Computational methods in string and field theory

Thesis is submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the University of the Witwatersrand, Faculty of Science, School of Physics, University of the Witwatersrand, Johannesburg, 2018Like any field or topic of research, significant advancements can be made with increasing computational power - string theory is no exception. In this thesis, an analysis is performed within three areas: Calabi–Yau manifolds, cosmological inflation and application of conformal field theory. Critical superstring theory is a ten dimensional theory. Four of the dimensions refer to the spacetime dimensions we see in nature. To account for the remaining six, Calabi-Yau manifolds are used. Knowing how the space of Calabi-Yau manifolds is distributed gives valuable insight into the compactification process. Using computational modeling and statistical analysis, previously unseen patterns of the distribution of the Hodge numbers are found. In particular, patterns in frequencies exhibit striking new patterns - pseudo-Voigt and Planckian distributions with high confidence and exact fits for many substructures. The patterns indicate typicality within the landscape of Calabi–Yau manifolds of various dimensions. Inflation describes the exponential expansion of the universe after the Big Bang. Finding a successful theory of inflation centres around building a potential of the inflationary field, such that it satisfies the slow-roll conditions. The numerous ways this can be done, coupled with the fact that each model is highly sensitive to initial conditions, means an analytic approach is often not feasible. To bypass this, a statistical analysis of a landscape of thousands of random single and multifield polynomial potentials is performed. Investigation of the single field case illustrates a window in which the potentials satisfy the slow-roll conditions. When there are two scalar fields, it is found that the probability depends on the choice of distribution for the coefficients. A uniform distribution yields a 0.05% probability of finding a suitable minimum in the random potential whereas a maximum entropy distribution yields a 0.1% probability. The benefit of developing computational tools extends into the interdisciplinary study between conformal field theory and the theory of how wildfires propagate. Using the two dimensional Ising model as a basis of inspiration, computational methods of analyzing how fires propagate provide a new tool set which aids in the process of both modeling large scale wildfires as well as describing the emergent scale invariant structure of these fires. By computing the two point and three point correlations of fire occurrences in particular regions within Botswana and Kazakhstan, it is shown that this proposed model gives excellent fits, with the model amplitude being directly proportional to the total burn area of a particular year.EM201

Firewall argument for acoustic black holes

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of requirements for the degree of Master of Science. June 8, 2015.We investigate the rewall paradox proposed by AMPS [1] by rst explaining the Information Paradox together with Hawking's derivation of the thermal radiation emitted from a evaporating black hole [28]. We then ask if one can apply arguments similar to that of Hawking and AMPS in the regime of uid mechanics, which was rst considered by Unruh [59]. We assume that a black hole, with a geometry conformal to the Schwarzschild metric, can be formed in a uid. The sonic hole or \dumb" hole, which is characterized by an acoustic event horizon, is the locus of points at which the background uid is traveling at the local speed of sound. Since sound disturbances are coupled to the background uid and travel at the speed of sound, the acoustic event horizon a ects sound disturbances in a manner analogous to how gravitational black holes a ect light [62]. Like a gravitational black hole, which evaporates by emitting Hawking radiation, we check if an acoustic black hole will emit in a similar kind of radiation in the form of phonons. This is done by constructing a massless scalar eld describing phonon propagation and treating the acoustic black hole just like a gravitational black hole. We apply the arguments put forth by Hawking and AMPS and see if there is any validity to an \acoustic rewall" as this would require certain physical phenomena emerging from sub-atomic scales

Bioengineering of Fetal Skin: Differentiation of Amniotic Fluid Stem Cells into Keratinocytes

PURPOSE Open fetal spina bifida repair has become a novel clinical standard of care. In very large spina bifida lesions, the skin defect cannot be covered primarily, asking for alternative solutions. We hypothesize that amniotic fluid stem cells (AFSC) could be differentiated into keratinocytes that could then be used to bioengineer autologous skin usable for in utero back coverage. METHODS To obtain human AFSC, amniotic fluid samples obtained from fetal surgeries were subjected to immunoselection for c-kit. C-kit-positive samples and controls were cultured with the additives morphogenetic protein 4 and vitamin C to induce differentiation towards keratinocytes. This process was monitored by measuring the expression of K8 and K14 via immunohistochemical staining, flow cytometry, and polymerase chain reaction. RESULTS After immunoselection and expansion, most cells were positive for K8, but none for K14. After completion of the differentiation protocol, cell colonies with keratinocyte-like appearance could be observed, but cells remained positive for K8 and negative for K14, indicating failed differentiation into keratinocytes. CONCLUSIONS Culturing of keratinocyte-like cells from AFSC, harvested intraoperatively, was not feasible in this setting. The reasons for failure must be investigated and eliminated, as bioengineering of fetal skin for clinical use during fetal surgery for spina bifida remains an attractive goal

Bioengineering and in utero transplantation of fetal skin in the sheep model: A crucial step towards clinical application in human fetal spina bifida repair

An intricate problem during open human fetal surgery for spina bifida regards back skin closure, particularly in those cases where the skin defect is much too large for primary closure. We hypothesize that tissue engineering of fetal skin might provide an adequate autologous skin substitute for in utero application in such situations. Eight sheep fetuses of four time-mated ewes underwent fetoscopic skin biopsy at 65 days of gestation. Fibroblasts and keratinocytes isolated from the biopsy were used to create fetal dermo-epidermal skin substitutes. These were transplanted on the fetuses by open fetal surgery at 90 days of gestation on skin defects (excisional wounds) created during the same procedure. Pregnancy was allowed to continue until euthanasia at 120 days of gestation. The graft area was analyzed macroscopically and microscopically. The transplanted fetal dermo-epidermal skin substitutes was well discernable in situ in three of the four fetuses available for analysis. Histology confirmed healed grafts with a close to natural histological skin architecture four weeks after in utero transplantation. This experimental study generates evidence that laboratory grown autologous fetal skin analogues can successfully be transplanted in utero. These results have clinical implications as an analogous procedure might be applied in human fetuses undergoing prenatal repair to facilitate primary skin closure. Finally, this study may also fertilize the field of fetal tissue engineering in general, particularly when more interventional, minimally invasive, and open fetal surgical procedures become available

Determining the origin of cells in tissue engineered skin substitutes: a pilot study employing in situ hybridization

Background: Definitive and high-quality coverage of large and, in particular, massive skin defects remains a significant challenge in burn as well as plastic and reconstructive surgery because of donor site shortage. A novel and promising approach to overcome these problems is tissue engineering of skin. Clearly, before eventual clinical application, engineered skin substitutes of human origin must be grafted and then evaluated in animal models. For the various tests to be conducted it is indispensable to be able to identify human cells as such in culture and also to distinguish between graft and recipient tissue after transplantation. Here we describe a tool to identify human cells in vitro and in vivo. Methods: In situ hybridization allows for the detection and localization of specific DNA or RNA sequences in morphologically preserved cells in culture or tissue sections, respectively. We used digoxigenin-labeled DNA probes corresponding to human-specific Alu repeats in order to identify human keratinocytes grown in culture together with rat cells, and also to label split and full thickness skin grafts of human origin after transplantation on immuno-incompetent rats. Results: Digoxigenin-labeled DNA probing resulted in an intensive nuclear staining of human cells, both in culture and after transplantation onto recipient animals, while recipient animal cells (rat cells) did not stain. Conclusion: In situ hybridization using primate-specific Alu probes reliably allows distinguishing between cells of human and non-human origin both in culture as well as in histological sections. This method is an essential tool for those preclinical experiments (performed on non-primate animals) that must be conducted before novel tissue engineered skin substitutes might be introduced into clinical practic

Optimizing in vitro culture conditions leads to a significantly shorter production time of human dermo-epidermal skin substitutes

Introduction: Autologous dermo-epidermal skin substitutes (DESS) generated in vitro represent a promising therapeutic means to treat full-thickness skin defects in clinical practice. A serious drawback with regard to acute patients is the relatively long production time of 3-4weeks. With this experimental study we aimed to decrease the production time of DESS without compromising their quality. Methods: Two in vitro steps of DESS construction were varied: the pre-cultivation time of fibroblasts in hydrogels (1, 3, and 6days), and the culture time of keratinocytes (3, 6, and 12days) before transplantation of DESS on nude rats. Additionally, the impact of the air-liquid interface culture during 3days before transplantation was investigated. 3weeks after transplantation, the macroscopic appearance was evaluated and histological sections were produced to analyze structure and thickness of epidermis and dermis, the stratification of the epidermis, and the presence of a basal lamina. Results: Optimal DESS formation was obtained with a fibroblast pre-cultivation time of 6days. The minimal culture time of keratinocytes on hydrogels was also 6days. The air-liquid interface culture did not improve graft quality. Conclusion: By optimizing our in vitro culture conditions, it was possible to very substantially reduce the production time for DESS from 21 to 12days. However, pre-cultivation of fibroblasts in the dermal equivalent and proliferation of keratinocytes before transplantation remain crucial for an equilibrated maturation of the epidermis and cannot be completely skippe

CD146 expression profile in human skin and pre-vascularized dermo-epidermal skin substitutes in vivo

Background CD146 is a cell adhesion molecule whose expression profile in human skin has not yet been elucidated. Here, we characterize CD146 expression pattern in human skin, in particular in blood endothelial cells (BECs) and lymphatic endothelial cells (LECs), which constitute human dermal microvascular endothelial cells (HDMECs), as well as in perivascular cells. Results We demonstrated that CD146 is a specific marker of BECs, but not of LECs. Moreover, we found CD146 expression also in human pericytes surrounding blood capillaries in human skin. In addition, we demonstrated that CD146 expression is up-regulated by the TNFα-IL-1β/NF-kB axis in both BECs and pericytes. Finally, we engineered 3D collagen hydrogels composed of HDMECs, CD146+ pericytes, and fibroblasts which developed, in vitro and in vivo, a complete microvasculature network composed of blood and lymphatic capillaries with pericytes investing blood capillaries. Conclusions Overall, our results proved that CD146 is a specific marker of BECs and pericytes, but not LECs in human skin. Further, the combination of CD146+ pericytes with HDMECs in skin substitutes allowed to bioengineer a comprehensive 3D in vitro and in vivo model of the human dermal microvasculature

Transglutaminases, involucrin, and loricrin as markers of epidermal differentiation in skin substitutes derived from human sweat gland cells

Background/Purpose: In a multi-project research line, we are currently testing whether a morphologically and functionally near normal epidermis can be cultured from human sweat gland (SG) cells and be used as a skin substitute. The present study focuses on the stratum corneum of the epidermis that assumes a vital barrier function for the skin. The main process in the formation of the cornified cell envelope in human epidermis, i.e. crosslinking of proteins and lipids, is catalyzed by several transglutaminases (TG). Therefore, we compared the expression patterns of various TG and their substrates in SG-derived versus keratinocyte-derived epidermal substitutes. Methods: Sweat gland cells, keratinocytes, and fibroblasts were isolated from human skin samples and cultivated separately to generate epidermal substitutes. These were transplanted onto the back of athymic rats. After 2weeks, the transplants were excised and analyzed histologically as well as by indirect immunofluorescence. We looked at the expression of TG1, 3, 5, and their substrates involucrin and loricrin (=markers of epidermal differentiation) in SG-derived and keratinocyte-derived skin substitutes as well as in normal skin. Results: The SG cell-derived epidermis was near normal anatomically, formed a cornified cell envelope and demonstrated TG1, 3, and 5 as well as involucrin and loricrin expression patterns similar to those found in keratinocyte-derived epidermis and normal control skin. Conclusion: These findings support the thesis that SG cells have the potential to form a near normal stratified epidermal analog that might be used as a skin substitute. The expression of TG1 and 3, not normally expressed in human SG, suggests the presence of re-programmed SG cells and/or stem cells capable of both de novo generating and maintaining an epidermi

Skingineering I: engineering porcine dermo-epidermal skin analogues for autologous transplantation in a large animal model

Background: Extended full thickness skin defects still represent a considerable therapeutic challenge as ideal strategies for definitive autologous coverage are still not available. Tissue engineering of whole skin represents an equally attractive and ambitious novel approach. We have recently shown that laboratory-grown human skin analogues with near normal skin anatomy can be successfully transplanted on immuno-incompetent rats. The goal of the present study was to engineer autologous porcine skin grafts for transplantation in a large animal model (pig study=intended preclinical study). Materials and methods: Skin biopsies were taken from the pig's abdomen. Epidermal keratinocytes and dermal fibroblasts were isolated and then expanded on culture dishes. Subsequently, highly concentrated collagen hydrogels and collagen/fibrin hydrogels respectively, both containing dermal fibroblasts, were prepared. Fibroblast survival, proliferation, and morphology were monitored using fluorescent labelling and laser scanning confocal microscopy. Finally, keratinocytes were seeded onto this dermal construct and allowed to proliferate. The resulting in vitro generated porcine skin substitutes were analysed by H&E staining and immunofluorescence. Results: Dermal fibroblast proliferation and survival in pure collagen hydrogels was poor. Also, the cells were mainly round-shaped and they did not develop 3D-networks. In collagen/fibrin hydrogels, dermal fibroblast survival was significantly higher. The cells proliferated well, were spindle-shaped, and formed 3D-networks. When these latter dermal constructs were seeded with keratinocytes, a multilayered and partly stratified epidermis readily developed. Conclusion: This study provides compelling evidence that pig cell-derived skin analogues with near normal skin anatomy can be engineered in vitro. These tissue-engineered skin substitutes are needed to develop a large animal model to establish standardized autologous transplantation procedures for those studies that must be conducted before "skingineering” can eventually be clinically applie

Rebuild, restore, reinnervate: do human tissue engineered dermo-epidermal skin analogs attract host nerve fibers for innervation?

Purpose: Tissue engineered skin substitutes are a promising tool to cover large skin defects, but little is known about reinnervation of transplants. In this experimental study, we analyzed the ingrowth of host peripheral nerve fibers into human tissue engineered dermo-epidermal skin substitutes in a rat model. Using varying cell types in the epidermal compartment, we wanted to assess the influence of epidermal cell types on reinnervation of the substitute. Methods: We isolated keratinocytes, melanocytes, fibroblasts, and eccrine sweat gland cells from human skin biopsies. After expansion, epidermal cells were seeded on human dermal fibroblast-containing collagen type I hydrogels as follows: (1) keratinocytes only, (2) keratinocytes with melanocytes, (3) sweat gland cells. These substitutes were transplanted into full-thickness skin wounds on the back of immuno-incompetent rats and were analyzed after 3 and 8weeks. Histological sections were examined with regard to myelinated and unmyelinated nerve fiber ingrowth using markers such as PGP9.5, NF-200, and NF-145. Results: After 3weeks, the skin substitutes of all three epidermal cell variants showed no neuronal ingrowth from the host into the transplant. After 8weeks, we could detect an innervation of all three types of skin substitutes. However, the nerve fibers were restricted to the dermal compartment and we could not find any unmyelinated fibers in the epidermis. Furthermore, there was no distinct difference between the constructs resulting from the different cell types used to generate an epidermis. Conclusion: Our human tissue engineered dermo-epidermal skin substitutes demonstrate a host-derived innervation of the dermal compartment as early as 8weeks after transplantation. Thus, our substitutes apparently have the capacity to attract nerve fibers from adjacent host tissues, which also grow into grafts and thereby potentially restore skin sensitivit