Single-cell behavior and population heterogeneity: Fluorescence microscopy-based inverse population balance modeling

Cell population balance models can account for the phenotypic heterogeneity that characterizes isogenic cell populations. To utilize the predictive power of these models, however, we must determine the single-cell reaction and division rates as well as the partition probability density function of the cell population. These functions (collectively called Intrinsic Physiological State or IPS functions) can be obtained through the Collins-Richmond inverse cell population balance modeling methodology, if we know the phenotypic distributions of (a) the overall cell population, (b) the dividing cell subpopulation and (c) the newborn cell subpopulation. This first part of this thesis presents the development of a novel assay that combines fluorescence microscopy and image processing to determine these phenotypic distributions. Morphological criteria were developed for the automatic identification of dividing cells and validated through direct comparison with manually obtained measurements. The newborn cell subpopulation was obtained from the corresponding dividing cell subpopulation by collecting information from the two compartments separated by the constriction. Finally, we applied the assay to quantify the heterogeneity of E. coli cells carrying the genetic toggle network with a green fluorescent marker. Our measurements for the overall cell population were in excellent agreement with the distributions obtained via flow cytometry. In the second part of the thesis, we develop and test a robust computational procedure for solving the inverse problem that yields the IPS functions. We employed numerical simulations in conjunction with a thorough parametric analysis to investigate the effect of various factors on the accurate recovery of the IPS functions. We also formulated and solved a minimization problem to obtain the bivariate partition probability density function (PPDF), which presents the most computational challenges of all three IPS functions. We successfully tested our method against uncertainty stemming from both finite sampling and measurements errors in the experimental data. We also investigated the feasibility of a more general solution for the PPDF and proposed methods to extend and solve the inverse problem in 2-D. Finally, we demonstrated the abilities and potential of our method by applying it to a model biological system involving E. coli cells carrying the toggle artificial regulatory network

Μικτό ηπατοκυτταρικό- χολαγγειοκαρκίνωμα: Ιστολογία, παθογένεια, κλινικά χαρακτηριστικά, θεραπευτική προσέγγιση

Τα δυο συχνότερα πρωτοπαθή επιθηλιακά καρκινώματα του ήπατος είναι το ηπατοκυτταρικό καρκίνωμα και το χολαγγειοκαρκίνωμα με αντίστοιχη κυτταρική προέλευση. Το μικτό ηπατοκυτταρικό καρκίνωμα- χολαγγειοκαρκίνωμα αποτελεί ένα ιδιαίτερο τύπο πρωτοπαθούς καρκινώματος ήπατος προσφάτως αναγνωρισμένο από τον ΠΟΥ, η παθογένεση του οποίου έχει συσχετισθεί με ηπατικά αρχέγονα κύτταρα. Στο εμβρυϊκό ήπαρ τα πολυδύναμα αρχέγονα κύτταρα είναι οι ηπατοβλάστες, από τους οποίους προκύπτουν τα ηπατοκύτταρα και τα χολαγγειοκύτταρα. Βασικοί δείκτες των ηπατοβλαστών είναι οι Dlk1, E-cadherin και EpCAM. Στο ήπαρ του ενήλικα κύτταρα ανιχνεύονται αρχέγονα/προγονικά με διττό ανοσοφαινότυπο, παρόμοια με τα ωοειδή κύτταρα των τρωκτικών. Ως πιθανότερη φωλεά των ηπατικών αρχέγονων κυττάρων θεωρούνται οι πόροι του Hering, χολαγγειόλια και μικρά χολαγγεία, ενώ τελευταία υποστηρίζεται η ύπαρξη και άλλων πιθανών φωλεών όπως κατά μήκος των μεγάλων χοληφόρων πόρων στα περιχολαγγειακά αδένια. Δείκτες ταυτοποίησης των κυττάρων αυτών είναι οι K19, EpCAM, CD133, Sox9, osteopontin, MIC1-1C3, Trop2, Foxl 1 και ο Lgr5. Άλλες πιθανές πηγές προέλευσης των ηπατικών αρχέγονων κυττάρων είναι τα χολαγγειοκύτταρα και τα ηπατοκύτταρα μετά από αποδιαφοροποίηση, ενώ ως εξωηπατική πηγή θεωρείται ο μυελός των οστών. Βασικά ρυθμιστικά σηματοδοτικά μονοπάτια ανάπτυξης των αρχέγονων κυττάρων του ήπατος είναι τα Wnt, Notch και FGF. Η επικρατούσα θεωρία προέλευσης του ΜΗΧΚ σχετίζεται με τα καρκινικά αρχέγονα κύτταρα και στηρίζεται κατά κύριο λόγο στην απομόνωση κυττάρων από πρωτοπαθείς όγκους του ήπατος με λειτουργικά χαρακτηριστικά σωματικών αρχέγονων καρκινικών κυττάρων, όπως αθανασία, αντίσταση στη θεραπεία και αποτελεσματική μεταμόσχευση, ενω η εξαλλαγή των ηπατικών αρχέγονων κυττάρων εχει πειραματικά ενοχοποιηθεί και για την ανάπτυξη ΗΚΚ και ΧΚ. Πιθανολογείται ότι σημαντικό ρόλο στην παθογένεση διαδραματίζει και η οδός της αποδιαφοροποίησης κακοήθων κυττάρων ηπατοκυτταρικού και χολαγγειοκυτταρικού τύπου σε κύτταρα με αρχέγονους χαρακτήρες. Στη διαδικασία εξαλλαγής των ηπατικών αρχέγονων κυττάρων σε καρκινικά εμπλέκονται τα μονοπάτια Wnt, Notch, Hedgehog, οι πρωτεΐνες της ομάδας PcG και η σηματοδοτική οδος TGF-β. Η συχνότητα του μικτού ηπατοκυτταρικού καρκινώματος- χολαγγειοκαρκινώματος (ΜΗΧΚ) ποικίλλει από 0.4% έως 14.2% στο σύνολο των όγκων του ήπατος και όπως και το ΗΚΚ αναπτύσσεται στα πλαίσια χρόνιας εξελικτικής ηπατικής νόσου. Αναγνωρίζονται δύο ιστολογικοί τύποι ΜΗΧΚ : το ΜΗΧΚ κλασσικού τύπου και το ΜΗΧΚ με χαρακτηριστικά αρχέγονων κυττάρων, το οποίο υποκατηγοριοποιείται σε 3 υποτύπους: στον τυπικό υπότυπο, τον ενδιάμεσο και τον χολαγγειολοκυτταρικό. Υπάρχουν ειδικοί δείκτες που προσδιορίζουν τον ανοσοφαινότυπο της καθε συνιστώσας του όγκου και έχουν διαγνωστική σημασία. Μια πιο πρόσφατη ταξινόμηση των πρωτοπαθών καρκινωμάτων του ήπατος είναι αυτή της Βrunt και συνεργατών σύμφωνα με την οποία εκτός από το συμβατικό ΗΚΚ και ΧΚ προτείνεται το διφαινοτυπικό ηπατοχολικό καρκίνωμα καθώς και οι τύποι του αμιγούς ΗΚΚ με εστιακή έκφραση χολαγγειοκυτταρικών δεικτών (ΗΚΚ θετικό για κερατίνη 19) και αμιγούς ΧΚ με ηπατοκυτταρικό ανοσοφαινότυπο εστιακά. Σε πειραματικές μελέτες έχει παρατηρηθεί ομοιογένεια μεταξύ των διαφορετικών συνιστωσών του όγκου σε μοριακό επίπεδο καθώς και ομοιότητα των γενετικών υπογραφών με το χολαγγειοκαρκίνωμα. Το ΜΗΧΚ δεν παρουσιάζει διαφορές στα κλινικά του χαρακτηριστικά με τα άλλα πρωτοπαθή καρκινώματα του ήπατος, ενώ διαφέρει ως προς τα απεικονιστικά πρότυπα. Η χειρουργική αφαίρεση του όγκου αποτελεί προς το παρόν τη θεραπεία επιλογής. Ασθενείς με ΜΗΧΚ που υποβάλλονται σε μεταμόσχευση ήπατος εμφανίζουν υψηλότερα ποσοστά 5-ετούς επιβίωσης από τους μη μεταμοσχευμένους. Η πρόγνωση του ΜΗΧΚ παραμένει δυσμενής με 5-ετή επιβίωση 12-18%.The two most common carcinomas of the liver are hepatocellular carcinoma and cholangiocarcinoma, which originate from hepatocytes and cholangiocytes respectively. Combined hepatocellular-cholangiocarcinoma is a new type of liver carcinoma, recently defined by WHO, which is pathogenetically related to hepatic stem cells. The multipotent stem cells of the fetal liver are hepatoblasts which give rise to hepatocytes and cholangiocytes. In the adult normal liver stem/progenitor cells HPCs (similar to oval cells in rodent) exist in low numbers around the periportal region. The hepatic progenitor cells (HPC) compartment comprises a spectrum of subtypes of variable differentiation including isolated progenitor cells and string-like ductular structures (DS) at the portal/parenchymal interface known as ductular reaction. The HPC immunophenotype includes hepatocytic, cholangiocellular, hepatoblastic and hematopoietic stem cell markers The most likely niche are the canals of Hering and terminal ductules, while recent work suggests the existence of other niches, such as interlobular bile ducts and peribiliary glands. Other sources of origin are hematopoietic stem cells which may be recruited by chemo-attractants from the bone marrow. Basic signaling pathways that regulate the development of hepatic progenitor cells are Wnt, Notch and FGF. Furthermore, there are sufficient data available suggesting that adult liver progenitors may also derive from mature hepatocytes and cholangiocytes through a dedifferentiation process in the context of acute and chronic liver injury. The most dominant theory regarding the origin of combined hepatocellular-cholangiocarcinoma is connected to cancer stem cells and is based on the isolation of cells from primary hepatic tumors with functional features of somatic cancer stem cells like immortality, resistance to treatment and effective transplantation. Furthermore it has been proven experimentally that the malignant transformation of hepatic stem cells leads to the development of hepatocellular carcinoma and cholangiocarcinoma. It has been postulated that the dedifferentiation of malignant hepatocellular and cholangiocellular cells into cell with stem like features plays a role in the pathogenesis of combined hepatocellular-cholangiocarcinoma. Cellular pathways involved in the malignant transformation of hepatic progenitor cells are Wnt, Notch, Hedgehog, the PcG group proteins and the signaling pathway TGF-β. Combined hepatocellular-cholangiocarcinoma accounts for 1.0% -14.2% of primary liver cancer cases and develops in the context of chronic hepatic disease. There are two histologic types of combined hepatocellular-cholangiocarcinoma: classical type combined hepatocellular-cholangiocarcinoma and combined hepatocellular-cholangiocarcinoma with stem cell features. The latter is divided into 3 subcategories: the typical subtype, intermediate subtype and cholangiocellular subtype. Specific markers define the immunophenotype of each tumor component, and are of diagnostic significance. A recent categorization of the primary hepatic carcinomas suggested by Brunt et al. apart from hepatocellular carcinoma and cholangiocarcinoma, the entity of biphenotypic hepatobiliary carcinoma is introduced. In addition, hepatocellular carcinoma with focal expression of biliary markers (HCC positive for CK 19) and cholangiocarcinoma with focal hepatocellular immunophenotype expression are recognized as separate types. Experimental studies have identified molecular homogeneity between the tumor components, as well as similarities of the genetic signatures with cholangiocarcinoma. Combined hepatocellular-cholangiocarcinoma shows no differences in clinical presentation compared to the other primary liver carcinomas, but it presents distinct imaging patterns. Surgical resection of the tumor is the treatment of choice a while patients that undergo liver transplantation exhibit a better 5-year survival compared to non-tranplants. The prognosis of combined hepatocellular-cholangiocarcinoma is poor with a 5-year survival of 12-18%

Φωτοαναγωγή του νερού με χρήση συμπλόκων του χαλκού(ΙΙ) ως καταλυτών

Η παρούσα πτυχιακή εργασία εκπονήθηκε στο Εργαστήριο Ανόργανης Χημείας του Εθνικού και Καποδιστριακού Πανεπιστημίου Αθηνών. Καλύπτει φωτοκαταλυτικά συστήματα παραγωγής υδρογόνου ομογενούς κατάλυσης. Έγινε προσπάθεια ανίχνευσης υδρογόνου στα εξής συστήματα: [Ru(bpy)3]Cl2/ [Cu(pq)2NO3]NO3*6H2O/ AscOH/AscONa, Fluorescein/ [Cu(pq)2NO3]NO3*6H2O/ TEOA. Επίσης, λήφθηκαν φάσματα UV-Vis στα παραπάνω συστήματα. Το σύμπλοκο [Ru(bpy)3]Cl2 και η οργανική ένωση Fluorescein χρησιμοποιήθηκαν ως φωτοευαισθητοποιητές (PS), το σύμπλοκο [Cu(pq)2NO3]NO3*6H2O λειτούργησε καταλυτικά στο σύστημα (cat) και το ασκορβικό οξύ και η τριαιθυνολαμίνη είχαν το ρόλο του δότη ηλεκτρονίων (D). Τέλος, το νερό (milli-Q/H2O) χρησιμοποιήθηκε ως διαλύτης. Είναι σημαντικό να αναφερθεί το γεγονός ότι τα παραπάνω συστήματα είναι από τα ελάχιστα που λειτουργούν σε υδατικές συνθήκες. Για να ανιχνευτεί το υδρογόνο χρησιμοποιήθηκε αέριος χρωματογράφος (GC).This work took place within the graduate thesis in the field of Inorganic Chemistry in the National and Kapodistrian University of Athens. This includes literature of photo-catalytic systems for hydrogen production in homogeneous catalysis. There has been conducted an effort to detect hydrogen in the following systems: [Ru(bpy)3]Cl2/ [Cu(pq)2NO3]NO3*6H2O/ AscOH/AscONa, Fluorescein/ [Cu(pq)2NO3]NO3*6H2O/ TEOA. Furthermore, we used UV-Vis spectroscopy in the above systems. The [Ru(bpy)3]Cl2 and Fluorescein were used as photosensitizers (PS), the [Cu(pq)2NO3]NO3*6H2O complex was used as catalyst and Ascorbic acid and Triethanolamine (TEOA) were used as electron donors. Also, water (milli-Q/H2O) was used as solvent in these systems. It is important to mention the fact that the aforementioned systems are just a few that can work in aquatic conditions. For hydrogen detection we used a gas chromatographer (GC)

Metabolic resting-state brain networks in health and disease

The delineation of resting state networks (RSNs) in the human brain relies on the analysis of temporal fluctuations in functional MRI signal, representing a small fraction of total neuronal activity. Here, we used metabolic PET, which maps nonfluctuating signals related to total activity, to identify and validate reproducible RSN topographies in healthy and disease populations. In healthy subjects, the dominant (first component) metabolic RSN was topographically similar to the default mode network (DMN). In contrast, in Parkinson\u27s disease (PD), this RSN was subordinated to an independent disease-related pattern. Network functionality was assessed by quantifying metabolic RSN expression in cerebral blood flow PET scans acquired at rest and during task performance. Consistent task-related deactivation of the DMN-like dominant metabolic RSN was observed in healthy subjects and early PD patients; in contrast, the subordinate RSNs were activated during task performance. Network deactivation was reduced in advanced PD; this abnormality was partially corrected by dopaminergic therapy. Time-course comparisons of DMN loss in longitudinal resting metabolic scans from PD and Alzheimer\u27s disease subjects illustrated that significant reductions appeared later for PD, in parallel with the development of cognitive dysfunction. In contrast, in Alzheimer\u27s disease significant reductions in network expression were already present at diagnosis, progressing over time. Metabolic imaging can directly provide useful information regarding the resting organization of the brain in health and disease

A Neural Approach to Ordinal Regression for the Preventive Assessment of Developmental Dyslexia

Developmental Dyslexia (DD) is a learning disability related to the acquisition of reading skills that affects about 5% of the population. DD can have an enormous impact on the intellectual and personal development of affected children, so early detection is key to implementing preventive strategies for teaching language. Research has shown that there may be biological underpinnings to DD that affect phoneme processing, and hence these symptoms may be identifiable before reading ability is acquired, allowing for early intervention. In this paper we propose a new methodology to assess the risk of DD before students learn to read. For this purpose, we propose a mixed neural model that calculates risk levels of dyslexia from tests that can be completed at the age of 5 years. Our method first trains an auto-encoder, and then combines the trained encoder with an optimized ordinal regression neural network devised to ensure consistency of predictions. Our experiments show that the system is able to detect unaffected subjects two years before it can assess the risk of DD based mainly on phonological processing, giving a specificity of 0.969 and a correct rate of more than 0.92. In addition, the trained encoder can be used to transform test results into an interpretable subject spatial distribution that facilitates risk assessment and validates methodology.Comment: 12 pages, 4 figure

Renin–angiotensin system gene polymorphisms among Saudi patients with coronary artery disease

BACKGROUND: Idiopathic REM sleep behavior disorder is a prodromal stage of Parkinson's disease and dementia with Lewy bodies. Hyposmia, reduced dopamine transporter binding, and expression of the brain metabolic PD-related pattern were each associated with increased risk of conversion to PD. The objective of this study was to study the relationship between the PD-related pattern, dopamine transporter binding, and olfaction in idiopathic REM sleep behavior disorder. METHODS: In this cross-sectional study, 21 idiopathic REM sleep behavior disorder subjects underwent (18) F-fluorodeoxyglucose PET, dopamine transporter imaging, and olfactory testing. For reference, we included (18) F-fluorodeoxyglucose PET data of 19 controls, 20 PD patients, and 22 patients with dementia with Lewy bodies. PD-related pattern expression z-scores were computed from all PET scans. RESULTS: PD-related pattern expression was higher in idiopathic REM sleep behavior disorder subjects compared with controls (P = 0.048), but lower compared with PD (P = 0.001) and dementia with Lewy bodies (P < 0.0001). PD-related pattern expression was higher in idiopathic REM sleep behavior disorder subjects with hyposmia and in subjects with an abnormal dopamine transporter scan (P < 0.05, uncorrected). CONCLUSION: PD-related pattern expression, dopamine transporter binding, and olfaction may provide complementary information for predicting phenoconversion. © 2017 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society

Independent Component Analysis of Resting State Activity in Pediatric Obsessive-Compulsive Disorder

Obsessive-compulsive disorder (OCD) is an often severely disabling illness with onset generally in childhood or adolescence. Little is known, however, regarding the pattern of brain resting state activity in OCD early in the course of illness. We therefore examined differences in brain resting state activity in patients with pediatric OCD compared with healthy volunteers and their clinical correlates. Twenty-three pediatric OCD patients and 23 healthy volunteers (age range 9-17), matched for sex, age, handedness, and IQ completed a resting state functional magnetic resonance imaging exam at 3T. Patients completed the Children\u27s Yale Brown Obsessive Scale. Data were decomposed into 36 functional networks using spatial group independent component analysis (ICA) and logistic regression was used to identify the components that yielded maximum group separation. Using ICA we identified three components that maximally separated the groups: a middle frontal/dorsal anterior cingulate network, an anterior/posterior cingulate network, and a visual network yielding an overall group classification of 76.1% (sensitivity=78.3% and specificity=73.9%). Independent component expression scores were significantly higher in patients compared with healthy volunteers in the middle frontal/dorsal anterior cingulate and the anterior/posterior cingulate networks, but lower in patients within the visual network. Higher expression scores in the anterior/posterior cingulate network correlated with greater severity of compulsions among patients. These findings implicate resting state fMRI abnormalities within the cingulate cortex and related control regions in the pathogenesis and phenomenology of OCD early in the course of the disorder and prior to extensive pharmacologic intervention. Hum Brain Mapp 35:5306-5315, 2014. (c) 2014 Wiley Periodicals, Inc

Automatic covariance pattern analysis outperforms visual reading of 18 F‐fluorodeoxyglucose‐positron emission tomography (FDG‐PET) in variant progressive supranuclear palsy

Background: To date, studies on positron emission tomography (PET) with F-18-fluorodeoxyglucose (FDG) in progressive supranuclear palsy (PSP) usually included PSP cohorts overrepresenting patients with Richardson's syndrome (PSP-RS). Objectives: To evaluate FDG-PET in a patient sample representing the broad phenotypic PSP spectrum typically encountered in routine clinical practice. Methods: This retrospective, multicenter study included 41 PSP patients, 21 (51%) with RS and 20 (49%) with non-RS variants of PSP (vPSP), and 46 age-matched healthy controls. Two state-of-the art methods for the interpretation of FDG-PET were compared: visual analysis supported by voxel-based statistical testing (five readers) and automatic covariance pattern analysis using a predefined PSP-related pattern. Results: Sensitivity and specificity of the majority visual read for the detection of PSP in the whole cohort were 74% and 72%, respectively. The percentage of false-negative cases was 10% in the PSP-RS subsample and 43% in the vPSP subsample. Automatic covariance pattern analysis provided sensitivity and specificity of 93% and 83% in the whole cohort. The percentage of false-negative cases was 0% in the PSP-RS subsample and 15% in the vPSP subsample. Conclusions: Visual interpretation of FDG-PET supported by voxel-based testing provides good accuracy for the detection of PSP-RS, but only fair sensitivity for vPSP. Automatic covariance pattern analysis outperforms visual interpretation in the detection of PSP-RS, provides clinically useful sensitivity for vPSP, and reduces the rate of false-positive findings. Thus, pattern expression analysis is clinically useful to complement visual reading and voxel-based testing of FDG-PET in suspected PSP. (C) 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society

Differences between Chronological and Brain Age Are Related to Education and Self-Reported Physical Activity

This study investigated the relationship between education and physical activity and the difference between a physiological prediction of age and chronological age (CA). Cortical and subcortical gray matter regional volumes were calculated from 331 healthy adults (range: 19-79 years). Multivariate analyses identified a covariance pattern of brain volumes best predicting CA (R(2) = 47%). Individual expression of this brain pattern served as a physiologic measure of brain age (BA). The difference between CA and BA was predicted by education and self-report measures of physical activity. Education and the daily number of flights of stairs climbed (FOSC) were the only 2 significant predictors of decreased BA. Effect sizes demonstrated that BA decreased by 0.95 years for each year of education and by 0.58 years for 1 additional FOSC daily. Effects of education and FOSC on regional brain volume were largely driven by temporal and subcortical volumes. These results demonstrate that higher levels of education and daily FOSC are related to larger brain volume than predicted by CA which supports the utility of regional gray matter volume as a biomarker of healthy brain aging