Дисертації з теми "3D skin model"

Skin is an organ with a complex structure which plays a crucial role in thebody’s defence against external threats and in maintaining major homeostatic functions. The need for in vitro models that mimic the in vivo milieu is therefore high and relevant with various applications including, among others, penetration, absorption, and toxicity studies. In this context, the choice of the biomaterial that will provide a 3D scaffold to the cultured cells is defining the model’s success. The FN-4RepCT silk is here suggested as a potent biomaterial for skin tissue engineering applications. This recombinantly produced spider silk protein (FN-4RepCT), which can self-assemble into fibrils, creates a robust and elastic matrice with high bioactivity, due to its functionalization with the fibronectin derived RGD-containing peptide. Hence it overcomes the drawbacks of other available biomaterials either synthetic or based on animal derived proteins. Additionally, the FN-4RepCT silk protein can be cast in various 3D formats, two of which are utilized within this project. We herein present a bilayered skin tissue equivalent supported by the FN-4RepCT silk. This is constructed by the combination of a foam format, integrated with dermal fibroblasts and endothelial cells, and a membrane format supporting epidermal keratinocytes. As a result, a vascularized dermal layer that contains ECM components (Collagen I, Collagen III, and Elastin) is constructed and attached to an epidermal layer of differentiated keratinocytes.The protocol presented in this project offers a successful method of evenly integrating cells in the FN-4RepCT silk scaffold, while preserving their ability to resume some of their major in vivo functions like proliferation, ECM secretion, construction of vascular networks, and differentiation. The obtained results were evaluated with immunofluorescence stainings of various markers of interest and further analysed, when necessary, with image processing tools. The results that ensued from the herein presented protocol strongly suggest that the FN-4RepCT silk is a promising biomaterial for skin tissue engineering applications.

Melanoma is a fatal form of skin cancer which progresses in an orchestrated pattern in human skin. Characterising these phases of melanoma in vitro can provide key insights into mechanisms of the disease progression. In this thesis, we investigate how in vitro three-dimensional (3D) model assays that recapitulate human skin can be used to identify key features underlying melanoma progression. In particular, we construct a 3D melanoma skin equivalent model using melanoma cells from the early and late phase of the disease. We further quantify melanoma cell migration, proliferation, invasion, as well as melanoma nest formation.

Background: Necatoriasis is a neglected tropical disease caused by the insidious parasite Necator americanus. This hookworm infects and reinfects approximately 500 million individuals worldwide, with a further 5.1 billion at high risk for acquiring the infection. Despite the high level of reinfection, no lasting immunological memory develops in the host. Albeit the profound health implications, chronicity and public health burden in developing countries, many aspects of human Necator americanus infection, particularly early events at the interface with the host immune system, are under researched. These figures and facts highlight the need for new research elucidating the molecular interactions between Necator americanus and the innate immune system. This will aid in the rational design of innovative and more efficient intervention strategies against hookworm infection, which is an essential measure for disease prevention. Objectives: In the context of Necatoriasis, this thesis studied the physical interaction between infective Necator americanus larvae (L3) with human dendritic cells (DCs) and epidermal keratinocytes, investigating the biological consequences. In addition, the development of a platform consisting of human keratinocytes, fibroblast and DCs on a 3D scaffold was constructed as an in vitro model of human skin. Results: The present thesis provides new insights into early immunological events at the interface of DCs and Necator americanus larvae and could explain how L3 affect immunity upon initial interaction with antigen presenting cells. For the first time, the data presented illustrates the sequestration of human DCs onto the sheath of L3 infective Necator americanus larvae, triggering the hookworm to exsheath. Intriguingly, the exposed cuticle of the larvae had negligible interaction with the free DCs. The findings also illustrate that the interaction between DCs and the larvae is mediated via a mandatory interaction with C-type lectin receptors, dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) and mannose receptor (MR). Blocking of either receptors with antibodies resulted in an inhibition of DC sequestration and aggregate formation. This demonstrates the biological relevance of previously identified lectin binding molecules on the Necator americanus larvae (L3) sheath in the context of interacting with DCs. These findings allude to a disparity between the surface chemistry of the sheath and larvae that could explain their differential ability to interact with DCs. While the exact nature of differences in the surface properties of the larvae and sheath are yet to be characterised, this data clearly indicates the presence of distinct chemical signatures on the cuticle sheath that attract DCs. However, this not only induces exsheathing but also enables larvae migration without being recognised or challenged by antigen presenting cells. A potential escape mechanism through which the larvae could bypass the immune cells, creating a possible site of ‘temporary immune privilege’. DCs incubated with viable axenic larvae exhibited an immature phenotype as evidenced by the low expression of the maturation markers CD80, CD83, CD86, CD40, and HLA-DR. Subsequently, the ability of DCs to acquire a mature phenotype in response to co-stimulation with lipopolysaccharide (LPS) in the presence of Necator americanus was assessed. These data show that DCs treated with the larvae will remain responsive to LPS stimulation. Additionally while the axenised larvae do not induce any cytokine production by DCs, they seem to suppress LPS induced cytokine expression, however these changes were not statistically significant (p value ≤0.3). Furthermore, the cell-free culture media from DCs, matured in the presence of LPS, had no visible effects on the larvae. Intriguingly, matured DCs in LPS-free culture media render the larvae non-viable through a lysing mechanism, alluding to a modified paracrine signalling response by mature immune cells in culture with the parasite. Interestingly, in the presence of epidermal keratinocytes, ex-sheathing was not mandatory to enable larval burrowing. In fact, only a small number of the larvae sheaths were recoverable from the apical surface of the keratinocyte layer; indicating preferential ensheathed larval burrowing. The data also illustrated the novel behavioural strategies promoting host invasion by Necator americanus larvae, in the presence of epidermal keratinocytes. Larvae were notably slower to exsheath in culture with keratinocytes and exhibited no vigorous movements as observed in DC cultures. This was thought to prevent early exsheathing, as the advantage of larvae maintaining their sheath during the initial stages of infection is in theory highly beneficial. Finally an immunocompetent tri-culture was developed on 3D layered PET scaffolds, encompassing epidermal keratinocytes and dermal fibroblasts, interspersed with DCs cultured at air liquid interface. A functional barrier was optimised, following which immune cell migration within the tri-culture system was observed successfully. Conclusion: Collectively, the sequestration of DCs onto the larvae sheath, suppression of maturation and cytokine expression, provides a possible explanation for the lack of a lasting immune response. These data provide novel insights into early immunological events at the interface of DCs, epidermal keratinocytes and Necator americanus larvae, which could explain how L3 evade immunity upon initial interaction with antigen presenting cells.

Includes bibliographical references
Burn injuries are among the most devastating of all injuries and a major global public health crisis, with fire related burns accounting for approximately 265 000 deaths annually. The African continent, most especially Sub-Saharan Africa, has the second highest mortality rates (15% of global mortality rates). In South Africa, 3.2 % of the total population sustains burn injuries, with 50 % of these cases as children under the age of20 years. Studies have also shown that most of these incidences are prevalent within the age groups of 0-5 years, and account for the 3rd most common cause of mortality in children under the age of 15 years. In depth knowledge and understanding of cellular facets of wound healing has allowed for a greater stance in the interventions aimed at circumventing problems associated with development of effective wound defects treatment regimen. Burn treatment options are largely dependent on the degree and extensiveness of burns. A wide body of literature exists with regards to traditional as well as current treatment options. These include, for instance the use of various forms of skin auto-grafts. Despite such great success with all kinds of innovative ideas surrounding the use of autologous skin grafting, lack of available donor sites for skin grafts still remains a problem, more so in cases where patients suffer burns spanning more than 70% TBSA. This therefore has inspired the design and use of bioengineered skin substitutes as well as cultured/non-cultured autologous epidermal cells. Unfortunately, to date, no tissue engineering technique has fully been able to recapitulate the anatomy and physiology of the skin, or has attained the biological stability as well as achieving the aesthetic outcome. Several hurdles are yet to be overcome to achieve this. Amongst many, inclusion of melanocytes, other skin appendages as well as potential progenitor cells is some of the attributes of an ideal 3D skin equivalent. Therefore pigmented 3D skin constructs are of great interest as they address not only the issues of complete wound healing, but also the aesthetic outcomes. In light of this, correct keratinocyte to melanocyte ratios are also of great importance in designing such pigmented 3D constructs. Therefore the major aim of this study was to isolate skin melanocytes and keratinocytes, and co-culture them at different ratios in order to attain optimal pigment production and/or consequent improved wound healing outcome. To determine the best keratinocyte to melanocyte ratio to use in developing pigmented3D skin constructs, the following co-culture ratios were used: 5:1, 10:1 and 20:1.Proliferation assays were employed to further elucidate the growth dynamics of both human skin melanocytes and keratinocytes in either mono- or co-culture system. Secondly, FACS was used to develop a reliable technique to be used to separate the two cell types from a co-culture system in order to perform downstream analyses. Thirdly, to establish the roles of the co-cultured cells in wound healing (with regards to proliferation and migration), scratch wound healing assays were employed. Lastly, FACS was used to infer the effect of such ratios on pigment production. Our results demonstrated that keratinocytes, compared to melanocytes mono-cultures have higher proliferation capacity. On the contrary melanocyte's proliferation is up-regulated by the presence of keratinocytes in a co-culture, whereas higher numbers of melanocytes in co-culture with keratinocytes resulted in less proliferative keratinocyte phenotype. The FACS separation technique worked excellently in identifying keratinocyte population from melanocytes, with an almost 100% accuracy. This is shown by melanocytes being sorted as 93% of MART-1 + cells in a mono-culture, followed by an approximately 5:1 separation of keratinocytes from melanocytes (77% Kc and 17% Mc). In vitro scratch assays demonstrated that none of the co-culture ratios was significantly superior with regards to wound healing capacities and pigment production, in the absence of fibroblast-conditioned medium. In conclusion, the 5:1 co-culture ratio seemed to yield a non-significant, yet best outcome with regards to wound healing capacity (only in the presence of fibroblast-derived factors), thus conferring it as a potential optimal ratio of keratinocytes to melanocytes, to be used in development of our pigmented 3D constructs.

This master’s thesis deals with the calculation of electrodynamic forces breaker BD250NE305. Main tasks in this semester project is to study the theoretical analysis of individual parts specified breakers. Processing theoretical analysis of these forces. Creating a 3D model current path and sheets quenching chamber single phase circuit breaker in Autodesk Inventor Professional 2012. Another challenge is the subsequent export the model into the simulation program ANSYS Maxwell. After simulation, the specified conditions must be processed and the results of the present work is to evaluate.

Acute skin irritation is the reversible inflammatory response of the epidermis to a topically applied irritant substance. A tissue engineered model of the epidermis is used to test chemicals. The degree of development of the model needs to be carefully judged in order to get the correct proportion of proliferating through to differentiated phenotypes for normal function. This judgement typically necessitates over sensitive models with an underdeveloped barrier functionality, as opposed to an insensitive model due to terminal differentiation and low numbers of basal keratinocytes. It has been reported that the lack of proliferative epidermal cells in cultures may be due to the absence of fibroblasts. Paracrine signalling in response to potential irritants is required for propagating an acute inflammatory response. The aim of this thesis is to develop a skin model using a Three Dimensional scaffold that accurately mimics the micro-environment at the DEJ, for supporting keratinocyte and fibroblast self-organisation. We hypothesise that it takes a full thickness skin model with a complete cascade of inflammatory stimuli and cytokine signalling to provide a real indication of irritation. Initial studies focused on Alvetex® (Reinnervate Ltd.), a highly porous polystyrene scaffold, with the aim of developing a skin model using the immortalised cell line HaCaT (human adult low calcium high temperature) keratinocytes or NhKs, in co-culture with dermal fibroblasts. Skin models using electrospun biodegradable polymer scaffolds made of Poly L-lactide (PLLA) and a Poly L-lactide/Polyhydroxybutyrate-co-hydroxyvalerate/Poly L-lactide (PLLA/PHBV/PLLA) composites were then developed. Issues with achieving epidermal-dermal separation in the Alvetex® scaffold due to keratinocyte entrapment lead to an Alvetex®-PHBV Bilayer. Concentration of the SDS needed to illicit an irritant response was deduced at 2D to be 0.1-0.15mM, 3D submerged to be 0.33-0.5mM and for 3D air-liquid models were at best unaffected by 8mM SDS with a Bilayer scaffold of PHBV-PLLA.

Thesis (Sc. D.)--Harvard-MIT Program in Health Sciences and Technology, 2012.
Page 157 blank. Cataloged from PDF version of thesis.
Includes bibliographical references (p. 145-156).
Space radiation poses a significant hazard to astronauts on long-duration missions, and the low fluences of charged particles characteristic of this field suggest that bystander effects, the phenomenon in which a greater number of cells exhibit damage than expected based on the number of cells traversed by radiation, could be significant contributors to overall cell damage. The purpose of this thesis was to investigate bystander effects due to signaling between different cell types cultured within 2D and 3D tissue architectures. 2D bystander signaling was investigated using a transwell insert system in which normal human fibroblasts (A) and keratinocytes (K) were irradiated with 1 GeV/n protons or iron ions at the NASA Space Radiation Laboratory using doses from either 2 Gy (protons) or 1 Gy (iron ions) down to spacerelevant low fluences. Medium-mediated bystander responses were investigated using three cell signaling combinations. Bystander signaling was also investigated in a 3D model by developing tissue constructs consisting of fibroblasts embedded in a collagen matrix with a keratinocyte epidermal layer. Bystander experiments were conducted by splitting each construct in half and exposing half to radiation then placing the other half in direct contact with the irradiated tissue on a transwell insert. Cell damage was evaluated primarily as formation of foci of the DNA repair-related protein 53BP1. In the 2D system, both protons and iron ions yielded a strong dose dependence for the induction of 53BP1 in irradiated cells, while the magnitudes and time courses of bystander responses were dependent on radiation quality. Furthermore, bystander effects were present in all three cell signaling combinations even at the low proton particle fluences used, suggesting the potential importance of including these effects in cancer risk models for low-dose space radiation exposures. Cells cultured in the 3D constructs exhibited a significant reduction in the percentages of both direct and bystander cells positive for 53BP1 foci, although the qualitative kinetics of DNA damage and repair were similar to those observed in 2D. These results provide evidence that the microenvironment significantly influences intercellular signaling and that cells may be more radioresistant in 3D compared to 2D systems.
by Sarah B. Lumpkins.
Sc.D.

Arbetet utforskar det liminala gränslandet mellan mode och arkitektur genom parallella jämförelser av fasadbeklädnad och kläder. Med utgångspunkt i den personliga sfären, undersöks arkitektoniska och bärbara strukturer utifrån och omkring kroppen. Detta är ett undersökande kollage över designpraktikens gränser som förenar det mest intima rummet; våra kläder med den mest intima arkitekturen; tältet. Både arkitektur och mode grundar sig i vikten av att skyla och skydda sig samt att uttrycka identitet. Vad finns det för rumslig potential i detta lager som vi adderar genom att klä, både mänsklig kropp och arkitektonisk huskropp? Dagens samhälle speglar en livsstil i ständig förändring och förflyttning vilket gör att vi behöver vara mer flexibla i våra rum. Det publika rummet ställer allt högre extroverta krav och gör alltmer anspråk på det privata rummet. För dagens samtida urbana nomad formas ett behov av en mobil, bärbar och konvertibel privat sfär.  Här följer en tolkning utifrån definitionen om arkitektoniskt och mänskligt skydd.

Solid tumours display varied oxygen levels and this characteristic can be exploited to develop new methodologies for detection. MCTS provides a useful model that mimic in vivo tumour microenvironment with varied metabolic gradient (oxygen, pH, glucose and ATP). Emission quenching of phosphorescence compounds by O2 is becoming a wide spread approach for sensing oxygen by optical method within biological model. The approach depends on the correlation of the lifetime of the phosphorescent probe with O2 pressure. The aim is to study the cell penetration and oxygen measurement potential of a novel phosphorescent PtLsCl probe in 2D and 3D biological models using a high resolution 1 and 2-photon emission Phosphorescence Lifetime Imaging Microscopy (PLIM). Quantitative analysis of fluorescence emission intensity and lifetime within cellular compartment showed preferred accumulation of PtLsCl in nucleoli of cells. Immunohistology experiments by Hypoxyprobe™ suggested hypoxia within MCTSs were dependent upon culture condition of MCTS (i.e. size and culture days). Thereafter, emission lifetime detected by 1 or 2-photon PLIM showed marked differences across the 3D MCTS and the variation in lifetime was dependent upon culture condition (size and culture days) of MCTS, suggesting varied oxygen concentration. The distribution of emission lifetime of PtLsCl in whole spheroids ranged from 0 to 12 microseconds with phosphorescence lifetime imaging revealing three distinct lifetime-related oxygen areas. Thereafter, emission lifetime of PtLsCl in the whole melanoma tissue engineered model was analysed. Distribution emission ranged from 0 to 13 microseconds, with phosphorescence lifetime imaging revealing three distinct areas – 1) a normal stromal region of 0 to 4.0 μsec; 2) a spheroid border 4.0 to 6.0 μsec and 3) an inner core of MCTS 6.0 to 13.0 μsec. A marked deviation in lifetime of PtLsCl across the melanoma tissue engineered model clearly demarcated tumour area within normal stroma. It is proposed that the depletion of O2 is known to increase the emission lifetime of the PtLsCl label described herein, and PtLsCl incorporated with 1 or 2 PE-PLIM system demonstrated an excellent potential for high-resolution mapping of oxygen concentration in multi-cellular tissue models. Furthermore, both 1 and 2P-PLIM of a highly sensitive PtLsCl label has shown the potential to detect changes in partial O2 pressure and related response (e.g. necrosis).

This thesis presents possibilities of 3D point cloud and true colored digital video fusion that can be used in the process of robot teleoperation. Advantages of a 3D environment visualization combining more than one sensor data, tools to facilitate such data fusion, as well as two alternative practical implementations of combined data visualization are discussed. First proposed alternative estimates view frustum of the robot's camera and maps real colored video to a semi-transparent polygon placed in the view frustum. The second option is a direct coloring of the point cloud data creating a colored point cloud representing color as well as depth information about an environment.

The main goal of this project was to familiarize readers with the techniques used in real-time animation of 3D characters. This work is focused on two types of animation: keyframe animation and skeletal animation. There are described algorithms for software and hardware accelerated model deformations, keyframe interpolations, animation blending, inverse kinematics and ragdoll. The result of this project is a framework, which consists of an animation library, examples demonstrating library functions and tools for export animations from 3D Studio Max and MilkShape 3D.

The aim of this research was to evaluate the utility of innovative in vitro techniques as an alternatives for human/animal tissues to study the transdermal uptake of organic flame retardants from indoor dust and consumer products. Firstly, we successfully designed and applied an in vitro physiologically based extraction test to provide new insights into the dermal bioaccessibility of various FRs from indoor dust. These investigations revealed the bioaccessible fraction for the brominated flame retardants (BFRs) α-, β-and γ- HBCD and TBBPA to 1:1 (sweat/sebum) mixture to be 41%, 47%, 50% and 40%, respectively, while for the phosphate flame retardants (PFRs) TCEP, TCIPP and TDCIPP, the values were 10%, 17% and 19%. With the exception of TBBP A, the presence of cosmetics had a significant effect (p < 0.05) on the bioaccessibility of our target FRs from indoor dust. The presence of cosmetics decreased the bioaccessibility of HBCDs from indoor dust, whereas shower gel and sunscreen lotion enhanced the bioaccessibility of target PFRs. Secondly, we developed a protocol for studying dennal uptake of legacy and novel brominated flame retardants using two 3D-HSE (three dimensional human skin equivalent tissue) models, EpiDerm™ and EPISKIN™ in compliance with the OECD guidelines 428. Overall, results showed a significant negative correlation between the permeability constant of FRs and their Log K_ow values. We also mimicked real life exposure scenarios by exposing the skin surface in turn to FR-containing dust, reference material plastics and upholstered fabrics. Our findings showed that under such scenarios dermal exposure to FRs was appreciable for UK adults and toddlers. For example, for dust exposure, our estimates of daily intake indicated toddlers to be 10 times more highly exposed than adults in the presence of sweat and sebum. This differential exposure is likely attributable to more dust adhering to toddler's skin and their higher exposed skin surface area to body weight ratio compared to adults.

Aujourd'hui, un grand nombre de nouvelles molécules peuvent être synthétisées à partir de la biomasse. Les tensioactifs dérivés de sucre sont notamment considérés comme une alternative aux tensioactifs fossiles en raison de leur biodégradabilité et de leur biocompatibilité. Cependant, les études associant la caractérisation physico-chimique et les propriétés biologiques de ce type de tensio-actifs sont limitées. Il est ainsi difficile de prédire les propriétés d'un tensioactif à partir de sa structure chimique. L'établissement d'une méthodologie permettant de relier la structure des surfactants à leurs propriétés apparait pertinent. Dans ce travail, quatre surfactants dérivés de sucre ayant chacun une chaîne C8 liée à une tête glucose ou maltose par un groupe amide ont été caractérisés par leurs propriétés tensio-actives dans différentes solutions (eau et milieu biologique). Leurs interactions avec des protéines ont également été analysées. Concernant l'évaluation des propriétés biologiques, des tests de cytotoxicité/irritation ont été effectués sur trois modèles in-vitro : 1) modèle cellulaire 20 (cellules L929 cultivées en monocouche), Il) modèle cellulaire 30 (cellules L929 cultivées dans un gel de collagène), Ill) épiderme humain reconstitué. Les résultats indiquent que les quatre surfactants synthétisés présentent de bonnes propriétés tensio-actives et trois d'entre eux sont moins cytotoxiques que des tensioactifs de référence. Plusieurs hypothèses permettant de relier la structure chimique des molécules à leurs propriétés physico-chimiques et biologiques ont été proposées. Des travaux futurs permettront d'enrichir la base de données sur les relations structure-propriétés des tensioactifs issus de la biomasse, et de l'utiliser pour synthétiser des surfactants présentant des propriétés adaptées aux applications envisagées
Nowadays, a wide variety of new molecules can derive from biomass. Among them, the family of sugar-based surfactants, which are considered as alternatives to fossil-based surfactants, due to their relatively high biodegradability and biocompatibility, exhibit interesting properties both in terms of their self-assembly and their ability to induce biological responses. In the study, for the purpose to analyse these properties, different methodologies have been established. In this work, physico-chemistry and cellular biology methodologies are associated to analyse the properties of pre-selected molecules characterized by gradua) structure modifications. Firstly, we have screened synthesized sugar-based surfactants according to their solubility and their ability to reduce surface tension of water. Four pre-selected molecules, with a C8 chain linked to a glucose or maltose head through an amide functional group, either under the form of carbamoyl (carbohydrate scaffold bearing the carbonyl) or alkylcarboxamide (the alkyl chain bearing the carbonyl), were then dissolved in water/ cell culture media for surface tension measurements. Their behaviors in solutions were characterized by Krafft points, Critical Micellar Concentrations or self-assembling properties through different methods. To evaluate the cytotoxic/ irritant effects of these molecules on cells and tissues, 3 in-vitro models were established: I) 2D cell culture mode! (L929 cell monolayer) II) 3D ce!! culture mode! (L929 cells embedded in collagen gel) and III) Reconstituted human epidermis (differentiated human keratinocytes). Corresponding experiments were carried out on these models with increasing complexity. Results show that the synthesized sugar-based surfactants, GlulamideC8, Glu6amideC8, Glu6amideC8' and MallamideC8 can reduce the surface tension of water solution to the came level as standard surfactants (Tween 20 and Hecameg). In the meantime, GlulamideC8, Glu6amideC8' and MallamideC8 present Iess cytotoxicity effects on L929 cells both in the monolayer model and the 3D mode! than Tween 20 and Hecameg. All synthesized and standard surfactants (GlulamideC8, Glu6amideC8, Gu6amideC8', MallamideC8, Tween 20 and Hecameg) have no significant cytotoxic/ irritant effects on reconstituted human epidermis at 1000 ig/mL after 48 h of topical application. Discussions have been made according to the results of experiments to establish possible structures/ physico-chemical properties - cytotoxicity relationships of these surfactants

La détection de peau consiste à détecter les pixels correspondant à une peau humaine dans une image couleur. Les visages constituent une catégorie de stimulus importante par la richesse des informations qu’ils véhiculent car avant de reconnaître n’importe quelle personne il est indispensable de localiser et reconnaître son visage. La plupart des applications liées à la sécurité et à la biométrie reposent sur la détection de régions de peau telles que la détection de visages, le filtrage d'objets 3D pour adultes et la reconnaissance de gestes. En outre, la détection de la saillance des mailles 3D est une phase de prétraitement importante pour de nombreuses applications de vision par ordinateur. La segmentation d'objets 3D basée sur des régions saillantes a été largement utilisée dans de nombreuses applications de vision par ordinateur telles que la correspondance de formes 3D, les alignements d'objets, le lissage de nuages de points 3D, la recherche des images sur le web, l’indexation des images par le contenu, la segmentation de la vidéo et la détection et la reconnaissance de visages. La détection de peau est une tâche très difficile pour différentes raisons liées en général à la variabilité de la forme et la couleur à détecter (teintes différentes d’une personne à une autre, orientation et tailles quelconques, conditions d’éclairage) et surtout pour les images issues du web capturées sous différentes conditions de lumière. Il existe plusieurs approches connues pour la détection de peau : les approches basées sur la géométrie et l’extraction de traits caractéristiques, les approches basées sur le mouvement (la soustraction de l’arrière-plan (SAP), différence entre deux images consécutives, calcul du flot optique) et les approches basées sur la couleur. Dans cette thèse, nous proposons des méthodes d'optimisation numérique pour la détection de régions de couleurs de peaux et de régions saillantes sur des maillages 3D et des nuages de points 3D en utilisant un graphe pondéré. En se basant sur ces méthodes, nous proposons des approches de détection de visage 3D à l'aide de la programmation linéaire et de fouille de données (Data Mining). En outre, nous avons adapté nos méthodes proposées pour résoudre le problème de la simplification des nuages de points 3D et de la correspondance des objets 3D. En plus, nous montrons la robustesse et l’efficacité de nos méthodes proposées à travers de différents résultats expérimentaux réalisés. Enfin, nous montrons la stabilité et la robustesse de nos méthodes par rapport au bruit
Skin detection involves detecting pixels corresponding to human skin in a color image. The faces constitute a category of stimulus important by the wealth of information that they convey because before recognizing any person it is essential to locate and recognize his face. Most security and biometrics applications rely on the detection of skin regions such as face detection, 3D adult object filtering, and gesture recognition. In addition, saliency detection of 3D mesh is an important pretreatment phase for many computer vision applications. 3D segmentation based on salient regions has been widely used in many computer vision applications such as 3D shape matching, object alignments, 3D point-point smoothing, searching images on the web, image indexing by content, video segmentation and face detection and recognition. The detection of skin is a very difficult task for various reasons generally related to the variability of the shape and the color to be detected (different hues from one person to another, orientation and different sizes, lighting conditions) and especially for images from the web captured under different light conditions. There are several known approaches to skin detection: approaches based on geometry and feature extraction, motion-based approaches (background subtraction (SAP), difference between two consecutive images, optical flow calculation) and color-based approaches. In this thesis, we propose numerical optimization methods for the detection of skins color and salient regions on 3D meshes and 3D point clouds using a weighted graph. Based on these methods, we provide 3D face detection approaches using Linear Programming and Data Mining. In addition, we adapted our proposed methods to solve the problem of simplifying 3D point clouds and matching 3D objects. In addition, we show the robustness and efficiency of our proposed methods through different experimental results. Finally, we show the stability and robustness of our methods with respect to noise

Bovine Dermatitis digitalis (DD) ist eine weltweit verbreitete Infektionskrankheit bei Rindern, die primär die plantare Haut über dem Kronenrand nahe des Zwischenzehenspalts der Hinterklauen betrifft. Schmerzhafte ulzero-proliferative Läsionen mit akuten und chronischen Erscheinungsformen führen zu Verhaltensänderungen und Lahmheit der Tiere. DD hat damit einen erheblichen Einfluss auf deren Wohl und ihre Leistungen. Zahlreiche Untersuchungen zur Ätiologie der Krankheit ergaben, dass es sich um das Zusammenspiel verschiedener Ursachen handelt. Einer synergistischen multifaktoriellen Infektion mit starker Beteiligung von Bakterien der Gattung Treponema kommt dabei besondere Bedeutung zu. Aspekte wie Tierhaltung, Hygienestandards und genetische Prädispositionen wurden ebenfalls intensiv untersucht. Nichtsdestotrotz bleiben Infektionsherde, Transmissionsrouten und Pathomechanismen weitgehend unklar. Zum besseren Verständnis der Ereignisse, die zu DD-Läsionen führen, sollte im Zuge dieser Arbeit ein organotypisches Zellkulturmodell der bovinen Haut erstellt werden, welches in späteren Versuchen mit dem Krankheitserreger zum Einsatz kommen soll. Verlässliche und reproduzierbare Techniken zur Isolation und Kultur von bovinen primären Keratinozyten und Fibroblasten wurden etabliert; geeignete Zellkulturmedien für die Langzeitkultivierung und –aufbewahrung der Hautzellen wurden identifiziert. Zur Erstellung des Hautmodells wurden zwei verschiedene Ansätze miteinander verglichen. Der zweite Ansatz, bei dem Keratinozyten direkt auf ein dermales Äquivalent, d.h. ein Pad aus bovinem Kollagen I mit eingesäten post-mitotischen Fibroblasten, gesät wurden, brachte ein vielversprechendes Hautmodell hervor. Die inkorporierten post-mitotischen Fibroblasten wiesen eine charakteristische Zellmorphologie mit intakten Nuklei auf. Die terminale Differenzierung der Keratinozyten auf dem dermalen Äquivalent wurde mittels Immunfluoreszenzfärbungen mit Antikörpern gegen die Markerproteine Keratin 14 und Desmoglein 1 gezeigt. Die Ergebnisse erster Experimente mit Treponema spp. verdeutlichen, dass das Hautäquivalent ein geeignetes Modell zur Untersuchung der Pathogenese der DD darstellt.
Bovine digital dermatitis (DD) is a worldwide occurring, infectious disease in cattle primarily affecting the plantar skin above the coronary band near the interdigital cleft on hind feet. Painful ulceroproliferative lesions with acute and chronic appearances lead to behavioral changes and lameness. Hence, DD has a major impact on animal welfare and performance. Substantial efforts in investigating the etiology of the disease revealed a synergistic origin with evidence for a multibacterial infection and the strong involvement of bacteria from the genus Treponema. As the interaction between host, pathogen and environment is not negligible, surrounding circumstances such as housing, general hygiene and genetic predispositions have been investigated intensively. Nevertheless, infection reservoirs, transmission routes and pathomechanisms remain widely unclear. To better understand the cellular and molecular events during Treponema-infection of bovine skin, it was the specific aim of this study to establish an organotypic in vitro skin model, which could be challenged with the causative agent of the disease. A technique to reliably and reproducibly isolate primary keratinocytes and fibroblasts from the site of infection was established. Appropriate cell culture media for the long-term cultivation and storage of bovine skin cells were identified. Two different methods to develop the skin model were compared. The second strategy in which keratinocytes were directly seeded on top of a dermal equivalent, i.e. a bovine collagen type I pad with embedded post-mitotic fibroblasts, gave rise to a promising organotypic skin equivalent. The incorporated post-mitotic fibroblasts showed a characteristic cell morphology with intact nuclei. The terminal differentiation of the keratinocytes on top of the dermal equivalent was shown with anti-K14 and anti-Dsg1 immunofluorescence stainings. The results of initial Treponema-experiments proved that the skin equivalent is a suitable model to investigate the underlying mechanisms during Treponema-infection of bovine skin and hence, the pathogenesis of DD.

Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2021
The skin is a complex organ mainly responsible for protecting the body from external threats and maintaining homeostasis. It is a complex three-dimensional structure that is composed of two main compartments, the dermis and the epidermis. Due to increasing ethical and legal pressure on animal usage in research, reconstructed 3D human skin models have been gaining popularity. These models mimic human skin architecture in vitro and allow relatively easy manipulation to meet specific needs. Some rare diseases remain poorly studied and could take advantage of this technology. One example is the Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) which is an early-onset neurological disease that was first described in Quebec, Canada, but cases have been reported worldwide. Patients suffer from spasticity and lack of coordination of muscle movements, resulting in an early wheelchair dependence and premature death. ARSACS is caused by loss-of-function mutations in the SACS gene, leading to a defective sacsin protein. Sacsin loss of function has been linked to mitochondrial dysfunction and abnormalities in the organization of intermediate filaments, but the complete picture is still unclear. Evidence of abnormalities in the skin of ARSACS patients has been reported, making this disease an interesting candidate to be studied using in vitro skin models. In this work, two different human keratinocyte cell lines (HaCaT and N/TERT-1) were used to create new human epidermal models using a polycarbonate inert matrix. The localization of different keratins and other markers (keratins 10, 14 and 15, and involucrin) were studied to characterize epidermal differentiation and stratification. Sacsin expression was analyzed in different cell lines and sacsin knockdown was attempted in HaCaT keratinocytes using lentiviral shRNAs. The HaCaT cell line was unable to recreate the normal multi-layer architecture of native skin nor the stratum corneum. This cell line expressed low amounts of the sacsin protein, and no difference was observed between the knockdown and the control by western blot. N/TERT-1 keratinocytes generated a stratified epidermis with all the normal layers present, including the stratum corneum. Complete epidermal differentiation was confirmed by the differential expression of epidermal markers. K14 expression was limited to the basal layer, while K10 was expressed in the upper layers, as expected. Involucrin was mostly expressed in the stratum granulosum and K15 expression was overall very low, indicating a successful differentiation. Sacsin expression was verified in different skin cells (HEKn, HDFn, and N/TERT-1), and N/TERT-1 expressed sacsin in amounts slightly lower than primary human keratinocytes. These findings suggest that the N/TERT-1 cell line has more potential to produce an epidermal skin model with an ARSACS phenotype, which can prove an important tool in future research. Despite the existing knowledge about sacsin structure and function, a lot is still unknown about this protein and how it causes the symptoms underlying ARSACS disease. Advances in this topic could contribute to the development of therapies that could cure or tackle some of ARSACS symptoms to ensure a better quality of life for the patients.