Molecular reponses of cell adhesion upon cell-cell contact initiation in a 2-D ultrasound standing wave trap

This thesis explores the interactions of cells and particles brought into contact in suspension in an ultrasound standing wave trap as aggregates or monolayers. The influence of CaCb , and hence modulation of electrostatic particle interaction force, on the growth and morphology of aggregates of 25 urn latex spheres was initially characterised by techniques including fractal dimension, and void analysis. At low CaCb additions highly hexagonally ordered structures were formed while with increasing CaCb additions the aggregate morphology gradually 'crossed over' from being essentially closely packed to a dendritic, highly disordered one. The physical environment of cells aggregating in the trap was then examined to assess the extent to which ultrasound might influence cell viability and function. The temperature rise was < 0.5 K. The drag due to acoustic streaming was smaller than the stress imposed by gentle preparative cell centrifugation. The attractive acoustic force between cells was small compared to the force required to reverse a single receptor- receptor bond and to the attractive van der Waals force. Acoustic spectrum analysis detected no cavitation activity in the suspensions. Fluorescent indicators showed that the number of viable cells (99 %) did not change during 1 h in the trap. The progression of cell-cell interactions i.e. length of membrane-membrane contact, and the integral intensities of stained cadherin/catenin complex molecules, the actin cytoskeleton and (for chondrocytes) gap junctions were examined over 60 min in primary chondrocytes, and in neural, prostate epithelial and prostate cancer cell lines. The aggregate form changed concurrently from hexagonally ordered cells to a continuous sheet of mostly quadrilateral and pentagonal cells. Rapid molecular responses to cell contact in other systems are reviewed. The cells in this work progressed from physical aggregation, through molecular adhesion, to displaying the intracellular consequences of receptor interactions. The ability to form mechanically strong confluent cell monolayers that can be monitored in situ or harvested from the trap provides a technique with general potential for monitoring the synchronous development of cell responses to receptor-triggered adhesion

Η γνώση και η αποδοχή της θεωρίας της Εξέλιξης ως παράγοντες που επηρεάζουν τις στάσεις των φοιτητών απέναντι στη διαφορετικότητα

Σκοπός της παρούσας μελέτης είναι η διερεύνηση του εάν και (εάν ναι) πώς η γνώση και η αποδοχή της Θεωρίας της Εξέλιξης μπορούν να επηρεάσουν τη στάση ενός ατόμου απέναντι στη διαφορετικότητα. Η εξελικτική θεωρία, η κεντρική ενοποιητική θεωρία στη Βιολογία, έχει την ιδιότητα να ερμηνεύει ταυτόχρονα την ποικιλομορφία και την ενότητα των ζωντανών οργανισμών. Πρόκειται για την επιστημονική θεωρία που ερμηνεύει την εμφάνιση των ανθρώπων στη Γη, το πώς διαφορετικές ομάδες ανθρώπων σχετίζονται μεταξύ τους και το πώς αποκτήσαμε πολλά από τα γνωρίσματά μας. Αντίστοιχα, η έννοια της διαφορετικότητας αναφέρεται στη συνύπαρξη ανθρώπων με διαφορετικά γενετικά ή επίκτητα χαρακτηριστικά όπως είναι: το φύλο, η ηλικία, η φυλή/χρώμα, η αναπηρία/πάθηση, η εθνική/εθνοτική καταγωγή και ο σεξουαλικός προσανατολισμός. Παρά το ότι δεν εντοπίσαμε στη βιβλιογραφία έρευνες σχετικά με την άμεση συσχέτιση των δύο εννοιών, ορμώμενοι από τη φράση «η σωστή κατανόηση της εξέλιξης, συμπεριλαμβανομένης της εκτίμησης της δυνητικής αξίας κάθε είδους στο περιβάλλον, θα έπρεπε να ενισχύει τους ηθικούς σκοπούς» (Blackwell et al., 2003), θεωρήσαμε μία ενδιαφέρουσα προσέγγιση για τη διπλωματική αυτή εργασία τη διερεύνηση των εξελικτικών παραμέτρων (Γνώση και Αποδοχή) που τυχόν επηρεάζουν τις στάσεις κάποιου απέναντι στη διαφορετικότητα. Η εξελικτική γνώση συνιστά τον βαθμό κατανόησης των εξελικτικών εννοιών, καθώς και την ύπαρξη τυχόν παρανοήσεων που παρεμποδίζουν την αντίληψη των εξελικτικών φαινομένων και της ιστορίας της ζωής στον πλανήτη. Πολλές εξελικτικές διαδικασίες είναι δύσκολο να κατανοηθούν λόγω των χρονικών και χωρικών διαστάσεων που εμπλέκονται, ενώ τα γνωστικά εμπόδια για την κατανόηση της εξέλιξης μέσω της φυσικής επιλογής περιλαμβάνουν εννοιολογικές δυσκολίες, μεθοδολογικά ζητήματα και προβλήματα συνοχής που απορρέουν από τη διαισθητικότητα των εναλλακτικών ιδεών των μαθητών. Από την άλλη, η θεωρία της εξέλιξης (ΘτΕ) αποτελεί ένα θέμα που συχνά απαιτεί τη διείσδυση στο σύστημα πεποιθήσεων ενός ατόμου προτού έλθει η αποδοχή της, με πλήθος θρησκευτικών, επιστημονικών, κοινωνικών, συναισθηματικών και επιστημολογικών παραγόντων να εμπλέκονται και να επηρεάζουν την εξελικτική αποδοχή. Παρομοίως, οι προσωπικές πεποιθήσεις που επηρεάζονται από το κοινωνικό περιβάλλον και οι προθέσεις ανοιχτής διάθεσης συνιστούν τις πιο καθοριστικές παραμέτρους για τη θέση που θα λάβει κανείς απέναντι στη διαφορετικότητα. Η έρευνα που πραγματοποιήθηκε ήταν ποσοτική με ερωτηματολόγιο σε πρωτοετείς ως επί το πλείστον φοιτητές του Τμήματος Βιολογίας και του ΠΤΔΕ από το ΕΚΠΑ (n=136), προκειμένου να αξιολογηθεί η στάση τους απέναντι στη ΘτΕ, όπως αυτή διαμορφώθηκε κατά κύριο λόγο μετά την ολοκλήρωση της Δευτεροβάθμιας Εκπαίδευσης. Το ερωτηματολόγιο διαμορφώθηκε από προϋπάρχουσες σταθμισμένες διεθνείς κλίμακες για την εξέταση κάθε παράγοντα, οι οποίες συνδυάστηκαν με ορισμένα δημογραφικά στοιχεία και διαμοιράστηκε σε αίθουσες διδασκαλίας και εργαστηρίων μέσω προσωπικών επισκέψεων της ερευνήτριας. Οι κλίμακες τις οποίες χρησιμοποιήσαμε ήταν: Η υπο-κλίμακα των 21 ερωτήσεων των Rutlege και Warden (2000) με στοιχεία που εξετάζουν τη γνώση σε ένα σύνολο εξελικτικών εννοιών, η κλίμακα ΜΑΤΕ (Measurement of Acceptance of Theory of Evolution) για την αξιολόγηση των επιπέδων αποδοχής της ΘτΕ και η κλίμακα της Εθνοπολιτισμικής Ενσυναίσθησης -Scale of Ethnocultural Empathy (SEE) για τη μέτρηση των στάσεων απέναντι στη διαφορετικότητα. Τα αποτελέσματα της έρευνας υποδηλώνουν μία χαμηλή γνώση και μία μέτρια προς υψηλή αποδοχή της εξελικτικής θεωρίας από τους φοιτητές του δείγματός μας, οι οποίες είναι στατιστικά υψηλότερες για τους φοιτητές του Τμήματος Βιολογίας και όσους φοιτητές δήλωσαν να έχουν προηγουμένως διδαχθεί σε ακαδημαϊκό μάθημα την εξελικτική θεωρία. Το μέσο σκορ Εθνοπολιτισμικής Ενσυναίσθησης των συμμετεχόντων βρέθηκε μέτριο προς υψηλό, όντας ένα από τα υψηλότερα σκορ που έχουν καταγραφεί με την κλίμακα SEE και χωρίς να διαφοροποιείται μεταξύ των επιμέρους φοιτητικών ομάδων. Ταυτόχρονα, οι τρεις εξεταζόμενες μεταβλητές συσχετίζονται θετικά ανά δύο μεταξύ τους, σε στατιστικά σημαντικό επίπεδο, για το σύνολο του δείγματος, ενώ οι επιμέρους συσχετίσεις διαμορφώνονται ως εξής: α) θετική συσχέτιση μεταξύ Γνώσης και Αποδοχής της ΘτΕ που διατηρείται και όταν το δείγμα μελετάται κατά ομάδες, β) θετική συσχέτιση μεταξύ Γνώσης της ΘτΕ και Εθνοπολιτισμικής Ενσυναίσθησης για τους φοιτητές που δήλωσαν να έχουν προηγουμένως κάποια επαφή με την Εξελικτική Θεωρία, γ) θετική συσχέτιση μεταξύ Εθνοπολιτισμικής Ενσυναίσθησης και Αποδοχής της ΘτΕ για τους φοιτητές του ΠΤΔΕ καθώς και δ) όσους φοιτητές δήλωσαν ότι δεν είχαν προηγούμενη επαφή με την ΘτΕ. Βασικά συμπεράσματα της εργασίας είναι η σχετικά χαμηλή εξελικτική γνώση που επιτυγχάνεται από την ελληνική Δευτεροβάθμια Εκπαίδευση, καθώς και η ανάγκη ενίσχυσης της εξελικτικής διδασκαλίας προς αύξηση, όχι μόνο των επιπέδων κατανόησης και αποδοχής της ΘτΕ από τους μελλοντικούς Έλληνες πολίτες, αλλά και των όποιων θετικών επιπτώσεων της εξελικτικής μόρφωσης στη στάση απέναντι στη διαφορετικότητα.The purpose of this study is to investigate whether and (if so) how the knowledge and the acceptance of the Evolutionary Theory can influence an individual's attitude towards diversity. Evolutionary Theory (ET), the central unifying theory in Biology, has the ability to simultaneously interpret the diversity and the unity of living organisms. It is the scientific theory that explains the appearance of humans on Earth, how different groups of people relate to each other and how we acquired many of our traits. Respectively, the concept of (cultural) diversity refers to the coexistence of people with different genetic or acquired characteristics such as: gender, age, race/color, disability/disease, ethnic/cultural origin and sexual orientation. Although we did not find any research in the literature on the direct correlation of the two concepts, driven by the phrase “a proper understanding of evolution, including appreciation of the potential value of every species in the environment, should enhance ethical purposes” (Blackwell et al., 2003), we considered investigating the evolutionary parameters (Knowledge and Acceptance) that may influence one's attitudes towards diversity to be an interesting approach for this dissertation. Evolutionary knowledge is the degree of understanding of the evolutionary concepts, as well as the possible existence of misconceptions that hinder the perception of evolutionary phenomena and the history of life on the planet. Many evolutionary processes are difficult to be understood due to the temporal and spatial dimensions involved, while cognitive barriers to understanding evolution through natural selection include conceptual difficulties, methodological issues and coherence problems arising from the intuition of students' alternative ideas. Evolution theory, on the other hand, is an issue that often requires penetration into an individual's belief system before it can be accepted, with a number of religious, scientific, social, emotional and epistemological factors involved and influencing evolutionary acceptance. Similarly, personal beliefs influenced by the social environment and openness in intentions are the most decisive parameters for one's position towards diversity. The research we conducted was a quantitative survey using a questionnaire, with the participation of (first-year mostly) students of the Department of Biology and the Pedagogical Department of Primary Education of the University of Athens (n = 136), as our purpose was to assess their attitudes towards ET the way they were formed during Secondary Education. The questionnaire was formulated from pre-existing weighted international scales examining each factor that were combined with certain demographics and it was distributed in classrooms and lab-classes through personal visits by the researcher. The scales we used were: The Rutlege and Warden (2000) sub-scale of 21 questions with elements that examine knowledge in a set of evolutionary concepts, the MATE scale (Measurement of Acceptance of Theory of Evolution) for assessing the levels of acceptance of ET and the Scale of Ethnocultural Empathy (SEE) for measuring attitudes towards diversity. Results of the research indicate a low knowledge and a moderate to high acceptance of evolutionary theory by the students of our sample, which are statistically higher for the students of the Department of Biology and those students who stated that they had previously been taught evolutionary theory in an academic course. The average score of Ethnocultural Empathy of the participants was found to be moderate to high, being one of the highest scores recorded with the SEE scale, with no differentiation between the student subgroups we examined. At the same time, the three studied variables were all positively correlated with each other, at a statistically significant level and for the entire students sample, while the subgroups correlations were: a) a positive correlation between the Knowledge and the Acceptance of the ET maintained when the sample was studied in groups, b) a positive correlation between ET Knowledge and Ethnocultural Empathy for students who stated they had some previous contact with ET, c) a positive correlation between Ethnocultural Empathy and ET Acceptance for the students of the Department of the Pedagogical Department of Primary Education, as well as d) the group of students denied any previous contact with ET. The main conclusions of the current study are the relatively low evolutionary knowledge achieved after the completion of the Greek Secondary Education, plus the need to strengthen the evolutionary teaching in order to increase, not only the levels of the knowledge and the acceptance of ET by the future Greek citizens, but also the possible effects that evolutionary education offers in the forming of positive attitudes towards diversity

Identification of Protein Biomarker Signatures for Acute Myeloid Leukemia (AML) Using Both Nontargeted and Targeted Approaches

Acute myeloid leukemia (AML) is characterized by an increasing number of clonal myeloid blast cells which are incapable of differentiating into mature leukocytes. AML risk stratification is based on genetic background, which also serves as a means to identify the optimal treatment of individual patients. However, constant refinements are needed, and the inclusion of significant measurements, based on the various omics approaches that are currently available to researchers/clinicians, have the potential to increase overall accuracy with respect to patient management. Using both nontargeted (label-free mass spectrometry) and targeted (multiplex immunoassays) proteomics, a range of proteins were found to be significantly changed in AML patients with different genetic backgrounds. The inclusion of validated proteomic biomarker panels could be an important factor in the prognostic classification of AML patients. The ability to measure both cellular and secreted analytes, at diagnosis and during the course of treatment, has advantages in identifying transforming biological mechanisms in patients, assisting important clinical management decisions.Peer reviewe

Next generation proteomics with drug sensitivity screening identifies sub-clones informing therapeutic and drug development strategies for multiple myeloma patients

With the introduction of novel therapeutic agents, survival in Multiple Myeloma (MM) has increased in recent years. However, drug-resistant clones inevitably arise and lead to disease progression and death. The current International Myeloma Working Group response criteria are broad and make it difficult to clearly designate resistant and responsive patients thereby hampering proteo-genomic analysis for informative biomarkers for sensitivity. In this proof-of-concept study we addressed these challenges by combining an ex-vivo drug sensitivity testing platform with state-of-the-art proteomics analysis. 35 CD138-purified MM samples were taken from patients with newly diagnosed or relapsed MM and exposed to therapeutic agents from five therapeutic drug classes including Bortezomib, Quizinostat, Lenalidomide, Navitoclax and PF-04691502. Comparative proteomic analysis using liquid chromatography-mass spectrometry objectively determined the most and least sensitive patient groups. Using this approach several proteins of biological significance were identified in each drug class. In three of the five classes focal adhesion-related proteins predicted low sensitivity, suggesting that targeting this pathway could modulate cell adhesion mediated drug resistance. Using Receiver Operating Characteristic curve analysis, strong predictive power for the specificity and sensitivity of these potential biomarkers was identified. This approach has the potential to yield predictive theranostic protein panels that can inform therapeutic decision making.Peer reviewe

Acoustic trapping in bubble-bounded micro-cavities

We present a method for controllably producing longitudinal acoustic trapping sites inside microfluidic channels. Air bubbles are injected into a micro-capillary to create bubble-bounded ‘micro-cavities’. A cavity mode is formed that shows controlled longitudinal acoustic trapping between the two air/water interfaces along with the levitation to the centre of the channel that one would expect from a lower order lateral mode. 7 µm and 10 µm microspheres are trapped at the discrete acoustic trapping sites in these micro-cavities. We show this for several lengths of micro-cavity

CLEC-2-dependent activation of mouse platelets is weakly inhibited by cAMP but not by cGMP

The present results demonstrate that platelet adhesion and activation on CLEC-2 ligands or LECs is maintained in the presence of PGI2 and NO

Flow-induced HDAC1 phosphorylation and nuclear export in angiogenic sprouting

Angiogenesis requires the coordinated growth and migration of endothelial cells (ECs), with each EC residing in the vessel wall integrating local signals to determine whether to remain quiescent or undergo morphogenesis. These signals include vascular endothelial growth factor (VEGF) and flow-induced mechanical stimuli such as interstitial flow, which are both elevated in the tumor microenvironment. However, it is not clear how VEGF signaling and mechanobiological activation due to interstitial flow cooperate during angiogenesis. Here, we show that endothelial morphogenesis is histone deacetylase-1- (HDAC1) dependent and that interstitial flow increases the phosphorylation of HDAC1, its activity, and its export from the nucleus. Furthermore, we show that HDAC1 inhibition decreases endothelial morphogenesis and matrix metalloproteinase-14 (MMP14) expression. Our results suggest that HDAC1 modulates angiogenesis in response to flow, providing a new target for modulating vascularization in the clinic

Pharmacological characterization of nanoparticle-induced platelet microaggregation using quartz crystal microbalance with dissipation: comparison with light aggregometry

Background: Engineered nanoparticles (NPs) can induce platelet activation and aggregation, but the mechanisms underlying these interactions are not well understood. This could be due in part to use of devices that study platelet function under quasi-static conditions with low sensitivity to measure platelet microaggregation. Therefore, in this study we investigated the pharmacological pathways and regulators of NP-induced platelet microaggregation under flow conditions at nanoscale using quartz crystal microbalance with dissipation (QCM-D) and compared the data thus obtained with those generated by light aggregometry. Methods: Blood was collected from healthy volunteers, and platelet-rich plasma was obtained. Thrombin receptor-activating peptide, a potent stimulator of platelet function, and pharmacological inhibitors were used to modulate platelet microaggregation in the presence/absence of silica (10 nm and 50 nm) and polystyrene (23 nm) NPs. Light aggregometry was used to study platelet aggregation in macroscale. Optical, immunofluorescence, and scanning electron microscopy were also used to visualize platelet aggregates. Results: Platelet microaggregation was enhanced by thrombin receptor-activating peptide, whereas prostacyclin, nitric oxide donors, acetylsalicylic acid, and phenanthroline, but not adenosine diphosphate (ADP) blockers, were able to inhibit platelet microaggregation. NPs caused platelet microaggregation, an effect not detectable by light aggregometry. NP-induced microaggregation was attenuated by platelet inhibitors. Conclusion: NP-induced platelet microaggregation appears to involve classical proaggregatory pathways (thromboxane A2-mediated and matrix metalloproteinase-2-mediated) and can be regulated by endogenous (prostacyclin) and pharmacological (acetylsalicylic acid, phenanthroline, and nitric oxide donors) inhibitors of platelet function. Quartz crystal microbalance with dissipation, but not light aggregometry, is an appropriate method for studying NP-induced microaggregation