Дисертації з теми "Motility and morphology"

In this thesis, we study mathematical models that describe the morphology of a generalized biological cell in equilibrium or under the influence of external forces. Within these models, the cell is considered as a thermodynamic system, where streaming effects in the cell bulk and the surrounding are coupled with a Helfrich-type model for the cell membrane. The governing evolution equations for the cell given in a continuum formulation are derived using an energy variation approach. Such two-phase flow problems that combine streaming effects with a free boundary problem that accounts for bending and surface tension can be described effectively by a diffuse interface approach. An advantage of the diffuse interface approach is that models for e.g. different biophysical processes can easily be combined. That makes this method suitable to describe complex phenomena such as cell motility and multi-cell dynamics. Within the first model for cell motility, we combine a biological network for GTPases with the hydrodynamic Helfrich-type model. This model allows to account for cell motility driven by membrane protrusion as a result of actin polymerization. Within the second model, we moreover extend the Helfrich-type model by an active gel theory to account for the actin filaments in the cell bulk. Caused by contractile stress within the actin-myosin solution, a spontaneous symmetry breaking event occurs that lead to cell motility. In this thesis, we further study the dynamics of multiple cells which is of wide interest since it reveals rich non-linear behavior. To apply the diffuse interface framework, we introduce several phase field variables to account for several cells that are coupled by a local interaction potential. In a first application, we study white blood cell margination, a biological phenomenon that results from the complex relation between collisions, different mechanical properties and lift forces of red blood cells and white blood cells within the vascular system. Here, it is shown that inertial effects, which can become of relevance in various parts of the cardiovascular system, lead to a decreasing tendency for margination with increasing Reynolds number. Finally, we combine the active polar gel theory and the multi-cell approach that is capable of studying collective migration of cells. This hydrodynamic approach predicts that collective migration emerges spontaneously forming coherently-moving clusters as a result of the mutual alignment of the velocity vectors during inelastic collisions. We further observe that hydrodynamics heavily influence those systems. However, a complete suppression of the onset of collective migration cannot be confirmed. Moreover, we give a brief insight how such highly coupled systems can be treated numerically using finite elements and how the numerical costs can be limited using operator splitting approaches and problem parallelization with OPENMP
Diese Dissertation beschäftigt sich mit mathematischen Modellen zur Beschreibung von Gleichgewichts- und dynamischen Zuständen von verallgemeinerten biologischen Zellen. Die Zellen werden dabei als thermodynamisches System aufgefasst, bei dem Strömungseffekte innerhalb und außerhalb der Zelle zusammen mit einem Helfrich-Modell für Zellmembranen kombiniert werden. Schließlich werden durch einen Energie-Variations-Ansatz die Evolutionsgleichungen für die Zelle hergeleitet. Es ergeben sie dabei Mehrphasen-Systeme, die Strömungseffekte mit einem freien Randwertproblem, das zusätzlich physikalischen Einflüssen wie Biegung und Oberflächenspannung unterliegt, vereinen. Um solche Probleme effizient zu lösen, wird in dieser Arbeit die Diffuse-Interface-Methode verwendet. Ein Vorteil dieser Methode ist, dass es sehr einfach möglich ist, Modelle, die verschiedenste Prozesse beschreiben, miteinander zu vereinen. Dies erlaubt es, komplexe biologische Phänomene, wie zum Beispiel Zellmotilität oder auch die kollektive Bewegung von Zellen, zu beschreiben. In den Modellen für Zellmotilität wird ein biologisches Netzwerk-Modell für GTPasen oder auch ein Active-Polar-Gel-Modell, das die Aktinfilamente im Inneren der Zellen als Flüssigkristall auffasst, mit dem Multi-Phasen-Modell kombiniert. Beide Modelle erlauben es, komplexe Vorgänge bei der selbst hervorgerufenen Bewegung von Zellen, wie das Vorantreiben der Zellmembran durch Aktinpolymerisierung oder auch die Kontraktionsbewegung des Zellkörpers durch kontraktile Spannungen innerhalb des Zytoskelets der Zelle, zu verstehen. Weiterhin ist die kollektive Bewegung von vielen Zellen von großem Interesse, da sich hier viele nichtlineare Phänomene zeigen. Um das Diffuse-Interface-Modell für eine Zelle auf die Beschreibung mehrerer Zellen zu übertragen, werden mehrere Phasenfelder eingeführt, die die Zellen jeweils kennzeichnen. Schließlich werden die Zellen durch ein lokales Abstoßungspotential gekoppelt. Das Modell wird angewendet, um White blood cell margination, das die Annäherung von Leukozyten an die Blutgefäßwand bezeichnet, zu verstehen. Dieser Prozess wird dabei bestimmt durch den komplexen Zusammenhang zwischen Kollisionen, den jeweiligen mechanischen Eigenschaften der Zellen, sowie deren Auftriebskraft innerhalb der Adern. Die Simulationen zeigen, dass diese Annäherung sich in bestimmten Gebieten des kardiovaskulären Systems stark vermindert, in denen die Blutströmung das Stokes-Regime verlässt. Schließlich wird das Active-Polar-Gel-Modell mit dem Modell für die kollektive Bewegung vom Zellen kombiniert. Dies macht es möglich, die kollektive Bewegung der Zellen und den Einfluss von Hydrodynamik auf diese Bewegung zu untersuchen. Es zeigt sich dabei, dass der Zustand der kollektiven gerichteten Bewegung sich spontan aus der Neuausrichtung der jeweiligen Zellen durch inelastische Kollisionen ergibt. Obwohl die Hydrodynamik einen großen Einfluss auf solche Systeme hat, deuten die Simulationen nicht daraufhin, dass Hydrodynamik die kollektive Bewegung vollständig unterdrückt. Weiterhin wird in dieser Arbeit gezeigt, wie die stark gekoppelten Systeme numerisch gelöst werden können mit Hilfe der Finiten-Elemente-Methode und wie die Effizienz der Methode gesteigert werden kann durch die Anwendung von Operator-Splitting-Techniken und Problemparallelisierung mittels OPENMP

Bdellovibrio bacteriovorus invade Gram-negative bacteria in a predatory process requiring type IV pili (T4P) at a single invasive pole. Bdellovibrio can encounter prey cells by flagellar motility in liquid culture, or by adventurous gliding motility on a solid surface. After T4P-mediated prey-invasion into the periplasm, the Bdellovibrio degrades the prey-cell nutrients to grow from both poles as a filament inside a now-rounded prey cell or ‘bdelloplast’. The growing Bdellovibrio cell must then partition DNA and proteins along a multi­nucleoid filament, before synchronous septation generates either an even or odd number of vibroid progeny. My thesis project investigated the control of cell morphology, polarity and surface motility in Bdellovibrio bacteriovorus. This study first focused on DivIVA, a protein involved in a variety of processes including polar peptidoglycan synthesis, control of cell morphology and control of septum formation at mid-cell in other bacteria. The Bdellovibrio DivIVA homologue was shown to have an important role in maintaining cell morphology, with fluorescent localisation and gene deletion studies suggesting that DivlVABd is likely to be involved in directing peptidoglycan synthesis, and could also play a role in septum biosynthesis. The product of a parA gene (bd3906), encoded adjacent to the Bdellovibrio ParB homologue and shown to be essential in Bdellovibrio by a previous study, was shown here to be involved in the ‘canonical’ Par role of Bdellovibrio chromosome partitioning. The function of ‘orphan’ ParA1 (Bd1326), so called because of the lack of a neighbouring parB gene, requires further investigation, though co-transcription of parA1 with neighbouring gene bolA, could implicate ParA1 as involved in the switch between Bdellovibrio growth and division. The ‘orphan’ ParA2 protein (Bd2331) was found by mutagenesis and fluorescent tagging to be involved in the regulation of reversals during Bdellovibrio adventurous gliding motility. When ParA2 was present at both poles of a Bdellovibrio cell, the cell was unable to glide on a surface, implicating ParA2 as involved in the inhibition of gliding motility. Concordant with this, deletion of parA2 resulted in cells which made more frequent reversals during gliding motility

Rho GTPases are important regulators of cell motility and morphology. Mammals have three highly homologous Rho proteins, RhoA, RhoB and RhoC. The aim of this thesis was to determine whether individual isoforms of Rho have unique functions in the regulation of cell motility using primary bone marrow-derived macrophages (BMM) as a model system. BMMs were shown to express RhoA, RhoB but not RhoC. BMMs were analysed to determine the role of RhoA and RhoB in motility and morphology. A comparison between RhoA and RhoB-null was carried out using a combination of RhoB-null BMMs, and BMM treated with the pan Rho inhibitor C3-transferase and the Rho kinase inhibitor Y27632. RhoB-null BMMs moved faster and had a smaller spread area than wild-types. Whereas BMM treated with C3-transferase and Y27632 had reduced migration. The RhoB-null BMMs did not change shape following CSF-1 withdrawal unlike wild-type cells. Analysis of BMM spreading and adhesion revealed that deletion of RhoB caused defects in the initial stages of cell spreading and adhesion. Interestingly in RhoB-null BMMs the GTPase activity of RhoA and Rac1 and phosphorylation of their targets LIMK and MLC was decreased less in response to withdrawal of CSF-1 than in wildtype cells. This suggests that RhoB may be acting as a 'brake' for RhoA activity.

Cancer is a worldwide disease and, in the UK, breast cancer is the most common. Compared to healthy cells, cancer cells migrate abnormally, associated with alterations in cell motility and morphology. The development of biomedical imaging techniques result in the production of large amounts of data. The analysis of such large data, the variety of cancer cell shapes and the potential links between cell motility and morphology present a challenge for cell migration study: how to analyse cell motility and morphology simultaneously. This thesis proposes a computational framework to address integrated cancer cell migration analysis. Firstly, automated tracking of cell boundaries is undertaken by a DWNA kinematic model of cell boundaries, described by B-spline active contours. The tracked cell states intrinsically links cell morphology to motility features. As a result, cell centroid and boundary dynamics are successfully tracked, followed by quantitative motility analysis. A module to quantitatively analyse cell morphology is proposed after tracking. Cell shapes are described by a 2D descriptor. Accordingly, cell morphodynamics are modelled as a hidden Markov process, along with three shape states: round, elongated and teardrop. In order to explore the potential interactions between cell shapes and motility, cell centroid motility characteristics are associated to the identified shape states. When the analysis was applied to breast cancer control cells, the identified shape states showed distinct motility characteristics. Finally, the proposed framework is adapted to the comparison of MDA-MB-231 cell behaviours with regulating migration-associated proteins: i) Blebbistatin and Y-27632, which are chemical inhibitors of two different proteins working on the same pathway, showed identical, but different degrees of effects on the motility and morphology characteristics of MDA-MB-231 cells. ii) The absence of FA-associated genes, including FAK, RhoE and beta-PIX, respectively showed distinct effects on cell migrations.

Field evaluation of frozen-thawed stallion semen has been limited to tests such as post-thaw motility and morphology that are not only subjective but also evaluate only a small population of cells. Flow cytometry has provided a quick, repeatable, objective method of evaluating a large number of cells, including spermatozoa. Two experiments were designed to first validate the use of several flow cytometric tests on frozen-thawed stallion semen and then determine a model that may best explain variation in fertility. Comparing samples that were live and freeze-killed validated the flow cytometric tests.

In experiment one, six ejaculates were collected from each of three stallions. The semen from each ejaculate was centrifuged and frozen in 0.5 ml polyvinyl chloride straws. Two straws from each ejaculate were thawed and evaluated. Semen was evaluated for post-thaw motility, morphology, mitochondrial activity using Rhodamine 123 (R123), plasma membrane integrity using propidium iodide (PI) and ethidium monoazide (EMA), and chromatin structure using the sperm chromatin structure assay (SCSA). Data was recorded as percentages for all but the SCSA for both experiment one and two. The extent of chromatin denaturation was calculated using the SCSA and the alpha-t population [at = red/(red +green) fluorescence]. From the alpha-t population, statistics were calculated such mean (Xat), standard deviation (SDat), percentage of cells outside (COMPat) the main alpha-t population and the mean green fluorescence (mean green) of the population.

Results from experiment one demonstrated that all flow cytometric tests except EMA were able to distinguish between live and freeze-killed samples (p < 0.0001). Also the stallion accounted for most of the variation in samples when compared to ejaculate and straw within an ejaculate. Therefore two straws could be chosen at random from a stallion and evaluated in experiment two.

In experiment two, twenty-nine stallions were evaluated using the same tests as experiment one excluding EMA. Fertility data was obtained from the 1998 or 1999 breeding season. Multiple linear regression was used to determine the best-fit model to predict overall pregnancy rate. SCSA and R123-PI assays accounted for the largest amount of variation in fertility (R² = 0.65, p < 0.0004). Within SCSA and the R123/PI assays Xat and PI staining had the highest contribution to this variation in fertility (R² = 0.11, R² = 0.47) respectively. The best-fit model for predicting fertility included the assay combination listed above and the interactions between SDat and mean green staining as well as R123 and mean green staining. Post-thaw motility and morphology did not account for significant variation in fertility (p = 0.22, p = 0.46) respectively.

Based on this project post-thaw motility and morphology are poor predictors of fertility in frozen-thawed stallion semen. However, through the addition of SCSA and R123-PI to the routine evaluation of frozen-thawed stallion semen time and money may be saved in advance by identifying those stallions with poor post-thaw fertility.
Master of Science

Bibliography
Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: Male infertility is often associated with impaired sperm motility and morphology (asthenoteratozoospermia) for which there is no specific therapeutic treatment. It has come to light that the modification and expression of human sperm proteins play a crucial role in sperm function. In the present study, we present proteomic data of human spermatozoa in the context of sperm dysfunction. Novel techniques have been used to successfully isolate and identify differences in protein expression on a cellular level associated with asthenoteratozoospermia. In the first part of the study, differences in protein expression within the total sperm proteome were investigated between immature and mature sperm populations. Semen was collected from healthy donors (n=23) and separated into mature and immature sperm populations by 3-layer Percoll gradient centrifugation. Cells were washed and motility and morphology were measured by computer assisted sperm analysis (CASA). For the proteomic investigation cells were lysed and proteins separated by means of two-dimensional gel electrophoresis (2D electrophoresis). PD-Quest was used to identify the differentially expressed proteins. The protein spots of interest were excised and subjected to in-gel digestion. Peptides were separated by High Pressure Liquid Chromatography (HPLC) analysis and amino acid sequences determined by mass spectrophotometry. Proteins were identified by Mascot, using the Swiss Prot database. The results show that the motility (immature; 26.1±1.75% total motile cells vs. mature; 60.93±3.24% total motile cells; p<0.001) and morphology parameters (immature; 64.1±2.75% normal head morphology vs. mature; 87.63±3.24% normal head morphology; p<0.001) of the two populations differed significantly. After 2D electrophoresis, 16 differentially expressed protein spots were identified within the total sperm proteome between the immature and mature sperm populations. 56% of the differentially expressed proteins were more abundant in the immature sperm population compared to the mature sperm population. Functions have been ascribed to these proteins of which only four proteins, namely Tubulin -3C/D chain, Tubulin -2C chain, Outer dense fibre protein 2 and A-Kinase anchoring protein 4 precursor, were directly related to sperm motility and morphology. In the second part of the study the expression of nuclear proteins in human spermatozoa was investigated between immature and mature sperm populations. Semen was collected from healthy donors (n=156) and further separated from the seminal plasma by PureSperm® gradient centrifugation. The immature and mature sperm populations were retrieved and used during further analysis. For the proteomic analysis of nuclear proteins, cells were fractionated into four different subcellular protein fractions, instead of analyzing the whole sperm proteome. The results show that the motility (immature; 32.33±0.51% total motile cells vs. mature; 88.67±0.85% total motile cells; p<0.0001) and morphology parameters (immature; 13.51±0.87% normal head morphology vs. mature; 20.89±1.20% normal head morphology; p<0.0001) of the two populations differ significantly. After 2D electrophoresis, 21 differentially expressed nuclear proteins were identified between the immature and mature sperm populations. 95% of the differentially expressed nuclear proteins were less abundant in the immature population compared to the mature population. Only one nuclear protein namely 78kDa Glucose regulated protein was more abundant in the immature population compared to the mature population. Functions ascribed to these individual proteins were directly related to sperm motility, morphology and energy metabolism. In conclusion,In conclusion, in the current study novel techniques have been employed to investigate protein differences between immature and mature sperm populations. From these results it is evident that protein expression in the total sperm proteome and nuclear protein fraction is significantly different and incomplete in the immature population, compared to mature population. Based on these findings, it is recommended that further studies should be done on human spermatozoa to validate the role of the individual proteins in sperm function. Proteomics is an ideal tool to identify idiopathic causes of male infertility, as it can help to identify novel receptors (and signal transduction pathways) that can be used in the screening of drugs to alleviate sperm dysfunction.
AFRIKAANSE OPSOMMING: Manlike infertiliteit word dikwels geassosieer met verlaagde sperm motiliteit en morfologie (asthenoteratozoospermia) waarvoor daar tot dusver nog geen spesifieke terapeutiese behandeling is nie. Dit het aan die lig gekom dat die modifisering en uitdrukking van menslike sperm proteïene ‘n belangrike rol speel in spermfunksie. In die huidige studie stel ons data voor van proteiene in menslike sperme in die konteks van abnormale spermfunksie. Unieke tegnieke was gebruik om verskille in proteïen uitdrukking op sellulêre vlak suksesvol te isoleer en identifiseer wat verband hou met asthenoteratozoospermia. Tydens die eerste deel van die studie was verskille in proteïen uitdrukking binne die totale spermproteoom tussen onvolwasse en volwasse spermpopulasies ondersoek. Sperme van gesonde skenkers (n=23) is geskei in twee spermpopulasies (onvolwasse en volwasse sperme) deur middel van ‘n 3-laag Percoll gradiënt sentrifugasie tegniek. Selle is gewas en sperm motiliteit en morfologie is gemeet deur rekenaar geassisteerde sperm analise (CASA). Vir proteomiese analise is selle geliseer en proteïene geskei deur twee dimensionele gel elektroforese (2D-elektroforese). PD-Quest sagteware is gebruik om statisties beduidende proteïen verskille aan te dui. Die proteïene van belang is uitgesny en onderwerp aan in-gel vertering. Peptiede is geskei met behulp van hoë druk vloeistof chromatografie (HPLC) analise en aminosuurvolgordes is bepaal deur massa spektrofotometrie. Proteïene is geïdentifiseer met behulp van Mascot deur van die Swiss Prot databasis gebruik te maak. Die resultate toon dat die sperm motiliteit (onvolwasse; 26.1±1.75% totale motiele selle vs. volwasse; 60.93±3.24% totale motiele selle; p <0,001) en morfologiese parameters (onvolwasse; 64.1±2.75% normale kop morfologie vs. volwasse; 87.63±3.24% normale kop morfologie; p <0,001) tussen die twee populasies beduidend verskil. Na 2Delektroforese is 16 proteïen kolle geïdentifiseer wat beduidend verskil het, tussen die totale sperm proteoom van onvolwasse spermpopulasies en volwasse spermpopulasies. 56% van die proteïene wat beduidend verskil het, was meer uitgedruk in die onvolwasse spermpopulasie ten opsigte van die volwasse sperm populasie. Funksies is toegeskryf aan hierdie proteïene waarvan net vier proteïene naamlik Tubulin -3C/D ketting, Tubulin -2C ketting, Buite digte vesel proteïen 2 en A-Kinase anker proteïen 4 voorloper direk verband hou met sperm motiliteit en morfologie. In die tweede deel van die studie is die uitdrukking van nukluêre proteïene in menslike spermatozoa tussen onvolwasse en volwasse spermpopulasies ondersoek. Sperme was van gesonde skenkers (n=156) versamel en verder geskei van seminale plasma deur middel van ‘n PureSperm® gradiënt sentrifugasie tegniek. Vir die proteomiese analise van nukluêre proteïene is selle gefraksioneer in vier verskillende sub-sellulêre proteïen fraksies, in plaas van analise van die totale spermproteoom. Die resultate toon aan dat die sperm motiliteit (onvolwasse; 32.33±0.51% totale motiele selle vs. volwasse; 88.67±0.85% totale motiele selle; p <0,001) en morfologiese parameters (onvolwasse; 13.51±0.87% normale kop morfologie vs. volwasse; 20.89±1.20% normale kop morfologie; p <0,001) tussen die twee populasies beduidend verskil. Na 2D-elektroforese is 21 kern proteïen kolle geïdentifiseer wat betekenisvol uitgedruk was tussen onvolwasse en volwasse spermpopulasies. 95% van die nukluêre proteïene wat beduidend verskil het, was minder uitgedruk in die onvolwasse spermpopulasie ten opsigte van die volwasse spermpopulasie. Slegs een kern proteïen naamlik 78kDa Glukose gereguleerde proteïen was meer uitgedruk in die onvolwasse spermpopulasie in vergelyking met die volwasse spermpopulasie. Funksies is toegeskryf aan hierdie proteïene wat direk verband hou met sperm motiliteit, morfologie en energie metabolisme. Ten slotte, in die huidige studie is unieke tegnieke geïmplementeer om proteïen verskille tussen onvolwasse en volwasse spermpopulasies te ondersoek. Uit hierdie resultate is dit duidelik dat proteïen uitdrukking in die totale sperm proteoom en in die kern proteïen fraksie beduidend verskil en onvolledig is in die onvolwasse spermpopulasie ten opsigte van die volwasse spermpopulasie. Op grond van hierdie bevindinge word aanbeveel dat verdere studies op menslike sperme gedoen moet word ten einde die rol van individuele proteïene in sperm funksie te kan bepaal. Proteomika is ‘n ideale tegniek om die iodiopatiese oorsake van manlike infertiliteit te identifiseer, aangesien dit kan help in die identifisering van unieke reseptore (en seintransduksie paaie) wat gebruik kan word om sperm disfunksie te verbeter deur farmaseutiese behandeling.

Testicular cancer (TC) is the most common cancer affecting men of reproductive age; however treatment with bleomycin, etoposide, and cis-platin (BEP) results in extremely high cure rates. The post-treatment quality of life of TC patients is therefore a major concern. The goal of this study was to determine the effects of BEP on sperm count, motility, and morphology in an animal model. Male Sprague Dawley rats were treated with BEP for 9 weeks. Rats were killed, and the numbers, motility, and morphology of the spermatozoa from the epididymides were analyzed. After BEP treatment, sperm counts decreased by almost 10-fold when compared to control (11.9x107 versus 1.65x107 sperm per epididymis). The percent of spermatozoa that were motile was > 30% lower in the treated group compared to control group. Morphological defects increased significantly in both the midpiece and principal piece of the flagella. These results indicate that BEP treatment has significant effects on spermatogenesis in the rat model.

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2010.
Bibliography
ENGLISH ABSTRACT: Sperm morphology remains an important parameter in the prediction of fertility, both in vivo and in vitro. However, there remains a considerable level of concern surrounding the true potential of this parameter due to the lack of standardization of differential staining techniques used for the evaluation of sperm morphology. This study aimed at investigating two commonly used staining techniques, Rapidiff® (RD) and Papanicolaou (PAP), along with a new commercially available stain, SpermBlue® (SB), in the evaluation of sperm morphometry and morphology. Results indicated that significant differences in sperm morphometry exist due to the use of the staining techniques. Findings further indicated that RD causes sperm head swelling while PAP causes sperm head shrinkage. Results obtained using the SB staining technique have indicated measurements closest to that which would be obtained through the evaluation of fresh, unstained sperm. The lack of standardization and the different effects various stains have on sperm structure and overall sperm morphology evaluation should raise a level of concern, particularly when evaluating patients with borderline morphology. Based on this, the use of the SB staining technique is recommended over RD and PAP for effective and accurate morphology evaluation. In further support of this technique, SB was shown to be quick and simple in method, and allowed for the easy detection of sperm by computer aided sperm analysis (CASA) systems such as the Sperm Class Analyzer (SCA®). The second aim of this study was to examine the concentration, morphology and motility of the resultant sperm populations following semen preparation using the PureSperm® density gradient and swim-up techniques. Semen preparation is an essential step in any fertility treatment protocol, and it is important that the sperm obtained following semen preparation has sperm morphology and motility characteristics capable of improving assisted fertility success rates. Currently, the PureSperm® density gradient and sperm swim-up are the most widely employed techniques in fertility clinics. Although there is sufficient evidence to suggest they are each effective at extracting sperm with improved quality from neat semen, there remains insufficient evidence to suggest which of these two techniques is superior. The present investigation revealed that both sperm preparation methods were effective at improving sperm morphology and motility, however to varying degrees. The swimup method yielded a population of sperm with superior motility and morphology when assessed according to World Health Organisation (WHO) criteria, while the PureSperm® density gradient technique isolated a higher percentage of normal sperm, according to both WHO and Tygerberg strict criteria, with motility better than that of neat semen. Although results obtained via the swim-up method suggest it would be best for use in in vitro fertilization (IVF), the very low concentration of sperm isolated via this method remains a significant draw-back. The PureSperm® density gradient separation technique on the other hand is capable of isolating larger quantities of sperm, which is likely to be of more benefit with fertility treatments requiring larger quantities of sperm. Based on these findings, the use of PureSperm® density gradient technique is recommended, due to its ability to isolate large quantities of good quality sperm. However, a swim-up may still be of use when performing fertility treatment using a sperm sample which possesses a high concentration and motility.
AFRIKAANSE OPSOMMING: Sperm morfologie bly ‘n belangrike parameter in die voorspelling van vrugbaarheid, beide in vivo en in vitro. Tog is daar nogsteeds ‘n aansienklike vlak van kommer rondom die ware potensiaal van hierdie parameter weens die gebrek aan standardisering van verskillende kleuringstegnieke wat gebruik word vir die evaluering van spermmorfologie. Hierdie studie is daarop gemik om ondersoek in te stel na twee algemeen gebruikte kleurings tegnieke naamlik, Rapidiff® (RD) en Papanicolaou (PAP), asook ‘n nuwe kommersiëel beskikbare kleurstof, SpermBlue® (SB), vir die evaluering van spermmorfometrie en morfologie. Resultate dui aan dat beduidende verskille in sperm morfometriese afmetings ontstaan as gevolg van die gebruik van die verskillende kleurstowwe. Bevindinge dui verder daarop dat RD swelling van die sperm se kop versoorsaak, terwyl PAP die spermkop laat krimp. Resultate wat verkry is met behulp van die SB kleuringstegniek dui daarop dat hierdie kleurstof aanleiding gegee het tot afmetings naaste aan die verkry tydens die beoordeling van vars, ongekleurde sperme. Die gebrek aan standardisasie en die uiteenlopende effekte wat verskillende kleurstowwe het op die spermstruktuur en die evaluering van sperm morfologie ingeheel is kommerwekkend, veral tydens die evaluering van pasiënte met grensgeval morfologie. Op grond van hierdie resultate, word die gebruik van die SB kleuringstegniek, bo die gebruik van RD en PAP, vir effektiewe en akkurate morfologie evaluering aanbeveel. Verdere ondersteuning vir die gebruik van die SB kleuringstegniek is die feit dat daar bevind is dat SB ‘n vinnige en eenvoudige metode is, wat toelaat vir maklike visualisering van sperme deur rekenaargesteunde sperm analise sisteme soos die Sperm Class Analyzer (SCA®). Die tweede doel van hierdie studie was om die konsentrasie, morfologie en die motiliteit van spermpopulasies te ondersoek, soos verkry tydens die voorbereiding van semen deur gebruik te maak van die PureSperm® digtheidsgradiënt en op-swem tegnieke. Die voorbereiding van semen is ‘n noodsaaklike stap in enige vrugbaarheidsbehandeling protokol, aangesien dit belangrik is dat die sperme wat deur hierdie prosesse verkry word oor die nodige morfologiese en motiliteit eienskappe beskik wat in staat is om die sukses van vrugbaarheidsbehandelings te verbeter. Huidiglik is die PureSperm® digtheidsgradiënt en op-swem tegnieke die mees algemeen gebruikte tegnieke in vrugbaarheidsklinieke. Alhoewel daar voldoende bewyse is wat voorstel dat elke tegniek effektief is vir die ekstraksie van sperme met beter kwaliteit vanuit semen, bly daar steeds onvoldoende bewyse wat daarop dui dat een van hierdie twee tegnieke beter is as die ander een. Huidige navorsing het getoon dat beide sperm voorbereidings metodes daarin geslaag het om sperme met normale morfologie en beter motiliteit te selekteer. Die opswem metode het ‘n spermpopulasie met beter motiliteit en verbeterde morfologie gelewer, soos getoets volgens die WGO kriteria, terwyl die PureSperm digtheidsgradiënt tegniek sperme met verbeterde morfologie, volgens beide die WGO en Tygerberg Streng Kriteria, en ‘n redelike verbetering in sommige motiliteits parameters geselekteer het. Hoewel die resultate wat verkry word via die op-swem metode voorstel dat dit die beste metode vir die gebruik tydens in vitro bevrugting sou wees, bly die baie lae konsentrasie van sperme wat met hierdie metode verkry word ‘n belangrike nadeel. Die PureSperm® skeidingstegniek laat egter toe vir die isolering van groter hoeveelhede sperme, wat waarskynlik meer voordelig sal wees vir bevrugtingsbehandelings wat meer sperme benodig. Gebaseer op hierdie bevindinge, word die gebruik van die PureSperm® digtheidsgradiënt tegniek aanbeveel, as gevolg van hierdie tegniek se vermoë om groot hoeveelhede goeie gehalte sperm te isoleer. Daar kan egter nogsteeds van op-swem metodes gebruik gemaak word tydens vrugbaarheidsbehandeling indien die semenmonster beskik oor ‘n hoë konsentrasie sperme met goeie beweeglikheid.

Desmoglein 3 (Dsg3) belongs to the desmoglein subfamily and functions as an adhesion molecule in desmosomes. Two pools of Dsg3 have been identified, detergent soluble and insoluble proteins. Recent studies show that DSG3 is upregulated in squamous cell carcinoma (SCC). However, its biological function in cancer remains poorly understood. The aim of this study was to investigate the extra-junctional functions of Dsg3, in particular its roles in signalling that regulates cell morphology and locomotion in cancer cells. This study adopted a unique cancer cell model with Dsg3 gain-of-function and has discovered two novel regulatory signal pathways that may play a crucial role in the control of cell invasion and metastasis in Dsg3 associated cancers. Firstly, Dsg3 regulates the phosphorylation of Ezrin at Thr567 in a PKCdependent manner that is crucial for its activation and regulation of actin based membrane projections and accelerated cell locomotion in SCC. Secondly, Dsg3 modulates the transcriptional activity of cJun:AP1 that is responsible for regulating a cohort of genes to confer an invasive phenotype. It is likely that these two pathways are closely linked in that the Dsg3-mediated activation of cJun:AP1 elicits PKCdependent Ezrin activation that in turn enable it to form a complex with Dsg3 at the plasma membrane to promote membrane projection and cell locomotion. Several lines of evidence support these conclusions: Dsg3 forms a complex with Ezrin at the plasma membrane and induces phosphorylation of Ezrin resulting in augmented membrane protrusions and cell migration. Dsg3 silencing inhibits junction formation concomitant with collapse of membrane protrusion. Furthermore, Dsg3 regulates the activity of cJun:AP1. Collectively, these findings provide new insight regarding Dsg3 in cancer, suggesting it acts as a key regulator of cell invasion and metastasis in SCC. Therefore, targeting Dsg3 could be a potential new strategy in the control of cancer progression and metastasis.

Little is known about the generation of Leishmania morphology and the function of morphology in trypanosomatids, despite every species having characteristic cell shapes and undergoing changes in morphology between life cycle stages. To address this I analysed morphogenesis of the cell body and flagellum through the cell cycle of the Leishmania insect (promastigote) life cycle stage using a novel method for determining cell cycle stage from cell size and DNA content. This showed cell body morphology is generated by growth and then remodelling of cell shape around mitosis and cytokinesis. Mathematical modelling of flagellum growth indicated flagellum length continues to increase over multiple cell cycles and does not reach a defined length. I also observed little link between the cell cycle and flagellum length regulation during differentiation to the mammalian macrophage-inhabiting (amastigote) life cycle stage. Analysis of motility showed the diverse flagellar lengths of promastigote Leishmania cells bestow different swimming abilities, and the capacity of Leishmania promastigotes for highly directional swimming differs sharply from trypomastigote Trypanosoma brucei. This difference did not arise from altered flagellar beating therefore appeared to be linked to morphology. Together these indicate the mechanisms of cell body morphogenesis, flagellum length regulation, life cycle stage differentiation and the swimming abilities of the cells the morphogenetic processes generate differ significantly between Leishmania and T. brucei. These insights motivated the programming of automated micrograph analysis tools based on a new DNA staining method to support similar future morphometric analyses. This is the first comprehensive comparison of morphogenesis and function of morphology in a promastigote and a trypomastigote and, by considering these new insights in the context of existing molecular biology and the morphological diversity across many trypanosomatid species, give insight into basic Leishmania biology, the shared molecular mechanisms underlying morphogenesis and the potential functions of the diverse morphologies which are seen in different trypanosomatid species and life cycle stages.

Thesis (MMed)--Stellenbosch University, 2001.
ENGLISH ABSTRACT: The aim of the study was to conduct a structured review of the literature published on the use of normal sperm morphology, as an indicator of male fertility potential in intrauterine insemination (M) programs. Published literature in which normal sperm morphology was used to predict pregnancy outcome in lUI during the period 1984 - 1998 was reviewed. Four hundred and twenty one articles were identified. Eighteen provided data that could be tabulated and analyzed. Eight of the analyzed studies provided sufficient data for statistical analysis. Six studies used the Tygerberg strict criteria and two the WHO guidelines (1987, 1992). A meta-analysis of the six studies in the strict morphology group yielded a risk difference (RD) between the pregnancy rates achieved in the patients below and above the 4% strict criteria threshold of -0.07 (95% CI: -0.11 to -0.03; p< 0.001). WHO criteria group (1987,1992) had insufficient data to be analysed. Meta-analysis showed a significant improvement in pregnancy rate above 4% threshold for strict criteria. Accurate evaluation of normal sperm morphology results should be an integral part of evaluating the male factor.
AFRIKAANSE OPSOMMING: Die doel van die studie was om 'n gestruktureerde literatuuroorsig van die gepubliseerde data oor normale sperm morfologie uit te voer om vas te stelof dit enige waarde het as voorspeller van manlike fertiliteitspotensiaal in intra uteriene inseminasie (lUI) programme. Gepubliseerde literatuur waar normale sperm morfologie gebruik IS om swangerskapsuitkoms te voorspel met IUI in die tydperk 1984 - 1998 is nagegaan. Vierhonderd een en twintig artikels is geïdentifiseer. Agtien het genoeg data gehad om te kan tabuleer en analiseer. Agt van die geanaliseerde studies het voldoende data gehad vir statistiese analise. Ses studies het die Tygerberg streng kriteria gebruik en twee die WGO (1987, 1992) riglyne. 'n Meta-analise van die ses studies in die streng kriteria groep het 'n risiko verskil tussen swangerskapstempo in pasiënte onder en bo die 4% streng kriteria afsnypunt, van -0,07 (95% betroubaarheidsindeks: -0.11 tot -0.03; p