Image and Video Processing Methods for Studying hiPSC-derived Cardiomyocyte Biomechanics

Cardiovascular disease (CVD) is the most common cause of death globally. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide a suitable model for studying CVD and developing new treatments. Using hiPSC-CMs in heart disease modelling has several benefits compared to using other cell types such as embryonic stem cells or animal cells. Many cardiovascular diseases are known to affect cardiomyocyte behaviour including their biomechanics. Therefore, studying the contractile machinery and force production of hiPSC-CMs is central in CVD research. Microscopy imaging methods are required for scientists to view cells, their contractile structures and study their force production. Namely fluorescence microscopy and traction force microscopy -based imaging methods are often used in studying cellular biomechanics. To quantify and extract information from microscopy images and videos, image and video processing methods are required. Therefore, image and video processing methods are a central part of studying hiPSC-CM biomechanics and CVD. The ever-increasing amount of imaging data requires high-throughput, fast, accurate and automated image, and video processing methods. The aim of this thesis is to provide a review of the state-of-the-art image and video processing methods and tools that can be used for studying hiPSC-CM biomechanics. The focus of this thesis is on machine learning -based and fully automated image and video processing methods. Moreover, this thesis acts as an introductory guide of the available methods and tools for researchers interested in image and video processing for studying hiPSC-CM biomechanics. This thesis is divided into three main sections. In the first section the working principles of fluorescence microscopy and traction force microscopy are introduced in addition to their applications in studying hiPSC-CM biomechanics. In the second and third sections image and video processing methods and tools for fluorescence microscopy and traction force microscopy are introduced respectively.Sydän- ja verisuonisairaudet ovat maailmanlaajuisesti merkittävin kuolinsyy. Ihmisen indusoiduista pluripotenteista kantasolu-johteisista soluista (engl. human induced pluripotent stem cell, hiPSC) voidaan erilaistaa sydänlihassoluja. hiPSC-johteiset sydänlihassolut (engl. hiPSC-derived cardiomyocyte, hiPSC-CM) ovat osoittautuneet hyödyllisiksi sydänsairauksien mallinnuksessa. Niiden käyttöön liittyy useita etuja verrattuna esimerkiksi alkion kantasoluihin tai eläinperäisiin sydänlihassoluhin. Sydänsairaudet vaikuttavat sydänlihassolujen käyttäytymiseen ja niiden biomekaniikkaan. Tämän vuoksi sydänlihassolujen voimaatuottavien rakenteiden ja voiman tuottamisen tutkiminen on keskeistä sydänsairauksien mallinnuksessa ja uusien hoitojen kehityksessä. Jotta soluja, niiden voimaa tuottavia rakenteita ja voimantuottoa voidaan tutkia, tarvitaan mikroskooppikuvantamista. Erityisesti fluoresenssimikroskopia ja siihen perustuvat muut kuvantamismenetelmät kuten traktiomikroskopia (engl. traction force microscopy, TFM) ovat osoittautuneet hyödyllisiksi hiPSC-CM-solujen biomekaniikan tutkimuksessa. Kuvista tai videoista saadun informaation kvantifioimiseksi puolestaan tarvitaan erilaisia kuvan- ja videonkäsittelyn menetelmiä. Tämän vuoksi kuvan- ja videonkäsittelyn menetelmät ovat keskeinen osa hiPSC-CM-solujen biomekaniikan ja sydänsairauksien tutkimusta. Jatkuvasti kasvava kuvantamisdatan määrä vaatii suurikapasiteettisia, nopeita, tarkkoja ja automatisoituja kuvan- ja videonkäsittelyn menetelmiä. Siksi tämän kirjallisuuskatsauksen tavoitteena on esitellä sekä huippuluokan että uusimpia kuvan- ja videonkäsittelyn menetelmiä hiPSC-CM-solujen biomekaniikan tutkimukseen keskittyen automatisoituihin ja koneoppimiseen perustuviin menetelmiin. Lisäksi tämä työ toimii oppaana saatavilla olevista kuvan- ja videonkäsittelyn menetelmistä sekä työkaluista hiPSC-CM-solujen biomekaniikan tutkimuksesta kiinnostuneille tutkijoille. Tämä työ jakautuu kolmeen pääosaan. Ensin lukijalle esitellään työn taustaa hiPSC-CM-soluista sekä kahdesta mikroskooppikuvantamismenetelmästä. Ensin käsitellään fluoresenssimikroskopian ja sitten traktiomikroskopian toimintaperiaatteita ja sovelluksia hiPSC-CM-solujen biomekaniikan tutkimuksessa. Seuraavassa osiossa perehdytään fluoresenssimikroskopialle soveltuviin kuvan- ja videonkäsittelyn menetelmiin ja työkaluihin keskittyen esikäsittelyyn, segmentointiin ja kvantitatiiviseen analyysiin. Kolmannessa osiossa esitellään traktiomikroskopialle soveltuvia kuvan- ja videonkäsittelyn menetelmiä ja työkaluja keskittyen traktiovoimien määritykseen

Le comportement pausal dans l'oral spontané chez les locuteurs du français langue étrangère

Ce mémoire de master se situe dans le champ d’étude de l’aisance en langue étrangère, à l’oral spontané. Plus précisément, nous avons réalisé une étude de cas sur le comportement pausal des locuteurs finnophones du français langue étrangère. Le groupe de participants de notre étude est constitué de 12 étudiants en langue française. Notre intérêt était de savoir si la durée du séjour linguistique dans un pays francophone que les participants de notre étude ont effectué corrèle positivement avec leur niveau d’aisance à l’oral spontané. Nous nous sommes notamment penchée sur la durée totale des pauses non sonores et sonores ainsi que sur leur positionnement syntaxique dans les productions des participants de notre étude. Les pauses sonores, quant à elles, nous ont également intéressée d’un point de vue qualitatif et nous avons voulu savoir si les participants ayant fait un séjour linguistique plus long produisent plus de pauses sonores qui sont vues comme caractéristiques au français que ceux ayant fait un séjour plus court. Lors de l’enregistrement des productions orales spontanées, nous nous sommes appuyée sur un livre fréquemment utilisé pour collecter des données dans le domaine, un livre qui ne contient que des images : « Frog where are you ? » (Mayer, 2003). Nous avons demandé aux participants de raconter l’histoire de ce livre en se basant sur les images. À la fin des enregistrements, les participants ont également rempli un questionnaire portant sur leur utilisation du français et sur leur parcours d’apprentissage du français et ont répondu à un test de vocabulaire. Pour ce qui est de l’analyse des productions orales, nous nous sommes servie du logiciel Praat, à l’aide duquel il nous a été possible de segmenter, identifier les différents types de pauses et mesurer leur longueur d’une manière précise. L’analyse du corpus nous a permis de montrer qu’il existe un lien entre la longueur des séjours effectués dans un pays francophone et le comportement pausal en français L2 spontané, voire le niveau d’aisance à l’oral. Il ressort de l’analyse que les différences se manifestent non seulement au niveau des durées totales des pauses mais également au niveau des positionnements syntaxiques des pauses. Par exemple, il nous semble que plus le séjour dans un pays francophone est long, moins l’apprenant produit de pauses non sonores à l’intérieur d’une phrase et plus les pauses sonores qu’il produit sont caractéristiques à la langue française. Toutefois, l’échantillon très limité de notre étude ne nous permet pas de généraliser les résultats de notre recherche

Miten motivoida kansalaisia syöpäseulontoihin?

Teema : motivaati

Machine Learning and Pathway Analysis-Based Discovery of Metabolomic Markers Relating to Chronic Pain Phenotypes

Recent scientific evidence suggests that chronic pain phenotypes are reflected in metabolomic changes. However, problems associated with chronic pain, such as sleep disorders or obesity, may complicate the metabolome pattern. Such a complex phenotype was investigated to identify common metabolomics markers at the interface of persistent pain, sleep, and obesity in 71 men and 122 women undergoing tertiary pain care. They were examined for patterns in d = 97 metabolomic markers that segregated patients with a relatively benign pain phenotype (low and little bothersome pain) from those with more severe clinical symptoms (high pain intensity, more bothersome pain, and co-occurring problems such as sleep disturbance). Two independent lines of data analysis were pursued. First, a data-driven supervised machine learning-based approach was used to identify the most informative metabolic markers for complex phenotype assignment. This pointed primarily at adenosine monophosphate (AMP), asparagine, deoxycytidine, glucuronic acid, and propionylcarnitine, and secondarily at cysteine and nicotinamide adenine dinucleotide (NAD) as informative for assigning patients to clinical pain phenotypes. After this, a hypothesis-driven analysis of metabolic pathways was performed, including sleep and obesity. In both the first and second line of analysis, three metabolic markers (NAD, AMP, and cysteine) were found to be relevant, including metabolic pathway analysis in obesity, associated with changes in amino acid metabolism, and sleep problems, associated with downregulated methionine metabolism. Taken together, present findings provide evidence that metabolomic changes associated with co-occurring problems may play a role in the development of severe pain. Co-occurring problems may influence each other at the metabolomic level. Because the methionine and glutathione metabolic pathways are physiologically linked, sleep problems appear to be associated with the first metabolic pathway, whereas obesity may be associated with the second.Peer reviewe

Machine Learning and Pathway Analysis-Based Discovery of Metabolomic Markers Relating to Chronic Pain Phenotypes

Recent scientific evidence suggests that chronic pain phenotypes are reflected in metabolomic changes. However, problems associated with chronic pain, such as sleep disorders or obesity, may complicate the metabolome pattern. Such a complex phenotype was investigated to identify common metabolomics markers at the interface of persistent pain, sleep, and obesity in 71 men and 122 women undergoing tertiary pain care. They were examined for patterns in d = 97 metabolomic markers that segregated patients with a relatively benign pain phenotype (low and little bothersome pain) from those with more severe clinical symptoms (high pain intensity, more bothersome pain, and co-occurring problems such as sleep disturbance). Two independent lines of data analysis were pursued. First, a data-driven supervised machine learning-based approach was used to identify the most informative metabolic markers for complex phenotype assignment. This pointed primarily at adenosine monophosphate (AMP), asparagine, deoxycytidine, glucuronic acid, and propionylcarnitine, and secondarily at cysteine and nicotinamide adenine dinucleotide (NAD) as informative for assigning patients to clinical pain phenotypes. After this, a hypothesis-driven analysis of metabolic pathways was performed, including sleep and obesity. In both the first and second line of analysis, three metabolic markers (NAD, AMP, and cysteine) were found to be relevant, including metabolic pathway analysis in obesity, associated with changes in amino acid metabolism, and sleep problems, associated with downregulated methionine metabolism. Taken together, present findings provide evidence that metabolomic changes associated with co-occurring problems may play a role in the development of severe pain. Co-occurring problems may influence each other at the metabolomic level. Because the methionine and glutathione metabolic pathways are physiologically linked, sleep problems appear to be associated with the first metabolic pathway, whereas obesity may be associated with the second.Peer reviewe

The costs of offering HPV-testing on self-taken samples to non-attendees of cervical screening in Finland

Background: Offering self-sampling to non-attendees of cervical screening increases screening attendance. Methods: We used observations from two Finnish studies on the use of self-sampling among the non-attendees to estimate in a hypothetical screening population of 100,000 women the possible costs per extra screened woman and costs per extra detected and treated CIN2+ with three intervention strategies; 1) a primary invitation and a reminder letter, 2) a primary invitation and a mailed self-sampling kit and 3) two invitation letters and a self-sampling kit. The program costs were derived from actual performance and costs in the original studies and a national estimate on management costs of HPV related diseases. Results: The price per extra participant and price per detected and treated CIN2+ lesion was lower with a reminder letter than by self-sampling as a first reminder. When self-sampling was used as a second reminder with a low sampler price and a triage Pap-smear as a follow-up test for HPV-positive women instead of direct colposcopy referral, the eradication of a CIN2+ lesion by self-sampling was not more expensive than in routine screening, and the addition of two reminders to the invitation protocol did not increase the price of an treated CIN2+ lesion in the entire screened population. Conclusions: As a first reminder, a reminder letter is most likely a better choice. As second reminder, the higher costs of self-sampling might be compensated by the higher prevalence of CIN2+ in the originally non-attending population.Peer reviewe

Role of the cystathionine beta-synthase / H2S pathway in the development of cellular metabolic dysfunction and pseudohypoxia in down syndrome

Background: Overexpression of the transsulfuration enzyme cystathionine-beta-synthase (CBS), and overproduction of its product, hydrogen sulfide (H2S) are recognized as potential pathogenetic factors in Down syndrome (DS). The purpose of the study was to determine how the mitochondrial function and core metabolic pathways are affected by DS and how pharmacological inhibition of CBS affects these parameters. Methods: 8 human control and 8 human DS fibroblast cell lines have been subjected to bioenergetic and fluxomic and proteomic analysis with and without treatment with a pharmacological inhibitor of CBS. Results: DS cells exhibited a significantly higher CBS expression than control cells, and produced more H2S. They also exhibited suppressed mitochondrial electron transport and oxygen consumption and suppressed Complex IV activity, impaired cell proliferation and increased ROS generation. Inhibition of H2S biosynthesis with aminooxyacetic acid reduced cellular H2S, improved cellular bioenergetics, attenuated ROS and improved proliferation. C-13 glucose fluxomic analysis revealed that DS cells exhibit a suppression of the Krebs cycle activity with a compensatory increase in glycolysis. CBS inhibition restored the flux from glycolysis to the Krebs cycle and reactivated oxidative phosphorylation. Proteomic analysis revealed no CBS-dependent alterations in the expression level of the enzymes involved in glycolysis, oxidative phosphorylation and the pentose phosphate pathway. DS was associated with the dysregulation of several components of the autophagy network; CBS inhibition normalized several of these parameters. Conclusions: Increased H2S generation in DS promotes pseudohypoxia and contributes to cellular metabolic dysfunction by causing a shift from oxidative phosphorylation to glycolysis.Peer reviewe

Human papillomavirus self-sampling with mRNA testing benefits routine screening

High risk human papillomavirus (hrHPV) based screening provides the possibility of vaginal self-sampling as a tool to increase screening attendance. In order to evaluate the impact and feasibility of opt-in self-sampling in the Finnish setting, we invited a randomized population of 5350 women not attending screening after age group invitation or after reminder, to attend HPV self-sampling-based screening in the autumn of 2018 in Helsinki. Out of those, 1282 (24.0%) expressed their interest and ordered the sampling package. Eventually 787 women (14.7% of the total invited population) took part in screening, 770 women by providing a vaginal sample within 2 months from invitation and 17 by providing a pap smear in the laboratory. Self-taken samples were collected in Aptima Multitest vials and tested using the Aptima HPV mRNA assay. A high proportion, 158/770 (20.5%) of the samples were positive in the Aptima HPV assay. One hundred and forty-one samples were further submitted to Aptima HPV Genotyping and extended genotyping by a Luminex based assay. Of those, 23 samples (16.3%) were HPV 16 positive and 7 (5.0%) were positive for HPV 18/45; extended genotyping revealed multiple high-risk and low-risk HPV genotypes. At follow-up seven cases of high-grade squamous intraepithelial lesion (HSIL) were diagnosed, which represents 4.4% of HPV positive women and 0.9% of screened women, whereas the rate was 0.5% in routine screening. Our findings suggest that self-sampling with HPV mRNA testing is a feasible approach to improve screening efficacy in a high-risk population among original nonattendees.Peer reviewe

Vaginal Microbiota Composition Correlates Between Pap Smear Microscopy and Next Generation Sequencing and Associates to Socioeconomic Status

Recent research on vaginal microbiota relies on high throughput sequencing while microscopic methods have a long history in clinical use. We investigated the correspondence between microscopic findings of Pap smears and the vaginal microbiota composition determined by next generation sequencing among 50 asymptomatic women. Both methods produced coherent results regarding the distinction between Lactobacillus-dominant versus mixed microbiota, reassuring gynaecologists for the use of Pap smear or wet mount microscopy for rapid evaluation of vaginal bacteria as part of diagnosis. Cytologic findings identified women with bacterial vaginosis and revealed that cytolysis of vaginal epithelial cells is associated to Lactobacillus crispatus-dominated microbiota. Education and socio-economic status were associated to the vaginal microbiota variation. Our results highlight the importance of including socio-economic status as a co-factor in future vaginal microbiota studies.Peer reviewe

Deciphering downstream gene targets of PI3K/mTOR/p70S6K pathway in breast cancer

<p>Abstract</p> <p>Background</p> <p>The 70 kDa ribosomal protein S6 kinase (<it>RPS6KB1</it>), located at 17q23, is amplified and overexpressed in 10–30% of primary breast cancers and breast cancer cell lines. p70S6K is a serine/threonine kinase regulated by PI3K/mTOR pathway, which plays a crucial role in control of cell cycle, growth and survival. Our aim was to determine p70S6K and PI3K/mTOR/p70S6K pathway dependent gene expression profiles by microarrays using five breast cancer cell lines with predefined gene copy number and gene expression alterations. The p70S6K dependent profiles were determined by siRNA silencing of <it>RPS6KB1 </it>in two breast cancer cell lines overexpressing p70S6K. These profiles were further correlated with gene expression alterations caused by inhibition of PI3K/mTOR pathway with PI3K inhibitor Ly294002 or mTOR inhibitor rapamycin.</p> <p>Results</p> <p>Altogether, the silencing of p70S6K altered the expression of 109 and 173 genes in two breast cancer cell lines and 67 genes were altered in both cell lines in addition to <it>RPS6KB1</it>. Furthermore, 17 genes including <it>VTCN1 </it>and <it>CDKN2B </it>showed overlap with genes differentially expressed after PI3K or mTOR inhibition. The gene expression signatures responsive to both PI3K/mTOR pathway and p70S6K inhibitions revealed previously unidentified genes suggesting novel downstream targets for PI3K/mTOR/p70S6K pathway.</p> <p>Conclusion</p> <p>Since p70S6K overexpression is associated with aggressive disease and poor prognosis of breast cancer patients, the potential downstream targets of p70S6K and the whole PI3K/mTOR/p70S6K pathway identified in our study may have diagnostic value.</p