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.

How do organisms attain the capacity to regenerate a structure, entire body, or not to regenerate? These are fundamental questions in biology for understanding how replicative systems are evolved to renew, age, and/or die. One outstanding question in regenerative biology that attracts attention is how and why the human central nervous system fails to regenerate after injury. Nervous system injuries are characterized by axonal damage and loss of synaptic function that contribute to debilitating neuronal dysfunctions. Although the molecular underpinnings of axon regeneration are well characterized, very little is known about how and what molecular pathways modulate reformation of synapses within regenerating axons to restore function. Thus, understanding the fundamental molecular and genetic mechanisms of functional axon regeneration (FAR), restoration of both axon and synapse, for the functional recovery of the nervous system remains elusive. In Chapter I, I outline the biology of regeneration and provide evolutionary perspectives of this phenomenon. Then, I provide clinical perspectives of central nervous system regeneration and therapeutic innovations. I next introduce the regulators of axon regeneration and how C. elegans as a genetic system allows detailed characterization of axon regeneration. In Chapter II, using C. elegans as a platform, I show how axon regeneration and synaptic reformation are controlled by distinct genetic pathways. I show how Poly-ADP ribose polymerase (PARP) pathway modulates functional restoration by regulating divergent genetic pathways leading to axon regeneration and synapse restoration. Finally, in Chapter III, I summarize the model of axon regeneration, evolutionary perspectives, and epistemic limitations of C. elegans axon regeneration.

Ce travail si concerne l`etude d`evolution du volume du fois apres l`hepatectomie majeure chez les porcs.Le planning se compose des imagairies sequentielle avant et apres le chirurgie, dans laquelle ablation d`une parite precis du fois est effectue. En suite, on etudie l`evolution de volume du fois et les facteures hemodynamic qui ont d`influence sur cet evolution. / The aim of this project is double. Firstly, relationships between liver function, blood flows and architecture along with liver volume after major hepatectomy will be carefully analyzed by combining experiments and modeling on multiple levels and scales. These results will be used by mathematicians to develop an alternative evaluation procedure based on innovative tools in order to improve identification of possible or likely post-operative liver failure as early as possible. In this way a critical liver function decrease can be duly treated. Preliminary studies in pigs indicate that patients in which liver failure is likely to occur, may significantly benefit from an implantable surgical device capable of modulating the diameter of the portal vein hence controlling the portal venous pressure into the liver.

We investigated the antibacterial effect of low concentrations of double antibiotic paste (DAP) loaded into a methylcellulose system against bacterial biofilms obtained from mature and immature teeth with necrotic pulps. Standardized radicular dentin specimens were randomly divided into six experimental groups (n = 20). Group 1: 5mg/mL DAP treatment. Group 2: 1mg/mL DAP treatment. Group 3: Calcium hydroxide (Ca(OH)2) treatment. Group 4: Methylcellulose. Group 5: No treatment. Group 6: No bacteria or treatment. Clinical bacterial isolates were obtained from mature and immature teeth with necrotic pulps indicated for endodontic regeneration or routine endodontic treatment, respectively. Specimens in each group were inoculated with either bacterial isolates (n = 10) and incubated anaerobically for 3 weeks. Specimens were then treated for one week with the assigned group treatment. Treatments were rinsed with sterile saline and biofilms were detached and spiral plated using biofilm disruption assays. Wilcoxon Rank Sum tests followed by pair-wise comparisons were used for statistical analyses. Treatment of infected dentin with 1 mg/ml of DAP, 5 mg/mL of DAP, and Ca(OH)2 demonstrated significant and substantial antibiofilm effects in comparison to untreated control groups or groups treated with placebo paste. Furthermore, 1 mg/mL of DAP caused complete eradication of biofilm obtained from mature tooth with necrotic pulp. However, the same concentration was not able to completely eradicate biofilm obtained from the immature tooth with necrotic pulp. Low concentrations of DAP (1-5 mg/mL) loaded into a biocompatible methylcellulose system demonstrated significant antibacterial effects against biofilm obtained from both mature and immature teeth with necrotic pulps.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20280号 / 医博第4239号 / 新制||医||1021(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 羽賀 博典, 教授 坂井 義治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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The factors affecting tree establishment in boreal forests after fire will help determine the community composition of the regenerating forest. These may have large consequences on the community dynamics for years after the fire disturbance. Factors such as burn severity and soil moisture among others have been shown to play a key role in influencing several facets of establishment. However, tree establishment after megafire in boreal forest in Europe has not yet been fully understood. Here I capitalise on a megafire in Sweden in 2014 to investigate the relative impact of different abiotic factors and preconditions on tree establishment six years after the fire. This study used a systematic survey of tree saplings (height >30cm) at 625 locations inside the nature reserve set up within the burnt area. Tested factors were: The number of dead trees lying down, slope and slope aspect, elevation, soil wetness, pre-fire standing volume, distance to fire perimeter, forest stand age, stand productivity index, previous stand dominant tree species, humus thickness after fire and depth of burn. Generalized Linear Mixed Models (GLMMs) were used to estimate the effect of these factors on specific tree species abundance. Strong influences from previous wood volume, soil wetness, elevation, and dead wood lying down had an effective influence on sapling abundance but were typically species-specific. Only elevation and wood volume had a consistent effect on all species’ abundances. Habitat context was important on a landscape scale. These results support the pattern of increasing boreal deciduousness caused by high burn severity and shorter disturbance intervals, in turn, caused by hotter, drier weather, which will have implications on the composition of boreal forests of tomorrow.

In this thesis project, different existing granular activated carbon regeneration methods/technologies are assessed based on existing literature. The project aims to identify and analyse the method with the highest on-site regeneration potential by using the Himmerfjärdsverket wastewater treatment plant as a reference and performing a cost estimation analysis.  Information is gathered about different methods from the literature study and then sorted into the following parts: working principle, technology readiness and cost, advantages and disadvantages, and references (case studies). The methods are then assessed and compared by a scoring and weightage system, where the factors which are regeneration efficiency, ease of implementation, sustainability, cost, and reliability are weighted and then scored for each method. Furthermore, the highest scoring method is then compared to the proposed regeneration method at Himmerfjärdsverket.  The results from my comparison and assessment show that chemical regeneration is the highest scored method, followed by microwave and wet-oxidation regeneration methods. On applying chemical regeneration at Himmerfjärdsverket, it is found out that it may indeed be cheaper and more sustainable than the proposed off-site regeneration method. However, thermal and biological regeneration are better alternatives at Himmerfjärdsverket than microwave and wet- oxidation.  From the above results, chemical regeneration has the highest potential for on-site regeneration of granular activated carbon in Sweden. / Läkemedel i vatten är ett stort hot mot miljö och hälsa. Kommunalt avloppsvatten består av avloppsvatten från hushåll, privata och offentliga institutioner och dagvattenavrinning. En viktig läkemedelskälla i avloppsvatten är ett läkemedel som kommer in via urin och / eller avföring. Olika tekniker finns för avlägsnande av farmaceutiska rester och andra mikroföroreningar från avloppsvatten. En sådan teknik är adsorptionen av dessa rester med hjälp av Granulärt Aktivtkor (GAC). Aktivtkor (AC) är ett kolhaltigt material med liten pordiameter, stora porvolymer och hög specifik yta rea vid bearbetning. Det anses vara det bästa adsorptionsmedlet för att adsorbera organiska, oorganiska och giftiga metalljoner som finns i avloppsvattnet. Det finns två typer av aktivtkol som används för att avlägsna farmaceutiska rester: Granulärt aktivtkol och pulveriserat aktivt kol.  Denna forskning syftar till att identifiera, jämföra och bedöma befintlig regenereringsteknik för att hitta den teknik som har störst potential och använda den på ett referensavloppsreningsverk. Det finns olika metoder för regenerering av använt aktivt kol (SAC). Dessa är termisk regenerering, kemisk regenerering, biologisk regenerering, elektrokemisk regenerering, mikrovågsregenerering och våt-oxidationsregenerering. Metoderna listas och förklaras med hjälp av deras arbetsprincip, beredskap, kostnad, fördelar, nackdelar och referenser till studier där de har använts.  Flera kriterier / faktorer beaktas för bedömning och jämförelse av olika regenereringsmetoder. Faktorerna poängsätts sedan med hjälp av ett viktat poängsystem. Var och en av de ovannämnda faktorerna görs mellan 0–5 och tilldelas en vikt mellan 1–3. En högre poäng betyder bättre prestanda i den givna faktorn. Medan en högre vikt betyder betydelsen av faktorn.  Från bedömningen visar sig kemisk regenerering vara den mest lämpliga metoden för regenerering av GAC på plats. Den minst lämpliga metoden är biologisk regenerering med en total poäng på 39. De två bästa regenereringsmetoderna på plats är kemisk och mikrovågsregenerering. I procent har kemisk regenerering och mikrovågsregenerering en rating på 93,3% och 90%. För att validera resultatet av bedömningen används Himmerfjärdsverket som referensavloppsverk.  Himmerfjärdsverket bygger om och expanderar till en högteknologisk anläggning. Den nya anläggningen kommer att bestå av modern reningsteknik och hög reningskapacitet. Den planerade nya anläggningen förväntas vara klar 2025, medan byggandet påbörjades i januari 2020.  I en studie genomförd av Syvab i samarbete med Ramboll, IVL och SU anges att det skulle behövas kolförbrukning på 15–20 g / m3 vatten. Den totala kostnaden per behandlat avloppsvatten skulle sannolikt öka med 20–30% till 2027 om läkemedelsreningen genomförs på Himmerfjärdsverket med hjälp av GAC och av regenereringsmetoden utanför anläggningen. Himmerfjärdsverket kommer att konsumera 3,92 ton kol dagligen eller 27,56 ton varje vecka. Detta kommer att kosta 28,7 miljoner SEK / år för 20 000 EBV (i värsta fall) och 11,5 miljoner SEK / år för 50 000 EBV.  Medan kostnaden för kemisk regenerering av GAC med högsta regenereringseffektivitet uppskattas till 27,4 miljoner SEK / år för användning av flytande NaOH-lösning och 17,7 miljoner SEK / år för användning av fast NaOH för 20 000 EBV. För mängden 573 t / år kol kommer NaOH-förbrukningen att vara 2083,5 t / år, vilket kommer att kosta 7,1 och 10,9 miljoner SEK / år för 50 000 EBV.  Eftersom kostnad är en av de viktigaste faktorerna som motverkar tillämpningen av metoder som är lika tillförlitliga som termisk regenerering. Om termisk regenerering implementeras på Himmerfjärdsverket kan installationen användas för att regenerera GAC från andra reningsverk från Stockholm. 2 GAC-bio filter i serieskapare bättre förutsättningar för biologisk regenerering av GAC samt ger låga föroreningskoncentrationer och höga syrekoncentrationer. För att lägga till det producerar Himmerfjärdsverket biogas som kan användas för att uppnå höga temperaturer som krävs enligt denna metod eller generera den erforderliga elen eller båda. I alla tre fall kommer kostnaden att minskas ytterligare.  Avslutningsvis har kemisk regenerering den högsta regenereringspotentialen på plats bland alla andra studerade metoder. Medan termisk regenerering är nära den andra på grund av kolförlusten. En pilotstudie krävs för att validera de regenereringseffektivitet som nämns i litteraturen och bearbetningsförhållandena och typerna av adsorbera vid Himmerfjärdsverket behöver utvärderas liksom behandlingsförhållandena.

Chronisch-entzündliche Darmerkrankungen wie Colitis ulcerosa (CU) sind Risikofaktoren für die Erkrankung an Kolorektalkarzinomen (KRK) (Eaden, Abrams et al. 2001, Lutgens, Vleggaar et al. 2008). Sowohl in CU (Hussain, Amstad et al. 2000, Takaku, Ajioka et al. 2001, Leedham, Graham et al. 2009, Kakiuchi, Yoshida et al. 2020) als auch in Colitis-assoziierten KRK (CA-KRK) (Yin, Harpaz et al. 1993, Brentnall, Crispin et al. 1994, Galandiuk, Rodriguez–Justo et al. 2012, Kobayashi, Tomita et al. 2017, Baker, Cross et al. 2019) treten vermehrt epitheliale Zellklone auf, die einen Verlust an physiologischem Tumorsupressor P53 Signaling haben. Diese Klone treiben vermutlich die Karzinogenese voran (Petitjean, Achatz et al. 2007). Bislang ist jedoch nicht verstanden, durch welchen Mechanismus diese Klone Wildtypzellen verdrängen könnten, um in der Mucosa fixiert zu werden. In der vorliegenden Studie nutze ich induzierbare Trp53 Knock-Out (KO) Mäuse, mithilfe derer ich die Auswirkungen eines Verlusts von P53 im Darmepithel im Colitis-Kontext sowohl in vivo als auch in vitro untersuchen kann. Ich zeige, dass während Trp53 für die Aufrechterhaltung der epithelialen Homöstase im Colon verzichtbar ist, es nötig ist, damit Epithelzellen nach der Schädigung durch eine chemische Colitis wieder in die Homöstase zurückkehren können. Epithel, dem P53 fehlt, verharrt nach der Schädigung in einem fetalen, regenerativen Status („locked regenerative state“), welcher durch eine andauernde Aktivierung des YAP-Signalweges charakterisiert ist. Alles in allem offenbart diese Arbeit die kontextabhängige Bedeutung des P53 Signalings im Kolonepithel im regenerativen Status nach colitis-assoziierter Schädigung und gibt erste Erklärungen für die erhöhte Häufigkeit von Zellklonen mit verringertem P53 Signaling in CU und CA-KRK. / Chronic inflammatory bowel diseases such as ulcerative colitis (UC) are risk factors for colorectal cancer (CRC) (Eaden, Abrams et al. 2001, Lutgens, Vleggaar et al. 2008). Both in UC (Hussain, Amstad et al. 2000, Takaku, Ajioka et al. 2001, Leedham, Graham et al. 2009, Kakiuchi, Yoshida et al. 2020) and UC-associated colorectal cancer (UC-CRC) (Yin, Harpaz et al. 1993, Brentnall, Crispin et al. 1994, Galandiuk, Rodriguez–Justo et al. 2012, Kobayashi, Tomita et al. 2017, Baker, Cross et al. 2019). Cell clones that lack tumor suppressor 53 (P53) signaling are observed at high frequency and are considered drivers of carcinogenesis (Petitjean, Achatz et al. 2007). The fixation of those clones could be due to a selective advantage of mutated cells specifically in the inflammatory context (Vermeulen, Morrissey et al. 2013), however mechanistic insights why and how mutant cells could outcompete wild type (WT) cells are lacking. In the present study, I have used inducible Trp53 knock-out (KO) mice through which I investigate the impact of loss of P53 signaling in colonic epithelium in the context of colitis, both in vivo and in vitro. I show that while Trp53 is dispensable for maintenance of epithelial homeostasis in the colon, it has a crucial function in epithelial cells to make them return to homeostasis after colitis-associated injury. Integrating proteome and transcriptome data, I discover that the colonic epithelium of Trp53 KO mice fails to reestablish the homeostatic crypt architecture and is locked in a “fetal-like” regenerative state that is characterized by activated Yes-associated protein 1 (YAP) signaling. This work discloses the context-dependent relevance of P53 signaling in the injury-induced regenerative state during colitis and gives reason for the higher abundance of clones lacking P53 signaling in UC and UC-CRC. It also demonstrates, how loss of P53 could contribute to chronification of the disease and a carcinogenic trajectory in affected epithelium.

Hintergrund: Im Rahmen vorangegangener Arbeiten wurden beim Menschen, Affen, Mäusen sowie Axolotln unterschiedliche regenerative Kapazitäten und stattfindende Regeneration nach Amputationsverletzungen im distalen Fingerendglied nachgewiesen. Immunhistochemische Studien zur Verteilung regenerationsfördernder Marker beim Menschen sind nicht bekannt. Hypothese: Diese Studie untersucht mittels immunhistochemischen Methoden die Verteilung von Bone Morphogenetic Protein 4 (BMP4) in den distalen Endphalangen von Menschen mit Polydaktylie und Affenföten der Gattung Pavian mit einem Gestationsalter von 125 Tagen im Kontext der unterschiedlichen regenerativen Kapazitäten der jeweiligen Spezies und deren Einfluss auf die Möglichkeit der Regeneration nach Amputationsverletzungen. Zusätzlich erfolgt die Aufarbeitung der Histoanatomie der Fingerbeere von Menschen, Affen, Axolotln sowie Mäusen durch diverse histologische Färbungen. Material und Methoden: Im Zeitraum von 2018-2022 wurden insgesamt jeweils zehn Menschen-, Affen-, Mäuse- und Axolotlfinger immunhistochemisch in einer Schnittdicke von sechs Mikrometern untersucht. Neben den immunhistochemischen Untersuchungen der BMP4-Expression bei Menschen und Affen wurden zudem zusätzlich Untersuchungen mittels Hämatoxylin-Eosin, Masson-Trichrom, Elastika van Gieson sowie Silberfärbung nach Von-Kossa durchgeführt, um die Histologie der Fingerbeere genauer zu untersuchen und Korrelationen zwischen BMP4-Positivität und der jeweiligen Areale herzustellen. Um eine Vergleichbarkeit hinsichtlich der Lokalisation herzustellen, wurden mediane mit lateralen Schnitten verglichen. Ergebnisse: Im menschlichen Fingerendglied zeigt sich in der Epidermis eine Abnahme der BMP4-Expression von proximal nach distal. Im Bereich der distalen Fingerkuppe ist im menschlichen Fingerendglied keine BMP4-Expression mehr nachweisbar. Im Bereich der Subcutis und oberflächlichen Dermis zeigen sich nervale Strukturen sowie Drüsen mit deutlicher BMP4-Positivität. Das menschliche Eponychium weist dasselbe Expressionsmuster wie die Epidermis auf und die BMP4-Expression nimmt von proximal nach distal an Intensität ab. Die dem Nagel zugewandte Seite des Eponychiums beim Menschen zeigt sich im Gegensatz zur dorsalen Epidermis des Eponychiums BMP4positiv. Die Nagelmatrixzellen weisen proximal der Lunulainzision keine BMP4Expression auf, wohingegen sich in distal gelegenen Nagelmatrixzellen BMP4Expression nachweisen lässt. Im Gegensatz zum menschlichen Fingerendglied zeigt sich im Fingerendglied beim Pavianfetus in der gesamten Epidermis der Endphalanx eine BMP4-Positivität. Die Epidermis des Mittelglieds proximal des distalen Interphalangealgelenks ist im Gegensatz zum Endglied BMP4-negativ. Hinsichtlich des Eponychiums ist beim Pavianfetus der dem Nagel aufliegende Teil BMP4-negativ, wohingegen der dorsale Teil ebenso wie die volare Epidermis eine positive BMP4-Expression aufweist. Die Nagelmatrix weist beim Pavianfetus sowohl proximal als auch distal eine durchgehende BMP4-Expression auf. Diese nimmt in ihrer Intensität jedoch nach distal zu. Schlussfolgerung: Es zeigt sich ein BMP4-Expressionsmuster bei menschlichen Hexadaktyliefingern sowie Affenföten, welches keine definitiven Rückschlüsse auf einen Zusammenhang zwischen BMP4-Expression und potenziell regenerativer Areale zulässt. In den durchgeführten Untersuchungen kann zudem keine auf BMP4Expression basierende Klassifikation zur Beurteilung der regenerativen Kapazitäten der menschlichen Endphalanx und daraus resultierter Therapieentscheidung etabliert werden. Zusammenfassend ist durch die alleinige Betrachtung das BMP4Expressionsmusters die regenerative Kapazität der Endphalanx nicht zu quantifizieren, weshalb weitere Studien zur Verteilung regenerationsfördernder Marker angeschlossen werden müssen.