statistical structure of temporal correlations for fluctuations in generated and forecast time series was used to quantify two types of forecast error: a timescale error ($e_{\tau}$) that quantifies the deviations between the high frequency components of the forecast and the generated time series, and a scaling error ($e_{\zeta}$) that quantifies the degree to which the models fail to predict temporal correlations in the fluctuations of the generated power. With no $a$ $priori$ knowledge of the forecast models, we suggest a simple memory kernel that reduces both the timescale error ($e_{\tau}$) and the scaling error ($e_{\zeta}$)

Contemporary Welfare Regimes in Baltic States: Adapting Post-Communist Conditions to Post-Modern Challenges

This article revises the conventional approach to welfare state development in the post-communist world, according to which the main challenge for the Eastern European states is to catch up with Western European welfare regimes. The article argues that adjustment to the new social risks and volatile markets is more important today than the catching-up scenario. Based on social and labour market statistics for Estonia, Latvia and Lithuania, the article analyses how the Baltic States are coping with this adjustment challenge. Adaptation to the post-modern conditions can be regarded as successful if the stability of welfare spending is accompanied by the expanding and flexible employment and by the stable or decreasing level of poverty. The findings suggest that the opportunities to increase the flexibility and equality of the labour market provided by the breakdown of the communist regime were not used. Instead, the Baltic welfare states continue to focus on protecting against the old social risks by combining neoliberal and post-communist principles. Poor performance in meeting new social risks poses a greater challenge for the post-communist welfare states than their lag in terms of gross welfare expenditure

Uriiniinkontinents ja sellega kaasnevad kulud

Uriiniinkontinents – tahtele allumatu uriini väljavool põiest – on suhteliselt sage haigusseisund, mida esineb eriti vanemaealistel isikutel. Haigele põhjustab see raskeid psühholoogilisi ja sotsiaalseid probleeme. Eristatakse põhiliselt kahte inkontinentsitüüpi: mööduv ja tõeline inkontinents. Viimane omakorda jaotub 5 alatüübiks: funktsionaalne, sund-, rõhk-, ülevoolu- ja segatüüpi inkontinents. Inkontinentsi põhjuste täpse väljaselgitamise tulemusel on võimalik patisenti edukalt aidata. Eesti Arst 2008; 87(6):433−43