The abundant cellular protein Cyclophilin A (CypA) was found to bind to HIV-1 capsid (CA) in 1993. Since that time, several complementary methods, including disruption of the binding interface by cyclosporine A, CA mutants, and CypA mutants, have been used to demonstrate that CypA acts within human target cells to promote HIV-1 infection. In contrast, in cells from non-human primates, CypA in target cells decreases HIV-1 infectivity, and it does so by promoting TRIM5α-mediated restriction. Using human cancer cell lines and the genetic methods available at the time, attempts to obtain evidence that CypA inhibits HIV-1 restriction by the human TRIM5α ortholog, let alone that human TRIM5α restricts HIV-1, were unsuccessful. Here we revisit the question of the mechanism by which CypA increases HIV-1 infectivity by exploiting lentiviral vectors optimized for primary human blood cells that serve as HIV-1 targets. Disruption of CA−CypA interaction is demonstrated to render HIV-1 vulnerable to endogenous human TRIM5α-mediated recognition and restriction, which occur prior to completion of reverse transcription. Identical findings were acquired with single-cycle vectors or with replication-competent viruses. Consistently, a previously identified, cyclosporine-resistant CA mutation A92E is also shown to confer resistance against restriction by human TRIM5α. Therefore, the results presented in this thesis reveal that HIV-1 exploits a host protein CypA bound to its CA to evade potent restriction by human TRIM5α. This finding not only answers a long-standing question regarding the role of CypA in HIV-1 infection, but also may reinvigorate the development of CypA inhibitors for treatment of HIV-1.

The field of hematology involves the analysis of blood and its components like platelets, red blood cells, white blood cells. The outcome of this analysis can be vital in determining the condition of the human body and it is important to obtain accurate results. A deep learning algorithm scans over the given input data for unique features and learns them. Then it identifies these features and correlates them to give the result. This can save a significant amount of time and manual work. In contrast, a traditional machine learning algorithm requires the developer to carry-out the feature engineering. This thesis involves the analysis of white blood cells (WBC) using deep learning techniques. In collaboration with a hematology company HemoCue AB based in Angelholm, we will be developing deep learning algorithms for the analysis of white blood cells in the HemoCue R WBC DIFF System. Predominantly, there are two stages in this thesis. The first stage is white blood cell identification, which is used to calculate the number of white blood cells in the given blood sample. The next stage is to identify the different types of white blood cells with which the concentration of each type of WBC in the given blood sample is calculated. We have explored different classification approaches like ’one vs all’ and ’4-class classifier’, and have developed two CNN architectural designs i.e. ’multi-input’ and ’multi-channel’. On comparing the performance of all these design approaches, a final integrated model is put forth for the analysis of WBCs in the company’s device. The proposed ’one vs all’ classification approach combined with a 3-class CNN classifier has yielded very promising results with a combined accuracy 95.45% in WBC identification and 90.49% in WBC differential classification.

<p>Prothrombinase is composed of a proteinase, factor Xa (Xa), its cofactor Va (Va), Ca²⁺ and a zymogen, prothrombin (II), assembled on a phospholipid surface. During coagulation, prothrombinase accelerates II to thrombin conversion; but during anticoagulation, it protects the proteinase from inhibition by antithrombin (AT) ± unfractionated heparin (UFH). Although the degree of Xa protection by prothrombinase varies according to the reports in literature, moderate to significant protective effects have been consistently reported by most investigators. To overcome the limitations of UFH, our laboratory has developed a covalent complex of AT and UFH (ATH) with superior anticoagulant responses. To further understand the mechanisms of enhanced anticoagulant activity of ATH, we proceeded to study inhibition of the prothrombinase complex<em> </em>on synthetic vesicles, activated platelets and red blood cells (RBCs). Using discontinuous inhibition assays, we determined the rate of inhibition of prothrombinase-complexed Xa compared to control Xa. With synthetic vesicles, Xa was protected from inhibition by AT+UFH when in prothrombinase, while only a mild protective effect was observed with ATH. Omission of various components of the prothrombinase led to a reduction in Xa protection for AT+UFH. However, an increased Xa protection against ATH was observed when II was omitted from the prothrombinase. In comparison to the synthetic vesicle system, activated platelets showed a similar trend for protection of Xa in reactions involving prothrombinase ± components, while no protection of Xa was observed for ATH reactions. Alternatively, RBCs showed differences relative to vesicles in that increased protection of Xa occurred with omission of II and Va for AT+UFH, whereas omission of Va increased protection against ATH inhibition. In addition, ATH had improved inhibition of thrombin generation, fibrin formation and plasma coagulation compared to AT+UFH. Studies of fluorescently labelled Xa and inhibitors detailed binding interactions with prothrombinase subunits. Overall, the results suggest that a covalent linkage between AT and heparin improves inactivation of prothrombinase complexed-Xa leading to down-regulation of prothrombinase function.</p> / Doctor of Philosophy (Medical Science)

Σκοπός της διδακτορικής διατριβής είναι η ανάπτυξη κατάλληλων μεθόδων ψηφιακής επεξεργασίας εικόνας και αναγνώρισης προτύπων με τις οποίες θα προσδιορίζονται βιομετρικές και διαγνωστικές παράμετροι μέσω της αλληλεπίδρασης φωτονίων στο ορατό και υπέρυθρο φάσμα. Πιο συγκεκριμένα επιλύεται ένα αντίστροφο πρόβλημα σκέδασης ΗΜ ακτινοβολίας από ένα ανθρώπινο, υγιές και απαραμόρφωτο ερυθρό αιμοσφαίριο. Παρουσιάζονται μέθοδοι εκτίμησης και αναγνώρισης των γεωμετρικών χαρακτηριστικών απαραμόρφωτων υγιών ερυθρών αιμοσφαιρίων με χρήση εικόνων που προσομοιώνουν φαινόμενα σκέδασης ηλεκτρομαγνητικής ακτινοβολίας που διέρχεται από προσανατολισμένα ερυθρά αιμοσφαίρια. Η διαδικασία της ανάκτησης της πληροφορίας περιλαμβάνει, εξαγωγή χαρακτηριστικών με χρήση δισδιάστατων μετασχηματισμών, κανονικοποίηση των χαρακτηριστικών και την χρήση νευρωνικών δικτύων για την εκτίμηση των γεωμετρικών ιδιοτήτων του ερυθροκυττάρου. Παράλληλα σχεδιάστηκε και αξιολογήθηκε σύστημα αναγνώρισης των γεωμετρικών χαρακτηριστικών των ερυθρών αιμοσφαιρίων. Οι εικόνες σκέδασης δημιουργήθηκαν προσομοιώνοντας το πρόβλημα εμπρόσθιας σκέδασης ενός επίπεδου ηλεκτρομαγνητικού (ΗΜ) κύματος, χρησιμοποιώντας την μέθοδο των συνοριακών στοιχείων, λαμβάνοντας υπόψη τόσο την αξονοσυμμετρική γεωμετρία του ερυθροκυττάρου όσο και τις μη αξονοσυμμετρικές οριακές συνθήκες του προβλήματος. Η επίλυση του εν λόγω προβλήματος πραγματοποιήθηκε στα 632.8 nm και εν συνεχεία επεκτάθηκε σε 12 διακριτά ίσου βήματος μήκη κύματος από 432.8 nm έως 1032.8 nm. Επίσης, προτάθηκε μία νέα πειραματική διάταξη για την απόκτηση πολλαπλών εικόνων σκέδασης και την εκτίμηση των γεωμετρικών χαρακτηριστικών των ερυθρών αιμοσφαιρίων, αποτελούμενη από μία πολυχρωματική πηγή φωτός (Led) και πολλαπλά χρωματικά φίλτρα. Επίσης κατασκευάστηκε μέθοδος επίλυσης του σημαντικού προβλήματος εύρεσης της περιεκτικότητας του διαλύματος σε ερυθρά αιμοσφαίρια διαφορετικών μεγεθών στην περίπτωση απόκτησης πολλαπλών εικόνων σκέδασης από διαφορετικές φωτοδιόδους και πολλαπλά χρωματικά φίλτρα. Στα πειράματα αξιολόγησης της μεθόδου που προτείνεται με εικόνες προσομοίωσης δείχνεται ότι είναι ικανή η εύρεση της αναλογίας των ερυθρών αιμοσφαιρίων με πολύ μεγάλη ακρίβεια ακόμα και στη περίπτωση όπου στις εικόνες έχει προστεθεί λευκός κανονικός θόρυβος. Η βασική μεθοδολογία που παρουσιάζεται στην παρούσα δια-τριβή μπορεί να χρησιμοποιηθεί για την αναγνώριση παθολογικών αιμοσφαιρίων ή να χρησιμοποιηθεί στην αναγνώριση μικροσωματιδίων σε υγρά ή αέρια. / The aim of this PhD thesis is the development of digital image processing and pattern recognition methods to estimate biometric and diagnostic parameters using scattering phenomena in the visible and infrared spectrum. More concretely, several reverse scattering problems of EM radiation from a human, healthy and undistorted Red Blood Cell (RBC) is solved. Methods of estimation and recognition of geometrical characteristics of healthy and undistorted RBCs using simulating images are presented. The information retrieval process includes, features extraction using two-dimensional integral transforms, features normalization, and Neural Networks for estimation of three major RBC geometrical proper-ties. Using the same features set, a recognition system of the geometric characteristics of RBCs was developed and evaluated. The scattering images were created simulating the forward scattering problem of a plane electromagnetic wave using the Boundary Element Method, taking into account both axisymmetric geometry of the scatterer and the non-axisymmetric boundary conditions of the problem. Initially, the problem is solved at 632.8 nm and consequently the same problem was solved at 12 different wavelengths, from 432.8 to 1032.8 nm equally spaced. Also, a new device for acquisition of scattering images from RBCs-flow, consisting of a multi-color light source (Led) was proposed, for RBC size estimation and recognition. Finally, a system for the estimation of different RBCs concentration was developed when scattering images acquired using multiple scattering images acquired from multiple Leds and color filters. The system was evaluated using additive white regular noise.

Human cells from acute myeloid leukemia (AML) patients are frequently transplanted into immune-compromised mouse strains to provide an in vivo environment for studies on the biology of the disease. Since frequencies of leukemia re-initiating cells are low and a unique cell surface phenotype that includes all tumor re-initiating activity remains unknown, the underlying mechanisms leading to limitations in the xenotransplantation assay need to be understood and overcome to obtain robust engraftment of AML-containing samples. We report here that in the NSG xenotransplantation assay, the large majority of mononucleated cells from patients with AML fail to establish a reproducible myeloid engraftment despite high donor chimerism. Instead, donor-derived cells mainly consist of polyclonal disease-unrelated expanded co-transplanted human T lymphocytes that induce xenogeneic graft versus host disease and mask the engraftment of human AML in mice. Engraftment of mainly myeloid cell types can be enforced by the prevention of T cell expansion through the depletion of lymphocytes from the graft prior transplantation.

Le travail a été réalisé en collaboration avec le laboratoire de mécanique acoustique de Marseille, France. Les simulations ont été menées avec les langages Matlab et C. Ce projet s'inscrit dans le champ de recherche dénommé caractérisation tissulaire par ultrasons. / Plusieurs études ont démontré une association entre l’agrégation érythrocytaire du milieu sanguin et plusieurs anomalies hémorhéologiques. Cette agrégation peut être quantifiée à l'aide du coefficient de rétrodiffusion ultrasonore. Pour décrire l’interaction entre l'onde ultrasonore et les tissus biologiques, on se sert de modèles. Ainsi, le modèle de facteur de structure (MFS) est utilisé pour évaluer le coefficient de rétrodiffusion des globules rouges agrégés. Toutefois, ce modèle numérique ne permet pas des mesures en temps réel du niveau d’agrégation et n'informe pas sur la structure du milieu sanguin comme par exemple la taille de l’agrégat. Pour pallier à ces difficultés, nous proposons un modèle où la théorie du milieu effectif est combinée au modèle de facteur de structure. Tout en permettant une mesure en temps réel de l’agrégation, ce modèle nommé TMEMFS fournit en plus deux indices structuraux de l’agrégation: le rayon de l’agrégat ainsi que sa compacité. Par le biais de simulations numériques en 3D, on a comparé le coefficient de rétrodiffusion suivant les modèles MFS et TMEMFS. Ceci dans le but de vérifier que dans la solution du problème direct, les propriétés acoustiques des globules rouges et les propriétés structurales du milieu agrégeant correspondaient à la réalité. Pour simuler des agrégats de globules rouges, une disposition hexagonale compacte a été utilisée. Les effets du rayon et de la compacité sur le coefficient de rétrodiffusion ont été étudiés. Basé sur la microstructure sanguine considérée, les résultats obtenus avec le modèle TMEMFS sont semblables à ceux du modèle MFS. Ce travail constitue un support théorique pour une mesure quantitative in vivo de l’agrégation érythrocytaire à des fins diagnostics. / Many studies have reported that an enhanced level of red blood cell aggregation is associated with the presence of hemorheological disorders. Pathological aggregation has been characterized by quantitative ultrasound based on the backscattering coefficient. In order to describe the interaction between the incident ultrasound and the interrogated biological tissues, mathematical models are used. Mathematical modeling is known to be the optimal way to describe the interaction occurring between ultrasound and tissues at the cellular level. The structure factor model (SFM), considered as the exact scattering model has been developed to predict the backscattering coefficient from blood. However, the numerical SFM cannot be applied in real time for practical measurements and does not provide aggregate size to assess the level of aggregation. Therefore, we come up with a new model based on the effective medium theory in order to tackle this difficulty. The effective medium theory combined with the structure factor model (EMTSFM) can be applied in real time and contrary to the SFM provides two indices of the aggregate state in vivo: aggregate size and compactness. Based on a 3D simulation study, the backscattering coefficients (BSCs) predicted by the effective medium theory combined with the Structure Factor Model (EMTSFM) are compared to the BSCs computed with SFM. Our aim here is to assess the accuracy of the EMTSFM against the SFM by comparing their BSC in the framework of a forward problem, i.e., the calculation of the BSC from the known acoustic and structure aggregate parameters. This was done in order to validate the proposed model. To simulate aggregates, RBCs are stacked following a hexagonal close packing scheme. The influences of the aggregate radius and compactness on the BSC are studied as well. The results showed good agreement between the SFM and the EMTSFM based on our simulated microstructure of RBC aggregates. Our work provides thus the theoretical background to assess locally the aggregation level for diagnosis purposes.

Despite recent advances in stem cell biology, immunotherapy and transplantation, substantial barriers still exist in the large-scale specific separation of a discrete population of human therapeutic cells from a cell suspension. The ideal purification technique should combine high cell purity, yield and function, with fast processing and affordability. Currently, fluorescence-activated cell sorting with flow cytometry (FACS) and magnetic activated cell sorting (MACS®) are the most used methods for cell separation and purification and have been employed extensively in molecular biology, diagnostic and cell sorting applications, because they are considered to be gentle, fast and scalable. However, these methods have several key disadvantages; they are invariably expensive, yield low log cell reduction (LCR) rates, and suffer from drawbacks when applied to niche cell populations, such as those requiring multiple tandem separation steps and/or involving combined positive and negative cell selection steps. To address this challenge, a new cell affinity selection system was developed. The selectivity is based on the reversible monomeric avidin biotin interaction and it is primary designed for positive selection. The initial studies were performed on flat, nonporous, glass coverslips and the technology was then successfully transferred on high grade smooth non-porous glass beads (with a diameter of 79.12 to 118.59 μm). The multi-step layer-by-layer deposition procedure culminating in dextran-coated supports bearing monomeric avidin was rigorously characterized and subsequently employed in packed bed chromatography experiments with human erythrocytes isolated from cord blood and B lymphocytes from cell lines. The developed affinity selection platform was highly selective, efficient and, most importantly, resulted in high yields, cell purity and viability comparable with MACS® technology. Additionally scale up is possible and could be easily transferred to another chromatographic matrix with the appropriate structure.

Neste estudo, foi determinado o dano celular em linfócitos periféricos após exposição ao 153Sm-EDTMP (Samário-153 etilenodiaminotetrametilenofosfonato) por meio da técnica de análise de micronúcleos e coloração diferencial. O 153Sm-EDTMP é um radiofármaco utilizado para alívio da dor em pacientes com metástase óssea. A análise da freqüência de micronúcleos em amostras sangüíneas de pacientes obtidas uma hora após a administração endovenosa do radiofármaco (41 MBq/kg) mostrou que não houve diferença estatística em relação aos valores basais em células binucleadas. Porém, a análise da distribuição do dano em células mononucleadas mostrou que os pacientes sem tratamento radioterápico prévio apresentaram um aumento significativo na freqüência de células com um micronúcleo e aqueles com tratamento radioterápico prévio, nas células com dois ou mais micronúcleos. Os experimentos in vitro realizados com exposição de sangue total a três concentrações radioativas de 153Sm-EDTMP (0,370; 0,555 e 1,110 MBq/mL) por uma hora mostraram um aumento na freqüência de micronúcleos e de células necróticas e apoptóticas com o aumento da dose de radiação. Foram construídas curvas dose-resposta para os indivíduos sadios e para os pacientes com metástases óssea sem prévio tratamento radioterápico. A comparação das curvas mostrou que os pacientes apresentaram uma radiossensibilidade mais alta que os indivíduos sadios tanto quanto a porcentagem de células com micronúcleos como de células mortas (necróticas e apoptóticas). / In this study the cellular damage in peripheral lymphocytes after exposure to 153Sm-EDTMP (Samarium-153 ethylenediaminetetrametylenephosphonate) was determined using the technique of micronuclei analysis and differential coloration. 153Sm- EDTMP is a radiopharmaceutical used for pain relief in patients with bone metastases. The analysis of the frequency of micronuclei in patient blood samples obtained one hour after endovenous administration of radiopharmaceutical (41 MBq/kg) showed no statistical difference in relation to basal values in binucleated cells. However the analysis of damage distribution in mononucleated cells, showed that the patients without previous radiotherapic treatment presented a significant increase in the frequency of cells with one micronucleus and in those who had taken previous radiotherapic treatment, in cells with two or more micronuclei. The in vitro experiments conducted with the exposition of total blood to three radiation concentrations of 153Sm-EDTMP (0.370, 0.555 and 1.110 MBq/mL) during one hour showed an increase in the frequency of micronuclei and necrotic and apoptotic cells with increasing radiation dose. Dose-response curves for healthy donors and patients with bone metastasis without previous radiotherapic treatment were constructed. The comparison of the curves showed that patients presented higher radiosensitivity, either micronuclei or dead cell (necrotic or apoptotic) percentages, than healthy donors.

La circulation sanguine joue un rôle vital en microcirculation, et ce pour le transport de l'oxygène, le dioxide de carbone et d'autres nutriments. Les globules rouges (GR) constituent la majorité des cellules du sang, c'est pourquoi par "écoulement sanguin", nous entendrons "écoulement d'une suspension de GR". Pendant longtemps l'écoulement sanguin était vu comme un phénomène passif où les GR sont considérés comme des cargos d'oxygène. La vision moderne est tout autre: l'écoulement sanguin est bel et bien un phénomène actif. Les GR ainsi que les cellules endothéliales (qui tapissent les faces internes des vaisseaux sanguins) sont impliquées dans un grand nombre de signalisations biochimiques induites par les contraintes hydrodynamiques, la route vers des régulations vasomotrices sans l'intervention du système nerveux. Par exemple, les GR ne transportent pas que l'oxygène, mais également de l'ATP (adenosine triphosphate), qui est libérée suite à des changements de conformation de protéines membranaires induite par les contraintes hydrodynamiques. Cette thèse est dédiée à la circulation sanguine et son couplage avec la signalisation biochimique ayant lieu en microcirculation. Plus précisément, les questions traités dans cette thèse sont i) la dynamique des GR, ii) le problème de la diffiusion-advection d'espèces chimiques au sein des écoulements sanguins, et iii) le rôle de la géométrie des réseaux vasculaires dans le processus de la signalisation biochimique mentionnés plus haut. Dans un premier temps nous analysons la dynamique de GR dans un écoulement de Poiseuille en présence de valeurs réalistes de contraste de viscosité. Dans un deuxième temps nous développons un modèle de diffusion-advection et le couplons aux écoulements sanguins en adoptant la méthode de Boltzmann sur réseaux; nous exploitons ensuite formulation en l'appliquant au problème de la libération de l'ATP par les GR sous écoulement. Enfin nous présentons des résultats préliminaires pour la problématique générale de l'écoulement sanguin mettant en jeu l'ATP libéré par les GR et la signalisation de calcium par les cellules endothéliales. Cette étude constitue un premier pas vers le problème général et ambitieux de la régulation locale mechano-biochimique impliquée dans la microcirculation. / Blood flow in microcirculation is vital for oxygen, carbon dioxide and nutrients transport. Most of blood cells are red blood cells (RBCs), so that by blood flow we mean flow of a suspension of RBCs. For long time blood flow has been mainly considered as a passive phenomenon, in which RBCs are viewed as passive carriers of oxygen. The modern view is completely different: blood flow is more active than we thought. The RBCs as well as vascular endothelial cells covering the internal walls of blood vessels are involved in a number of biochemical signaling processes that are triggered by shear stress eliciting a number of biochemical events, and ultimately resulting into vasomotor regulation without participation of the nerve system. For example, RBCs do not only carry oxygen but also ATP (adenosine triphosphate) , the release of which occurs thanks to changes of RBC membrane protein conformations caused by shear stress. Released ATP reacts with some endothelial membrane receptors leading to vasodilation. This thesis is devoted to blood flow and its coupling to biochemical signaling. More precisely, we investigate i) the dynamics of RBCs, ii) the advection diffusion of chemicals in blood flow and the role of iii) the geometry of vessel networks, in the mentioned signaling processes in microcirculations. Firstly, we study the RBC dynamics in a pipe flow with realistic viscosity contrast values, where a link between shape dynamics and rheology is established. Secondly, we develop an advection-diffusion solver that can handle general moving curved boundaries based on lattice-Boltzmann method (LBM); we then implement it for the study of the problem of ATP release from RBCs under shear flow. Membrane tension and deformation induced by shear stress together with vessel network geometry contribute to ATP release. Finally we demonstrate the capability of applying our model and our numerical tool to the complete problem of blood under flow involving ATP release from RBCs and endothelial calcium signaling as a preliminary step to the ambitious task of mechano-involved local regulation events in microcirculation.

Les mégacaryocytes sont des cellules de la moelle osseuse, à l’origine de la production des plaquettes sanguines. Quand elles arrivent à maturité, elles grossissent et émettent des prolongements de cytoplasme à travers la paroi des vaisseaux irriguant la moelle. Dans la circulation sanguine, ces prolongements, soumis aux forces de l’écoulement, s’allongent et se rompent pour former des plaquettes. Des techniques microfluidiques capables de produire des plaquettes in vitro existent et sont une alternative prometteuse au don. Mais le rendement reste à améliorer. Pour cela, il est nécessaire de mieux comprendre la fragmentation des mégacaryocytes en plaquettes. Ce travail de doctorat s’inscrit dans ce contexte et sera développé en deux axes principaux dans ce manuscrit. Dans une première partie nous développons une méthode pour trier des cellules en fonction de leur déformabilité, afin de savoir si les propriétés mécaniques d’un mégacaryocyte sont liées à leur stade de maturité. La méthode a d’abord été mise au point avec des microcapsules. Leurs propriétés mécaniques sont déterminées par analyse inverse à partir de la mesure de leur forme en écoulement dans des constrictions droites. Puis le dispositif utilisé a été miniaturisé pour s’adapter à la taille des cellules. Pour la caractérisation de leurs propriétés mécaniques, deux outils ont été utilisés: l’analyse inverse et la microscopie à force atomique sans pointe. Une deuxième partie porte sur l’étude de l’élongation et de la rupture de mégacaryocytes soumis écoulement. Nous avons quantifié les variations spatiotemporelles du taux d’élongation et développé un protocole d’ablation laser pour étudier les mécanismes de rupture de cellules en élongation. / When they mature in the bone marrow, the precursors of platelets, called megakaryocytes, grow and extend protrusions able to join blood circulation. There these protrusions elongate and break into platelets. Microfluidic techniques for in vitro platelet production represent a promising alternative to donation. In order to enhance platelet production and match the needs of clinical applications such as transfusion, we need to better understand the fragmentation of megakaryocytes into platelets. Our contribution will be described in this manuscript in two main axes. First, in order to know if mechanical properties of megakaryocytes can indicate their maturity stage, we develop a cell sorting method based on deformability. The method is first validated with microcapsules. Their mechanical properties are determined by inverse analysis from their shape under flow in straight microchannels. Then the device is downscaled. The characterization of cell mechanical properties are performed using inverse analysis and tipless atomic force microscopy. Second, we study megakaryocyte elongation and rupture in a microfluidic device. We quantify the spatial and temporal variations of the elongation rate and develop a laser ablation protocol to trigger and study the rupture of elongating cells.