The potential application for stem cell therapy is vast. Despite a limited understanding of their mechanisms of action, clinical trials assessing stem cells in human stroke are underway. Colony stimulating factors (CSF) such as granulocyte colony stimulating factor (G-CSF), which have been used to mobilise haematopoietic stem cells (HSC), also show promise in treating stroke. Preclinical experiments evaluating the effect of G-CSF in stroke were meta-analysed; G-CSF significantly reduced lesion size in transient but not permanent models of ischaemic stroke. Further studies assessing dose-response, administration time, length of ischaemia and long-term functional recovery are needed. Tracking iron-labelled cells with MRI may help to establish migratory patterns following transplantation. Our systematic review of iron-labelled stem cells in experimental stroke suggests that compounds already licensed for humans (ferumoxide and protamine) may potentially be used in clinical trials. In a phase IIb single-centre randomised controlled trial (n=60), the safety of G-CSF in recent stroke was assessed (STEMS-2). G-CSF appears safe when administered subacutely and may reduce stroke lesion volume. Phase III trials are required to test efficacy. An updated Cochrane review on CSFs in stroke shows that G-CSF was associated with a non-significant reduction in early impairment but had no effect on functional outcome in 6 small studies. In two trials, erythropoietin therapy was significantly associated with death by the end of the trial. It is too early to know whether G-CSF could improve functional outcome in stroke. In 8 recruits randomised into STEMS-2, mobilised CD34+ HSCs were paramagnetically labelled, re-infused and tracked with serial T2* MRI. Post-stroke HSC labelling appears safe and feasible. There is suggestive evidence in one patient that labelled HSCs migrate to the ischaemic lesion. Our in vitro evaluation of CD34+ HSCs has revealed that uptake of superparamagnetic iron oxide (SPIO) is enhanced but not dependent upon a transfection agent. Iron labelling of CD34+ cells in this manner did not affect cell viability or inhibit growth. This methodology could be applied to clinical trials. We have established the expression of G-CSF protein, its receptor (G-CSFR) and CD34 antigen in post-mortem brain tissue from participants recruited to STEMS-2. Areas of angiogenesis and expression of G-CSFR in acute and chronic infarction suggest potential targets for therapy. There are many preclinical studies reporting the effects of stem cells in treating stroke (with a noticeable lack of neutral or negative articles). Despite the wealth of literature there remain many unanswered questions and patients should not undergo stem cell therapy unless it is as part of a well designed clinical trial.

India is rapidly becoming established as a major player in the stem cell sector. However, concerns have been raised about the use of unproven stem cell therapies and the exploitation of parents for cord blood banking. This study aims to explore the nature of stem cell activities, how key stakeholders generate expectations around them and frame the ethical issues they raise, and why the biomedical governance system is unable to regulate these emerging practices. The study involved a survey, documentary analysis and qualitative interviews with key scientists, clinicians, representatives of firms and policymakers. The thesis observes that, unlike international commentaries which largely focus on embryonic stem cell treatments, in India it is adult and cord blood stem cells which are dominant in research and clinical settings. Expectations are configured on the basis that stem cells have the potential to: solve the problem of organ shortage; help patients with ailments; provide affordable health care; and establish India as a global player. The creation of expectations is ethically problematic given the potential health risks and economic exploitation of both native and international patients. However, the ethically contested activities are justified by clinicians on the basis that the Helsinki Declaration allows to use an experimental therapy; there are many 'desperate patients' demanding these treatments; and adult stem cells are safe. To date, the government of India appears to be unable to prevent these activities. Contrary to suggestions in previous literature and by some informants that new legislation is needed to address the problem, this thesis finds that state-led mechanisms for biomedical governance lack the ability to implement existing oversight measures. This implementation gap is partly because other forms of governance are not strong enough and partly because there are high expectations at state level aimed at establishing India as a global player in the stem cell sector.

During development, polarity is a common feature of many cell types. One example is the polarisation of whole fields of epithelial cells within the plane of the epithelium, a phenomenon called planar cell polarity (PCP). It is widespread in nature and plays important roles in development and physiology. Prominent examples include the epithelial cells of external structures of insects like the fruit fly Drosophila melanogaster, polarised tissue morphogenesis in vertebrates and sensory hair cells in the vertebrate ear. In this work we focus on the wing and the abdomen of Drosophila, where PCP becomes obvious in the alignment of hairs and bristles. The underlying dynamics are not fully understood yet, but two distinct protein networks centred around the transmembrane proteins Frizzled and Dachsous, respectively, have been shown to play essential roles. We will present and analyse five models for different aspects of the process of planar cell polarisation. The first two models assess the nature of PCP in a generic setting, ensuring that the results are valid for whole classes of PCP models. Models three and four are existing more complex models that include detailed assumptions about the underlying protein interactions of the Frizzled system in the Drosophila wing. Model five considers the Dachsous system in the Drosophila abdomen. We describe the features of the different types of mechanisms and determine the conditions under which they can yield polarity. All five models can establish wild-type polarity for a wide range of parameter values. We find, however, that for model one, three and four an inhomogeneous pattern exists for the same parameter values as the polarised state. Therefore, in these cases either specific initial conditions, which are unlikely in nature, or a global bias are necessary to ensure correct polarisation. Furthermore, we present the effects of clonal clusters of cells on the polarity of the surrounding cells in our models and relate them to the phenotypes observed in experiments. Model one and five show the largest discrepance between the numerical and the experimental results. We discuss the biological relevance of these findings and indicate outstanding questions.

The eukaryotic cell cycle primarily consists of five phases, namely a resting state, G0, and four cycling phases G1, S, G2 and M phase, with cells progressing in this order before dividing into two cells back in phase G1. Understanding how a drug affects the cell cycle can give insight into the drug's mechanism of action and may assist research into potential treatment strategies. The pentacyclic acridinium salt RHPS4 (3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl] acridinium methosulfate) is an attractive agent because it is potentially cell-cycle specific and inhibits the activity of telomerase, an enzyme known for its role in cellular immortalisation in human cancer. The precise mechanism of action of the drug on the cell cycle dynamics, however, remains unclear. We have devised experiments, collected experimental data and formulated a mathematical model describing the cell cycle dynamics of cancer cells and their time- and dose-dependent modulation by RHPS4 to investigate how the compound affects cells in each stage of the cell cycle. In addition to a control case, in which no drug was used, we treated colorectal cancer cells with three different concentrations of the drug and fitted simulations from our models to experimental observations. We have shown that the model is "identifiable", meaning that, at least in principle, the parameter values can be determined from observable quantities. Our fitting procedure also generates information on the sensitivity of parameters in the model. We found that RHPS4 caused a marked concentration-dependent cell death in treated cells, which is well modelled by allowing the rate parameters corresponding to cell death to be sigmoidal functions of time. Since the drug uptake into the nucleus is rapid (saturation within 5 hours), the observed delay effect of 5 days of the compound is unexpected and is a novel finding of our research into this compound. Our results show that, at low concentrations, RHPS4 primarily affects the cells in the G2/M phase, and that the delay decreases at larger doses. We propose that secondary effects lead to the induction of observed cell death and that changes in the molecular structure of the non-coding DNA sequences at chromosome ends, called telomeres, might be a precursor of delayed cell death. We therefore investigated the dynamics of telomere length in different conformational states, that is, t-loops, G-quadruplex structures and those being elongated by telomerase. By formulating differential equation models we studied the effects of various levels of telomerase and RHPS4 concentrations on the distribution of telomere lengths and analysed how these effects evolve over large numbers of cell generations. As well as calculating numerical solutions, we use quasicontinuum methods to approximate the behaviour of the system over time, and predict the shape of the telomere length distribution. We showed that telomere length maintenance is tightly regulated: too high levels of telomerase lead to continuous telomere lengthening, and large concentrations of RHPS4 lead to progressive telomere erosion. Our results suggest different effects of RHPS4 dependent on the drug concentration used: low concentrations reduce telomere length, but do not impair the equilibrium of the system, and high concentrations destabilise the system leading to chromosome degradation and senescence and/or cell death. Moreover, our models predict a positively skewed distribution of telomere lengths at equilibrium, and our model predictions are in good agreement with experimental data.

The Schizosaccharomyces pombe cdc21⁺ gene product is related to the Mcm2-3-5 family of replication proteins. By phylogeny analysis of their protein sequences and screening for cdc21⁺-related sequences using molecular probes I have suggested that at least six types of cdc21⁺-related genes may be present in the yeast genome. The isolation of interaction suppressors of the cdc21^ts mutant was attempted by overexpression of an S. pombe cDNA library. Two cDNAs were isolated, ts11⁺ and dom1⁺, whose overexpression specifically affected the viability of cdc21^ts cells under certain conditions. The predicted dom1 protein is 60% identical to the budding yeast HMG-like Nhp2 protein. I have studied the phenotype of S. pombe cells overexpressing the cdc21⁺ gene and amino-terminal truncations of it. Overexpression of the cdc21⁺ gene caused cell elongation but cells were not significantly affected in growth rate. Cells overexpressing the carboxyl-terminal part of cdc21⁺ arrested in S phase and also entered mitosis in the absence of nuclear division. The possibility that chromosomes in cdc21^ts arrested cells may be damaged was investigated by pulsed field gel electrophoresis. No differences could be found compared to wild-type chromosomes. I have also studied the arrest phenotype of cdc21 rad1 and cdc21 cdc2.3w double mutants. Both strains entered mitosis at the restrictive temperature indicating that cdc21^ts cells arrest in S phase and may contain DNA damage. I have generated two new mutant alleles of cdc21⁺. The first allele was made by deleting most of the cdc21⁺ open reading frame (ORF). The second allele was constructed by placing the cdc21⁺ ORF under control a regulatable promoter. The resulting construct was used to complement the cdc21 deletion. Both mutants were inviable under appropriate conditions arresting in S phase as elongated cells, although a proportion of them (15-20%) entered mitosis in the absence of nuclear division.

The intracellular transport of organelles and vesicles is thought to utilise both microtubules and actin filaments, which mediate long and short-range transport, respectively. Melanosomes, synthesised in melanocytes, are a convenient model organelle to study intracellular transport, since they are visible using brightfield microscopy. They are believed to be transported from the perinuclear area to the actin cortex along microtubules, and then captured by the myosin-Va/melanophilin/Rab27a complex which traffics them along actin filaments to the plasma membrane. In contrast, data presented here demonstrate that anterograde melanosome transport relies only upon the actin cytoskeleton. Myosin-Va null melanocytes were used to test the importance of microtubules and actin on long-range organelle transport. In these cells, melanosomes cluster around the perinuclear area, but disperse into peripheral dendrites upon reintroduction of the myosin-Va gene. When this assay was repeated in the absence of microtubules, melanosomes still dispersed indicating that microtubule-based motors are not necessary for long-range anterograde trafficking. However, depolymerising F-actin, or freezing actin dynamics with latrunculin A or jasplakinolide inhibited the dispersion of pigment granules in myosin-Va null cells melanocytes and induced a clustered phenotype in WT melanocytes. This effect was abolished if microtubules were absent, suggesting that microtubules are only required for retrograde transport whilst dynamic actin is essential for anterograde melanosome transport. Moreover, when Kif5B was forcibly recruited to the melanosome membrane via an inducible dimerisation system, the melanosomes dispersed abnormally. An siRNA knockdown screen of over 120 actin binding proteins identified several proteins including formin-1, Arpc1b, cofilin-1, gamma-actin and spire1/2, which appear to be necessary for maintaining peripherally dispersed melanosomes. This evidence further underlines the importance of the actin cytoskeleton, rather than the microtubule network as previously thought, for the anterograde trafficking of melanosomes.

Dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Master of Science in Anatomical Sciences. / Due to paucity of free respiratory macrophages (FRMs), compared with mammals, birds have been alleged to be more susceptible to pulmonary infections and affliction. The goal of this study was to question or validate this speculation. Twenty-two mature healthy chickens, 24 domestic ducks and 20 rats were used in various experiments. After pulmonary lavage, FRMs were stained with trypan blue for cell count and with neutral red and trypan blue to assess cell longevity. The cell dynamics was determined by counts made on serial lavages. The morphological attributes of the FRMs and that of the respiratory “phagocytic epithelium” were quantified stereo logically. The rat had a significantly greater number of macrophages and the surface density of the filopodia of the FRMs was higher than that of the birds. The volume density of the vesicular bodies of the macrophages in the three groups of animals was not significantly different. Putative cell flux onto the respiratory surface was observed in the birds and not in the mammal. The surface area of the “phagocytic epithelium” of the birds was very extensive. Compared with mammals, in general, FRMs are fewer in birds. Despite this, the pulmonary defensive status in birds may not necessarily be compromised: functionally, avian FRMs appear to be more efficient. Moreover, their defense function is complimented particularly by the phagocytic activity of the epithelium immediate to the blood-gas barrier, the most vulnerable site of the lung-air sac system. / WHSLYP2017

To improve the design of prostheses surfaces, our research group investigates how biocompatible polyelectrolyte multilayers (PEMUs) can be constructed to serve as coatings for biomedical implants, providing a versatile, inexpensive, and potentially efficient solution to create anti-bacterial, anti-inflammatory, and biologically selective surfaces. More specifically, this dissertation research investigates how individual cells and cell sheets adhere and migrate on PEMUs constructed to have uniform and gradients of modulus and how individual cells and gram negative bacteria, Escherichia coli, adhere to PEMUs constructed to have an anti-adhesive surface chemistry. In this investigation, PAH/PAA PEMUs are shown to be biocompatible compared to the soluble polycation PAH at concentrations above 0.1mM. Soluble PAH concentrations at 1 and 10mM cause irreversible damage to the plasma membrane of smooth muscle, A7r5, and bone, U2OS, cells. Additionally, adhesive and motile responses of cells are dependent on PEMU surface chemistry. Cells on PEMUs terminated with the polycation PAH relocalize their focal adhesions to their cell periphery and are highly motile compared to cells cultured on PAA terminated PEMUs and uncoated glass coverslips. To investigate effects of PEMU modulus on cell adhesion and motility, PEMUs were made with the polyanion PAA (poly(acrylic acid)) modified with a photosensitive 4-(2-Hydroxyethoxy) benzophenone (PAABp) and the polycation PAH (poly(allylamine hydrochloride)). UV irradiating PAH/ PAABp PEMUs forms covalent bonds between PE layers and consequently increases its Young's Elastic Modulus, while retaining innate surface chemistry. Individual cells and cell sheets detect differences in PEMU modulus and respond by varying morphology and behavior. These PAH/PAABp PEMUs modulate the adhesion, spreading, and migration of individual cells, specifically smooth muscle, bone, and fibroblast cells. PAABp containing PEMUs were constructed to have either a shallow (~5MPa mm-1) or a steep (~50MPa mm-1) modulus gradient. Only smooth muscle cells durotax along steep modulus gradients toward increasing modulus and orient toward increasing modulus on shallow modulus gradients. In contrast, bone cells discriminately adhere to the stiffest region of both steep and shallow modulus gradients and fibroblasts show no difference in behavior along any region of the gradients. Epithelial sheets, isolated as primary explants of fish epithelial tissue from the scales of fish Poecilia sphenops (Black Molly) and Carassius auratus (Comet Goldfish), orient toward increasing modulus on steep modulus gradient. Cell sheets collectively durotax near the ~90MPa region of the gradient toward increasing modulus. Surfaces with substantial zwitterionic functionality (possessing a net neutral surface charge due to equal contribution of both positive and negative charges in polymer side groups) have been shown to effectively prevent cell and protein attachment. PEMUs built with PAH (poly(allylamine hydrochloride)) and PAA (poly(acrylic acid)) containing the AEDAPS zwitterionic group 3-(2-(acrylamido)-ethyldimethyl ammonio) propane sulfonate (PAH/PAA-co-AEDAPS PEMUs) and a new benzophenone crosslinker to stiffen the thin film were shown to prevent rat aortic smooth muscle (A7r5) and mouse fibroblast (3T3) cells attachment, but failed to prevent irreversible attachment of biofilm-forming gram-negative bacteria Escherichia coli, strain ATCC-8739. AEDAPS containing PEMUs are hydrophilic and have increased nanoroughness of ~10nm. 'Super soaking' AEDAPS PEMUs incorporates more zwitterions into the PEMU and significantly maximizes the surface presentation of PAA-co-AEDAPS, which promotes early attachment of bacteria, but eventually, causes a gradual decrease in bacteria attachment with increasing incubation time. This investigation provides further insight into the possible application of PEMUs as bioselective thin film coatings, which may have potential for use in biomedical applications. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the Doctor of Philosophy. / Fall Semester 2015. / October 26, 2015. / Biocompatibility, Cell Adhesion, Durotaxis, Epithelial Cell Sheet, Motility, Polyelectrolyte Multilayers / Includes bibliographical references. / Thomas C. S. Keller, III, Professor Directing Dissertation; Joseph B. Schlenoff, University Representative; Laura R. Keller, Committee Member; Teng Ma, Committee Member; Steven Lenhert, Committee Member.

Sulfur, an essential element required by the cell for the biosynthesis of crucial compounds, such as amino acids, co-factors and vitamins, is found most abundantly in the form of sulfate. In order to cross the lipid bilayer for cell usage, sulfate, being a negatively charged ion, requires an energetically `friendly' passageway. This could be accomplished via an ion channel that allows the flow of sulfate into the cell along its concentration gradient, or via an active transport system that allows sulfate to be taken up by the cell when it is in scarcity in the environment, against its concentration gradient, like the ABC transporter and sodium or proton-coupled symporters. The subject of this dissertation, CysZ, is a bacterial sulfate transport protein that does not belong to any known superfamily of canonical transporters or channels with no distinguishing features that could hint at its functional mechanism. We have undertaken a structural and functional approach to further understanding the role of CysZ in sulfate transport. In this dissertation, we report the structure of CysZ, its functional role in sulfate uptake and have suggested a mechanistic hypothesis for the passage of sulfate through CysZ along with the exciting future directions that might follow.

Within the past thirty years, the discovery and characterization of the microtubule-associated motor proteins, kinesins and cytoplasmic dynein, has radically expanded our understanding of intracellular trafficking and motile phenomena. Nevertheless, the mechanisms by which eukaryotic cells integrate motor functionality and cargo interactions over multiple subcellular domains in a spatiotemporally controlled way remain largely mysterious. During transport within the neuronal axon, dynein and the kinesins run in opposite directions along uniformly polarized microtubule tracks, so that each motor must switch between active transport and being, itself, a cargo in order to be properly positioned and carry out its function. The axon thus represents a model system in which to study the regulatory mechanisms governing intracellular transport, especially under conditions when it must be modulated in response to changing environmental cues, such as during axon outgrowth and development. Recently, the localization of certain messenger RNAs and their local translation to yield protein has emerged as a critical process for the development of axons and other neuronal compartments. I observed that transcripts encoding the dynein cofactors Lis1 and dynactin are among those localized to axons, so I hypothesized that stimulus-dependent changes in axonal transport may occur via local synthesis of dynein cofactors. In these studies, I have shown that different conditions of nerve growth factor signaling on developing axons trigger acute changes in the transport of various axonal cargoes, contemporaneous with rapid translational activation and production of Lis1 and dynactin’s main subunit, p150Glued, within the axons themselves. Differential synthesis of these cofactors in axons was confirmed to be required for the observed stimulus-dependent transport changes, which were completely prevented by axon-specific pharmacologic inhibition of protein synthesis or RNA interference targeted against Lis1 and p150Glued. In fact, Lis1 was, in an apparent paradox, locally synthesized in response to both nerve growth factor stimulation and withdrawal. I demonstrated that this is due to the fact that Lis1 is produced from a heterogeneous population of localized transcripts, differentiated chiefly by whether they interact with the RNA-binding protein APC. Preventing the binding of APC to Lis1 transcripts thus inhibited axonal synthesis of Lis1 and its resultant transport effects under conditions of nerve growth factor stimulation, while having no bearing on the similar phenomena seen during nerve growth factor withdrawal. This demonstrates that association with RNA-binding proteins can functionally distinguish sub-populations of localized messenger RNAs, which, in turn, provides a foundation for mechanistically understanding how localized protein synthesis is coupled to specific stimuli. Axonally synthesized Lis1 also was shown to have a particular role in mediating transport of a retrograde death signal originating in nerve growth factor-deprived axons, as neurons exhibited greatly reduced cell death when axonal synthesis of Lis1 was blocked. Through the application of pharmacologic agents inhibiting different steps in the propagation of this pro-apoptotic signal, I established that the signal depends upon effective endocytosis and the activity of glycogen synthase kinase 3β. It is therefore likely that the retrogradely transported signaling cargo in question is a glycogen synthase kinase 3β-containing endosome or multivesicular body—a type of large cargo consistent with Lis1’s known role in adapting the dynein motor for high-load transport. Preliminary results further indicate that axons exposed to another type of degenerative stress, in the form of toxic amyloid-β oligomers, may also employ local synthesis of Lis1 as a means of regulating transport and survival signaling. These findings establish a previously undescribed mechanism of regulating dynein activity and cargo interactions through local synthesis of its cofactors, allowing for rapid responses to environmental cues and stimuli that are especially relevant during the development of the nervous system. In addition to illustrating a regulatory principle that may be generally applicable to subcellular compartments throughout polarized cells, these studies provide new insights into intracellular transport disruptions that occur in lissencephaly, neurodegeneration, and other human disease states.