Akademisk avhandling--Uppsala. / Extra t.p., with thesis statement, inserted. Bibliography: p. 73-76.

Akademisk avhandling--Uppsala. / Extra t.p., with thesis statement, inserted. Bibliography: p. 73-76.

Thesis (M.S.)--Duquesne University, 2004. / Title from document title page. Abstract included in electronic submission form. Includes bibliographical references (p. 55-60) and index.

Thesis (M.S.)--University of Michigan, 2002. / Includes bibliographical references.

Thesis (M.S.)--University of Michigan, 2001. / Includes bibliographical references.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Pterocarpus angolensis is one of the timber tree species that regenerates naturally in the dry forest of Namibia, mainly assisted by the influence of forest fires. Tree development goes through a prolonged suffrutex stage to reach the sapling stage and then, finally, the bole tree stage. This study focused on assessing the main factors facilitating the development of Pterocarpus angolensis seedlings through the suffrutex stage to the sapling stage in Namibia dry forests. To achieve the study objectives three study locations (Okongo and Ncumcara Community Forests and Caprivi State Forest) were selected, representing a rainfall gradient. Within each study location, two different fire history treatments (recently burnt (RB) and recently unburnt (RU)) were selected, and four plots were randomly selected from each fire history treatment. Face to face individual interviews was conducted with community members surrounding the three forests to obtain indigenous knowledge information about Pterocarpus angolensis tree development. Seedlings and saplings found in all plots were counted and measured (tree height and diameter at breast height (DBH)) while trees more than 3 m high were only counted and measured for DBH. Laboratory analysis was performed to determine basic soil texture and nutrient status. In addition, destructive sampling was done on individual trees in the seedling and sapling stages at each study location. The destructive samples allowed for estimation of biomass in above and below ground components, determination of carbohydrate storage in the taproots and estimation of tree age by counting growth rings on the neck disc of the taproot sample. These measures could shed light on the tree development through the suffrutex stage. The main agents causing Pterocarpus angolensis tree damage and stand disturbances observed are drought, fires, insects, diseases, temperature, lightning, wind, animals and humans. Forest fires were found to be one of the major disturbances in all the study locations, particularly damaging to seedlings when fire intensity is high. Likewise, the most important factors influencing the tree development from seedlings to sapling and sapling to bole tree stages are soil water, soil fertility, plant competition, sunlight and fires. Through counting growth rings of taproot neck discs, it is estimated that the ages of seedlings most commonly range from 5 to 12 years in the dry forests of Okongo, Ncumcara and Caprivi. The soil texture in the three forests is dominated by sand, with the soil reaction usually being moderately acidic while the soils have low levels of organic carbon, phosphorus and exchangeable base cations. This study revealed that Caprivi State Forest (location with the highest rainfall) has the highest stand density followed by Okongo Community Forest and Ncumcara Community Forest with the lowest. Trees were grouped into different DBH and height classes. The highest numbers of trees are found in DBH class 0 – 10 cm and in height class 0.6 – 1.0 m at Okongo Community Forest but at Ncumcara and Caprivi many of the trees are in height class 1.1 – 1.5 m. The mean DBH difference is significant between locations but not significant between fire history treatments. A higher abundance of mature trees are found at Okongo Community Forest while a greater abundance of saplings occur at Ncumcara Community Forest which shows a significant difference between study locations. Seedling abundance is the same across study locations and fire history treatments. The difference in stand structure between study locations appears to be strongly influenced by different management regimes on the three locations. A majority of respondents from all the study locations alleged soil water followed by soil fertility as the main influential factors to Pterocarpus angolensis development. Again, most of the respondents revealed that seedling takes 4 – 7 years to reach sapling stage and their main environmental disturbance is fire. Tree cutting by members of the community was also perceived by the respondents as an important non-environmental disturbance. The most abundant tree development stage perceived by respondents was mature trees while seedlings rated the sparsest stage. Based on the respondents no silvicultural practices are performed to promote Pterocarpus angolensis growth. It follows that the Kiaat trees are currently growing without human intervention that might enhance their development. A combination of social survey (interview) and ecological survey provided reliable information on ecological processes. A weak positive significant correlation relationship existed between shoot mass (aboveground biomass) and taproot mass (belowground biomass), meaning when the taproot mass increases the shoot mass also increases. Analysis of non-structural carbohydrates (NSC) storage in taproots showed that both sugar and starch contents in the taproots could facilitate the survival of the tree during suffrutex stages and its rapid growth thereafter. Based on this study Pterocarpus angolensis regeneration in these three dry forests is poor because seedling abundance is the lowest compared to saplings and mature trees. These study findings can be used as the basis for further studies to predict Pterocarpus angolensis natural regeneration in the dry forests, as well as input when management regimes are being developed for the dry forests of North Namibia. / AFRIKAANSE OPSOMMING: Pterocarpus angolensis (Kiaat) is een van die boomspesies wat natuurlik verjong in die droë bosveld van Namibië, met die hulp van bosbrande. Die boom ontwikkel deur ʼn lang semi-struik stadium waartydens die boompies as saailinge bekendstaan. Daarna ontwikkel dit deur die jongboom stadium tot dit uiteindelik die kroon stadium bereik. Hierdie studie fokus op die faktore bydra tot die ontwikkeling van Pterocarpus angolensis van die semi-struik stadium na die jongboom stadium in die droë bosveld van Namibië. Om die doelstellings van die tesis te bereik is drie studiegebiede gekies langs ʼn reënvalgradiënt (naamlik Okongo en Ncumcara gemeenskapsbosse asook Caprivi Staatsbos). Binne elke studiegebied is twee behandelings met verskillende brandgeskiedenis gekies (gebrand of nie-gebrand in die onlangse verlede). Vier persele is ewekansig uit elk van hierdie behandelings gekies vir eksperimentering. Persoonlike onderhoude is gevoer met gemeenskapslede wat in die omgewing woon ten einde inheemse kennis en inligting te versamel oor die ontwikkeling van die jong Pterocarpus angolensis bome. Alle saailinge en jongbome wat voorkom in die persele is getel en gemeet (boomhoogte en deursnee op borshoogte (DBH)) terwyl bome wat hoër as 3 m is, slegs getel en vir DBH gemeet is. Laboratoriumtoetse is gedoen op grondmonsters ten einde ‘n basiese beskrywing van die grondtekstuur en voedingstofstatus te verkry. Verder is destruktiewe bemonstering toegepas op bome in beide die saailing en jongboom stadium op elke studiegebied. Hierdie bemonstering het dit moontlik gemaak om bogrondse en ondergrondse biomassa te skat, om die opberging van koolhidrate in die penwortels te bepaal, en ook om die boom ouderdom te skat vanaf jaarringe in die nek van die penwortel monster. Hierdie metings kon lig werp op die boomontwikkeling deur die semi-struik stadium. Die faktore wat skade aan Pterocarpus angolensis bome veroorsaak asook versteuring van die opstande waarin die bome voorkom is droogte, brande, insekte, siektes, temperatuur uiterstes, weerlig, wind, diere en mense. Die bevindinge dui op bosbrande as een van die belangrikste versteuringsfaktor in al drie studiegebiede; dit is veral skadelik vir saailinge in die semi-struik stadium wanneer die vuurintensiteit hoog is. Die faktore wat die boomontwikkeling van saailing, na jongboom en kroonstadium beïnvloed is hoofsaaklik grondwater, grondvrugbaarheid, plantkompetisie, sonlig en brande. Die ouderdom van saailinge (bepaal vanaf jaarring tellings in die nek van penwortel monsters) van die meeste saailinge én jongbome is na raming tussen 5 en 12 jaar vir die droë bosse in die studiegebiede van Okongo, Ncumcara en Caprivi. Die grondtekstuur van hierdie studie se drie bosgebiede is hoofsaaklik sanderig, met ’n effens suur grondreaksie terwyl die gronde lae vlakke van organiese koolstof, fosfor, en uitruilbare basiese katione bevat. Die studie het aangedui dat Caprivi staatsbos (met die hoogste reënval) die digste opstande huisves, gevolg deur Okongo en dan Ncumcara gemeenskapsbos, met die laagste digtheid. Bome is gegroepeer in verskillende DBH en hoogte klasse. Die meeste bome kom voor in die DBH klas van 0-10 cm en in die hoogteklas van 0.6 – 1.0 m by Okongo, maar by Ncumcara en Caprivi is daar meer bome in die hoogteklas van 1.1 - 1.5 m. Die gemiddelde DBH verskil is betekenisvol tussen studiegebiede, maar is nie betekenisvol verskillend tussen brandgeskiedenis behandelings nie. ’n Hoër voorkoms van volwasse bome is by Okongo aangetref, terwyl ’n hoër voorkoms van jongbome by Ncumcara waargeneem is, en hierdie verskil was statisties betekenisvol. Die voorkoms van saailinge is soortgelyk oor alle studiegebiede en brandgeskiedenis behandelings heen. Die verskil in die struktuur van die opstande op die drie studiegebiede word skynbaar sterk beïnvloed deur verskillende bestuurspraktyke wat in elke gebied toegepas word. Die meerderheid van respondente van al drie studiegebiede beweer dat grondwater, gevolg deur grondvrugbaarheid die belangrikste faktore is wat P. angolensis ontwikkeling beïnvloed. Meeste van die respondente onthul dat saailinge 4 tot 7 jaar neem om die jongboom stadium te bereik en dat die belangrikste versteuringsagent bosbrande is. ʼn Belangrike nie-omgewingsfaktor wat verantwoordelik is vir versteuring in die bosse is mense wat bome, lote en/of takke afsaag. Respondente is van mening dat volwasse bome die grootteklas met die mees algemene voorkoms is, terwyl saailinge die skaarsste grootteklas uitmaak. Die respondente het aangedui dat geen boskultuurpraktyke toegepas word om die groei van P. angolensis aan te help nie. Die gevolgtrekking is dus dat die Kiaatbome tans groei sonder menslike ingryping om hul ontwikkeling te verbeter. Die kombinasie van persoonlike onderhoude en ʼn ekologiese opnames het betroubare inligting rakende ekologiese prosesse opgelewer. ’n Swak positiewe, maar betekenisvolle korrelasie bestaan tussen die massa van die bogrondse lote en die penwortelmassa, wat beteken dat die lote se massa toeneem met toenemende wortelmassa. Analise van opgebergde nie-strukturele koolhidraatreserwes in die penwortel toon dat beide suiker- én styselinhoud in die penwortels die oorlewing van die boom in die struikstadium aanhelp, asook sy vinnige groei na die struikstadium. Die feit dat die saailinge minder volop is as jongbome en volwasse bome in hierdie studie dui aan dat verjonging van Pterocarpus angolensis in hierdie droë bosse maar swak is. Die bevindinge van die studie bevat inligting wat gebruik kans word (a) as die grondslag van verdere studies op die natuurlike verjonging van Pterocarpus angolensis in droë bosse, en (b) as inset wanneer bestuursaanbevelings vir die droë bosse van Noord Namibië ontwikkel word.

1. A brief historical introduction is given. It deals with the general features of wound healing in which epithelial participation is the dominant feature. 2. In Section A the influence of ascorbic acid on healing of skin wounds in rats is discussed. 3. Second wounds were inflicted at the same site as the initial wounds one month after the latter had healed. 4. In Section B the influence of ascorbic acid on healing of skin wounds in guinea pigs is investigated. 5. In order to discover the action ascorbic acid has on ear wounds, the tips of guinea pigs' ears were cut off. 6. The influence of ascorbic acid on the regeneration of corneal epithelium in guinea pigs is discussed. 7. The effect of ascorbic acid on healing of muco-periosteum was studied. 8. The influence of pantothenic acid on healing of skin wounds in rats was studied. 9. Pantothenic acid (Bepanthen-Salbe) ointment was applied directly to skin wounds.

The cell is the central functional unit of life. Cell behaviors, such as cell division, movements, differentiation, cell death as well as cell shape and size changes, determine how tissues change shape and grow during regeneration and development. However, a generally applicable framework to measure and describe the behavior of the multitude of cells in a developing tissue is still lacking. Furthermore, the specific contribution of individual cell behaviors, and how exactly these cell behaviors collectively lead to the morphogenesis and growth of tissues are not clear for many developmental and regenerative processes. A promising strategy to fill these gaps is the continuing effort of making developmental biology a quantitative science. Recent advances in methods, especially in imaging, enable measurements of cell behaviors and tissue shapes in unprecedented detail and accuracy. Consequently, formalizing hypotheses in terms of mathematical models to obtain testable quantitative predictions is emerging as a powerful tool. Tests of the hypotheses involve the comparison of model predictions to experimentally observed data. The available data is often noisy and based on only few samples. Hence, this comparison of data and model predictions often requires very careful use of statistical inference methods. If one chooses this quantitative approach, the challenges are the choice of observables, i.e. what to measure, and the design of appropriate data-driven models to answer relevant questions. In this thesis, I applied this data-driven modeling approach to vertebrate morphogenesis, growth and regeneration. In particular, I study spinal cord and muscle regeneration in axolotl, muscle development in zebrafish, and neuron development and maintenance in the adult human brain. To do so, I analyzed images to quantify cell behaviors and tissue shapes. Especially for cell behaviors in post-embryonic tissues, measurements of some cell behavior parameters, such as the proliferation rate, could not be made directly. Hence, I developed mathematical models that are specifically designed to infer these parameters from indirect experimental data. To understand how cell behaviors shape tissues, I developed mechanistic models that causally connect the cell and tissue scales. Specifically, I first investigated the behaviors of neural stem cells that underlie the regenerative outgrowth of the spinal cord after tail amputation in the axolotl. To do so, I quantified all relevant cell behaviors. A detailed analysis of the proliferation pattern in space and time revealed that the cell cycle is accelerated between 3-4 days after amputation in a high-proliferation zone, initially spanning from 800 µm anterior to the amputation plane. The activation of quiescent stem cells and cell movements into the high-proliferation zone also contribute to spinal cord growth but I did not find contributions by cellular rearrangements or cell shape changes. I developed a mathematical model of spinal cord outgrowth involving all contributing cell behaviors which revealed that the acceleration of the cell cycle is the major driver of spinal cord outgrowth. To compare the behavior of neural stem cells with cell behaviors in the regenerating muscle tissue that surrounds the spinal cord, I also quantified proliferation of mesenchymal progenitor cells and found similar proliferation parameters. I showed that the zone of mesenchymal progenitors that gives rise to the regenerating muscle segments is at least 350 µm long, which is consistent with the length of the high-proliferation zone in the spinal cord. Second, I investigated shape changes in developing zebrafish muscle segments by quantifying time-lapse movies of developing zebrafish embryos. These data challenged or ruled out a number of previously proposed mechanisms. Motivated by reported cellular behaviors happening simultaneously in the anterior segments, I had previously proposed the existence of a simple tension-and-resistance mechanism that shapes the muscle segments. Here, I could verify the predictions of this mechanism for the final segment shape pattern. My results support the notion that a simple physical mechanism suffices to self-organize the observed spatiotemporal pattern in the muscle segments. Third, I corroborated and refined previous estimates of neuronal cell turnover rates in the adult human hippocampus. Previous work approached this question by combining quantitative data and mathematical modeling of the incorporation of the carbon isotope C-14. I reanalyzed published data using the published deterministic neuron turnover model but I extended the model by a better justified measurement error model. Most importantly, I found that human adult neurogenesis might occur at an even higher rate than currently believed. The tools I used throughout were (1) the careful quantification of the involved processes, mainly by image analysis, and (2) the derivation and application of mathematical models designed to integrate the data through (3) statistical inference. Mathematical models were used for different purposes such as estimating unknown parameters from indirect experiments, summarizing datasets with a few meaningful parameters, formalizing mechanistic hypotheses, as well as for model-guided experimental planning. I venture an outlook on how additional open questions regarding cell turnover measurements could be answered using my approach. Finally, I conclude that the mechanistic understanding of development and regeneration can be advanced by comparing quantitative data to the predictions of specifically designed mathematical models by means of statistical inference methods.

Focal cartilage defects (FCD) of the knee joint remain a difficult area of treatment for orthopaedic surgeons, as they often progress to generalized osteoarthritis (OA). Osteochondral autograft transfer (OAT) to the damaged cartilage area has shown promise, but this has been associated with pain and bleeding at the site of graft harvest. The use of mesenchymal stem cells (MSCs) in a matrix to regenerate articular cartilage has been proposed. This work describes a prospective case-control series comparing OAT with a novel, MSC-seeded scaffold graft in the stifle joints of healthy merino sheep. The triphasic grafts were composed of a beta-tricalcium phosphate osseous phase, an intermediate activated plasma phase and a collagen I hydrogel cartilage phase. The osseous and cartilage phases were seeded with autologous MSCs. All sheep underwent creation of a full-thickness, 4.0 mm diameter FCD (n=20) followed by six weeks of unrestricted activity, allowing the defects to degenerate naturally. At six weeks, half of the lesions were treated with OAT and half with the triphasic engineered grafts. At 6-month and 12-month follow-up, no significant differences were noted between groups with regard to overall histological scores. Macroscopic and biomechanical analysis at 12 months showed no significant differences between groups. In summary, autologous MSC-seeded implants showed comparable repair quality to OAT without the associated donor site morbidity.

Regeneration is a process that renews damaged or lost cells, tissues, or even of entire body structures, and is a phenomenon which is widespread in the animal kingdom. Urodeles such as newts and salamanders have a remarkable regeneration ability. They can regenerate organs such as gills, lower jaws, retina, appendages like fore- and hind limbs, and also the tail including the spinal cord. The regeneration process requires the use of resident stem cells or somatic cells, which have to be reprogrammed. In both cases the reprogrammed cells are less differentiated, meaning the cell would have the ability to form any kind of fetal or adult cell which rose from the three different germ layers, the ectoderm, mesoderm and endoderm. Artificial reprogramming of differentiated mammalian somatic cell had been reported previously. It was shown that four pluripotency factors, OCT4 (also called POU5f1), SOX2, c-MYC and KLF4 are sufficient to generate an induced pluripotent stem (iPS) cell. It has been shown that some of these factors are also involved in regenerating processes. In newt limb and lens tissue, Sox2, c-Myc and Klf4 mRNA levels were upregulated in the beginning of blastema formation when compared to non-amputated tissue. Oct4 mRNA however, was not detected. During xenopus tail regeneration, Sox2 and c-Myc were expressed, while the xenopus Pou homologs Pou25, Pou60, Pou79, Pou91 were not detected. In regenerating zebrafish fin tissue, Sox2, Pou2, c-Myc and Klf4 mRNA were not upregulated. The mammalian transcription factor OCT4, a class V POU protein, is responsible in maintaining pluripotency in gastrula stage embryos. It was reported that mouse OCT4 is also expressed in the caudal node of embryos having 16 somites. It is further known that progenitors exist in mouse tailbud, which give rise to neural and mesodermal cell lineage. This suggests that the OCT4 expressing cells in caudal node might be a stem cell reservoir. Oct4 was detected in axolotl during embryonic development, and prior to my work we found Oct4 when screening the axolotl blastema cDNA library. In addition, we also identified Pou2, another class V POU gene. Phylogenetic analysis showed a clear distinction of both genes in the axolotl. We determined the mRNA pattern of Pou2 during embryogenesis and compared it to Oct4 mRNA and protein. Both genes are expressed in the primordial germ cells and the pluripotent animal cap region of the embryo. Apart from this similarity, both genes have a different expression pattern in the embryo. We are interested in the involvement of OCT4, POU2, as well as the transcription factor SOX2 in regenerating axolotl spinal cord. We asked whether the cellular pluripotent character conferred by POU factors is limited to mammals or if it is an ancient characteristic of lower vertebrates. To answer the question we performed in vitro and in vivo studies. Hence this thesis is separated into two chapter. By in vitro studies we investigated the pluripotent PouV orthologs from different species. Therefore, we performed reprogramming experiments using mouse or human fibroblasts and transduced them with axolotl Oct4 or Pou2, in combination with human or axolotl Sox2, c-Myc and/or Klf4. The generated iPS cells with the different sets of factors had similar endogenous pluripotency gene expression profiles to embryonic stem cells. Further, iPS cells expressed the pluripotency markers like OCT4, NANOG, SSEA4, TRA1-60 and TRA1-81. Another evaluation of the iPS cells was the formation of embryoid bodies. Immunouorescence staining showed that tissue from all three germ layers was formed after induction. We observed a positive staining for the endoderm marker !-FEROPROTEIN, the mesoderm marker !-SMOOTH MUSCLE ACTIN and the ectoderm marker \"III TUBULIN in the generated cells. This indicated that the iPS cells generated using axolotl Oct4 and Sox2 in combination with mammalian Klf4 and with or without c-Myc, as well as iPS cell generated with axolotl Pou2 and mammalian Sox2 and Klf4 and with or without c-Myc have a pluripotent potential. In addition, the axolotl factors are able to form heterodimers with the mammalian proteins. Furthermore, we compared the reprogramming ability with POU factors from mouse, human, zebrash, medaka and xenopus. We showed that xenopus Pou91, as the only non-mammalian example, is nearly as efficient as mouse and human Oct4 cDNAs in inducing GFP expressing cells. Also axolotl Pou2, axolotl Oct4 and medaka Pou2 showed reprogramming character however at a much lower efficiency. In contrast, zebrash Pou2 is not able to establish iPS cells. This indicates that a reprogramming ability to a pluripotent cell state is an ancient trait of Pou2 and Oct4 homologs. By in vivo studies we investigated the role of Oct4, Pou2 and Sox2 gene expression in regenerating spinal cord tissue. Performed in situ hybridizations and antibody staining studies in the regenerating spinal cord showed that Oct4, Pou2 and Sox2 were expressed during spinal cord regeneration. Knockdown experiments in regenerating spinal cord using morpholino showed that Pou2-morpholino does not have an effect. In contrast, SOX2 was required for spinal cord regeneration but to a lesser extent, than OCT4, which decreased the regenerated length signicantly compared to control. Even though, with Sox2-morpholino we did not observe the phenotype as a significantly shorter regenerated spinal cord, about 45% of SOX2 knocked down cells were not cycling and proliferating anymore. This indicates that axolotl SOX2 has an effect in regeneration. Therefore we wanted to know whether spinal cord cells would also have a pluripotent character in vivo and form other tissue types. Regenerating cells of the spinal cord are only able to form the same cell type and thus they keep their cell memory. However, when we performed transplantations of OCT4/SOX2 expressing spinal cord cells into somite stage embryos, we could show the formation of muscle cells. This shows that the spinal cord cells have the potential to change their fate in an embryonic context, where the normal environment of spinal cord has changed. However, our data do not indicate whether muscle is formed directly from the spinal cord or whether spinal cord cells fuse to developmental myoblasts, a cell type of embryonic progenitors, which give rise to muscle cells. To clearly state whether regenerating OCT4/SOX2 expressing spinal cord cells are pluripotent we have to perform OCT4 knock down in spinal cord and transplant these less proliferating cells into embryos, observing their cell fate.