Background: A significant portion of lactating women in less developed countries have vitamin A (VA) deficiency. Lactation has substantial effects on a mother’s metabolism and VA is known to be needed in multiple biological processes, including growth, vision, immunity, and reproduction. Objective: The objective of this pilot study was to utilize metabolomics profiling to conduct a broad, exploratory assessment of differences in plasma metabolites associated with low VA status versus adequacy in lactating women. Methods: Plasma samples from lactating women who participated in a survey in Samar, Philippines, were selected from a cross-sectional study based on plasma retinol concentrations indicating low (VA-; n=5) or adequate (VA+; n=5) VA status (plasma retinol <0.7 or >1.05 µmol/L). The plasma results collected from six metabolomics assays (oxylipins, endocannabinoids, bile acids, primary metabolomics, aminomics, and lipidomics) were compared by group, using liquid chromatography mass spectrometry. Results: Twenty-eight metabolites were significantly different in the VA- versus VA+ status, with 24 being lipid mediators (p<0.05). The lipid mediators demonstrated lower concentrations of the arachidonic acid- and eicosapentaenoic acid-derived oxylipins, as well as lysophospholipids and sphingolipids, in the VA- group (p<0.05). Chemical similarity enrichment analysis identified HETEs, HEPEs, and DiHETEs as significantly different oxylipin clusters (p<0.0001, false discovery rate (FDR) p<0.0001), as well as sphingomyelins, saturated lysophosphatidylcholines, phosphatidylcholines, and phosphatidylethanolamines (p<0.001, FDR p<0.01). Conclusions: The multi-assay nutritional metabolomics profiling of low VA status compared with adequacy in lactating women demonstrated reduced lipid mediator concentrations. Future studies with stronger study designs and a large and more diverse population are needed to validate these preliminary results.

In recent years, deficiencies in construction documents has become a common problem in the construction industry. In most cases this leads to financial consequences, both for clients and building contractors. The risk of financial consequences can be minimized if the deficiencies and errors are eliminated before the construction phase or if it’s detected in an early stage of a project. Within Peab Skellefteå, a lot of time, energy and money are spent to investigate the deficiencies and errors in construction documents. This mostly occur in the construction phase, since that is where the deficiencies and errors usually are discovered. Previous studies concerning the same area indicate that financial consequences is a result of inadequate construction documents and alteration and additional works, but none of the studies includes further investigation concerning how it practically appears in projects and in what way it affects the client’s and building contractor’s economy. Based on this, the purpose of the report has been formulated: The purpose of the master thesis is to investigate and understand how inadequate construction documents affects individual construction projects in terms of economy. Based on the results from the interviews, recommendations will be proposed regarding how the risk of financial consequences can be reduced to help entrepreneurs avoid these types of situations. The content of this report is essentially based on interviews with nine participants, either site managers, construction engineers or purchasers from Peab Skellefteå. Theory from previous studies is also used and the report is limited to traditional contracts. The results of the interview study revealed that both the building contractor and the client have been negatively affected in terms of economy in project 1–6. It is hard to report accurate numbers, since inadequate construction documents might affect the next work stage, contract rate, time that should have been spent doing something else and the time spent to investigate deficiencies and errors, which can be difficult to prove. Some costs could be deduced to the discovered deficiencies and errors, see Table 1. Generally, it’s stated that deficiencies in construction documents entails financial consequences for both entrepreneurs and clients. The respondents explain that the drawings are the most problematic construction document and that’s where most errors are discovered. The parts of the drawings that’s usually inadequate is piercings, fittings, inner walls, details, attachments and contradictory information. Deficiencies typically connected to financial consequences for the building contractor is piercings, reinforcement, fire sealing and time spent investigating the deficiencies. For the client, typical deficiencies connected to financial consequences are lack of foundation- and ground investigation, quantifying errors, when incorrect dying times leads to extra costs and additional work. The respondents’ suggestions for improvement in collaboration with the author’s own thoughts is the basis of the given recommendation. The main potential improvements for designers are to review construction documents more accurate, increase their knowledge about production and materials, work with BIM-coordination and be receptive to feedback. Clients should check the coordination, increase their knowledge about work steps and materials and have consistent solutions through the project. Building contractors should have coordination meetings with designers, clients and installers, make an appropriate number of tender calculations, provide feedback to designers and clients, use the question/answer-method, design a useful contract administration and increase the review of contract documents for the often-deficient parts.

Bakgrund Målet med palliativ omvårdnad är att lindra lidande, främja välbefinnande och ge patienter ökad livskvalitet. Det är sjuksköterskan som har det övergripande ansvaret att se till att dessa delar efterföljs. Tidigare forskning belyser patienters och anhörigas upplevelser av palliativ omvårdnad där det framkommer att flertalet brister finns. Syfte Att beskriva sjuksköterskors erfarenheter av palliativ omvårdnad. Metod Litteraturstudie med beskrivande syntes. Resultat I analysen framkom tre teman och sju subteman. Första temat, Att erfara känslomässigt engagemang, genererade subteman Meningsfullhet i yrket och Personlig påverkan. Det andra temat, Hinder för god palliativ omvårdnad, genererade subtemanKunskapsbrist och känslan av otillräcklighet och Brister i omvårdnadsarbetet. Tredje temat, Möjligheter för god palliativ omvårdnad, genererade subteman Kunskap i omvårdnadsarbetet, Samarbete i omvårdnadsteamet samt Betydelsen av kommunikation. Slutsats Sjuksköterskor upplevde hinder i den palliativa omvårdnaden genom avsaknad av utbildning inom palliativ omvårdad samt brist i kommunikationen med patienter och derasanhöriga. Även brist på sjuksköterskor upplevdes vilket medförde en känsla av otillräckligheti professionen. Patienter och anhöriga ansåg att sjuksköterskorna var omhändertagande och att omvårdnaden var god, men uppmärksammade även brister i kommunikationen samt atttidsbristen sänkte omvårdnadens kvalitet. / Background The goal of palliative care is to alleviate suffering, promote well-being and give patients an increased quality of life. The nurse has the overall responsibility to ensure that these parts are being followed. Previous research sheds light on patients’ and relatives’ experiences of palliative care, where a lot of deficiencies appears. Aim To describe nurses’ experiences of palliative care. Method Literature study with descriptive synthesis. Results The analysis revealed three themes and eight subthemes. The first theme, To experience emotional commitment, generated subthemes Meaning in the profession and Personal influence. The second theme, Obstacles to good palliative care, generated subthemes Lack of knowledge and the feeling of inadequacy and Deficiencies in the nursing work. The third theme, Opportunities for good palliative care, generated subthemesKnowledge in nursing, Collaboration in the nursing team and Supportive communication. Conclusion Nurses experienced obstacles in palliative care due to a lack of training in palliative care and a lack of communication with patients and their relatives. There was also a shortage of nurses, which led to a feeling of inadequacy in the profession. Patients and relatives considered that the nurses were caring and that the care was good, but also noticed shortcomings in communication and that the lack of time reduced the quality of care.

Le système immunitaire est particulièrement dépendant des mécanismes de réparation de l’ADN, en effet le développement du système immunitaire adaptatif nécessite certains mécanismes de réparation de l’ADN, lors de la recombinaison V(D)J et lors de la commutation de classe des immunoglobulines. De plus, le système hématopoïétique est par sa nature très sensible aux lésions spontanées de l’ADN. Il existe chez l’homme de nombreux déficits immunitaires directement liés à un défaut de réparation de l’ADN. L’identification du gène responsable est importante pour un conseil génétique familial approprié et pour la prise en charge médicale. Nous avons accès aujourd’hui à de puissants outils de dépistage génétique grâce au séquençage à haut débit et la liste des gènes responsables d’un déficit immunitaire s’allonge de plus en plus en rapidement. La première partie de ce travail porte sur la mise au point d’un nouvel outil de dépistage rapide des déficits de la réparation de l’ADN, en particulier dans le cas de déficit immunitaires. Ce test est fondé sur l’observation d’un biais du répertoire du TCRdes lymphocytes T circulants lorsque les thymocytes ont une durée de vie diminuée, or un défaut de réparation de l’ADN entraîne une diminution de la survie thymocytaire. Nous avons mis au point deux techniques, par biologie moléculaire et par cytométrie en flux, pour détecter un éventuel biais du répertoire du TCRα et évaluer la pertinence de ce test dans les déficits immunitaires liés à un défaut de réparation de l’ADN. Un biais a notamment été détecté dans les cas de déficit en facteur du NHEJ et en ATM. Nous avons également établi en collaboration avec le service d’Immunologie Clinique de l’hôpital Saint-Louis une cohorte de patients atteints de déficit immunitaire commun variable (DICV) dont la présentation clinique est évocatrice d’un défaut de réparation de l’ADN. Une série de test fonctionnels de dépistages de déficit de la réparation de l’ADN ainsi que des analyses génétiques (CGH array, séquençage complet de l’exome) ont été fait chez ces patients afin d’identifier de nouveaux gènes impliqués dans les DICV. Parmi les 18 patients analysés, dans 5 cas on retrouve une sensibilité cellulaire accrue aux agents génotoxiques et chez 15 patients, un gène candidat a été identifié. Ces résultats sont encore préliminaires et la caractérisation génétique et fonctionnelle des mutations identifiées sera poursuivie par notre équipe. Pour finir, nous avons entrepris l’exploration génétique et fonctionnelle de deux mutations identifiées chez une jeune patiente atteinte de déficit immunitaire combiné (CID) associé à un syndrome lymphoprolifératif et une auto-immunité, et chez qui une hypersensibilité cellulaire à la Mitomycine C, agent pontant de l’ADN, a été détectée. La première mutation a été identifiée dans le gène ELKS qui code pour un facteur impliqué dans la réparation de l’ADN. La complémentation fonctionnelle de ce gène prouve l’implication de cette mutation dans l’hypersensibilité des cellules de la patiente à la MMC. Nous avons développé un modèle murin KO conditionnel de ce gène dans les cellules hématopoïétiques qui n’a pas montré de défaut de développement du système immunitaire. La deuxième mutation identifiée se situe dans le gène BACH2 codant pour un répresseur transcriptionnel très impliqué dans le développement du système immunitaire. Les souris KO pour ce gène ont un phénotype proche du déficit immunitaire décrit chez cette patiente. Les investigations de cette mutation sont en cours chez elle et chez les membres de sa famille également porteurs de la mutation. / The immune system is particularly dependent on DNA damage response (DDR) pathways. The development of the adaptive immune system requires certain DDR mechanisms, in particular during the V(D)J recombination and during class switch recombination (CSR), furthermore, the hematopoietic system is very sensitive to spontaneous DNA lesions. Therefore, there are many immune deficiencies in human directly related to a DDR deficiency. The identification of the responsible gene is important for appropriate genetic counseling. Today, we have access to powerful genetic screening tools, in particular next generation sequencing (NGS) and the list of genes responsible for immune deficiency is growing rapidly. The first part of this work focuses on the development of a new screening tool for DDR defects, in particular in the case of immune deficiency, and evaluation of clinical interest. This test is based on the observation of a bias of the TCRα repertoire in circulating T lymphocytes when thymocytes lifespan is diminished and we know that DDR defect causes decreased thymocyte survival. We have developed two techniques, by molecular biology and by flow cytometry, to detect a potential bias of the TCRα repetoire and assess the suitability of this test in some immunodeficiencies linked to a DDR defect. A significant bias was detected in the case of ATM and NHEJ factor deficiency. Furthermore, we have established a cohort of patients suffering from common variable immunodeficiency (CVID) with a clinical presentation highly suggestive of DDR defect, in collaboration with the Clinical Immunology Service of Hôpital Saint-Louis (Paris). Functional test for DDR defect and genetic analysis (CGHarray, whole exome sequencing) were performed in these patients to identify new genes involved in CVID. Among the 18 patients analyzed until now, five cases of cellular sensitivity to genotoxic agents have been detected and a candidate gene was identified in 15 of them. These results are still preliminary and our team will pursue genetic and functional characterization of the identified mutations. Finally, we undertook genetic and functional exploration of two mutations identified in a young patient with combined immunodeficiency (CID) associated with a lymphoproliferative disease and autoimmunity, and in whom a cellular hypersensitivity to mitomycin C, a DNA crosslinking agent, was detected. The first mutation was identified in the ELKS gene, which codes for a factor involved in DNA repair. Functional complementation of this gene demonstrates the involvement of this mutation in the hypersensitivity of patient’s cells to MMC. We have developed a conditional knockout mouse model of this gene in hematopoietic cells that did not show any defect in development of the immune system. The second mutation was identified in BACH2 gene encoding a transcriptional repressor involved in the development of the immune system. Knockout mice for this gene have a similar phenotype to the immune deficiency described in this patient. Investigations on this mutation are ongoing in the patient and among family members that also carry the mutation.

D. Tech. (Food Service Management, Department of Hospitality, Tourism and PR Management, Faculty of Human Sciences)|, Vaal University of Technology| / Background South Africa is one of the developing countries faced with the co-existence of undernutrition and overnutrition, often experienced within the same household. On the one hand, hunger, undernutrition and micronutrient deficiencies are linked to poverty and household food insecurity. Simultaneously, South Africans are exposed to ‘nutrition in transition’, where overweight and chronic diseases of lifestyle, such as diabetes mellitus, cardiovascular diseases and cancer are on the rise as part of the overnutrition profile. Sound nutrition is important throughout the lifecycle to combat under- and overnutrition and nutrition-related diseases. In urban areas, the grandmother or another senior female family member is often responsible for caring for the children in the household during the day. This includes physical, emotional and nutritional care. It is therefore essential for the caregiver to have good nutrition knowledge, to provide not only in her own needs, but also in those of the children. A nutrition education programme is one strategy for improving the nutrition knowledge of caregivers of children. Objective The primary objective in this study was to develop, tailor, implement and evaluate a nutrition education programme (NEP) for Sesotho-speaking females and caregivers of children in the Boipatong Township in the Vaal Region of South Africa and to test its impact in the short and longer term. Nutrition knowledge regarding four South African food-based dietary guidelines (FBDGs) was empirically tested before and after the intervention and then compared to a control group. In addition, dietary intake was assessed to detect any changes after the intervention in the medium term. Methodology In this both quantitative and qualitative methodologies were applied. Two frameworks, the United Nations Children’s Fund (UNICEF) Framework of Malnutrition (2004) and the Food and Agriculture Organisation (FAO) Framework for Nutrition Education (1997), gave structure to the planning, implementation and evaluation of the research project. This study’s nutrition education programme was based on a socio-ecological model to guide behavioural change in terms of healthy food choices. In the preparation phase, a situational analysis was performed by means of a cross-sectional analytical design using descriptive statistics. Socio-demographic and self-reported health information was obtained. Nutrition knowledge, based on the South African food-based dietary guidelines (FBDGs), was measured, and dietary intake was assessed and compared with the estimated average requirements (EARs) for their age categories. Phase two, the formulation phase, used an experimental design. The acceptability and understanding of the existing national nutrition education (NE) material for individuals with low living standards (LSM) was investigated in this phase of the nutrition education programme (NEP). A culturally tailored booklet was developed in English, translated into Sesotho, pilot tested and implemented as part of the nutrition education programme. Lesson plans were developed and pilot tested. A non-randomised control trial was used in the implementation and evaluation phases. The effect of the nutrition education programme on nutrition knowledge was quantitatively measured in a pre- and post-test design at each discussion session in the short term, using paired t-tests in the experimental group of caregivers. The evaluation phase tested the impact of the nutrition education in the longer term. Nutrition knowledge was measured quantitatively in a before-after intervention test design by means of a self-completed structured questionnaire, based on the four South African FDBGs included in the programme. A control group who was not subjected to any intervention was used to complete the same questionnaire before and after the intervention in the same time period as the experimental group. In the experimental group, dietary intake was assessed before and after the intervention to detect changes in dietary intake. No dietary intake was measured in the control group, as changes could be attributed to influences beyond the control of this study. Two randomly selected focus groups of the experimental group were run to provide deeper insight into their perceptions regarding the acceptability and understanding of the NEP and NE material. Results The situational analysis of the preparation phase revealed a poor community that displayed typical patterns of nutrition in transition, at risk of malnutrition. The median age of the sample of caregivers was 44 years (IQR 32-62). Income and consumption poverty was confirmed by 80.5 percent of households spending R300 or less on food, with 75 percent of households having between four and seven people living in the dwelling. Dietary results were indicative of food poverty and poor food choices, possibly due to monetary constraints. A low energy intake (median 5323 kJ/day; IQR 3369-7949), was observed. Only 13.9 percent reached the estimated energy requirement (EER) of 7855 kJ per day for their age category. The overall mean average requirements of the diet was 0.7 but the possiblity of micronutrient deficiencies could not be excluded with a MAR of 0.6 for vitamins and minerals respectively. The median nutrition knowledge was 50 percent (IQR 42-54%) The lowest score was obtained for the FBDG ‘Enjoy a variety of food’ (33.4%; 95% CI 1.1), followed by the FBDG on animal protein (40.3%; 95% CI 1.0). It was decided to augment these two FBDGs with the plant protein FBDG (54.3%; 95% CI 1.8). Despited a relatively good knowledge measured in the caregivers, none of the plant protein food items appeared in the top 20 food items most frequently consumed. The formulation phase included the testing of existing nutrition education material. Messages were well understood (60.5%). However, caregivers found the images contained in the pamphlets and the design of the pamphlets confusing. A definite need was identified for new, culturally acceptable NE material in the caregivers’ home language, Sesotho (74%). During the implementation phase the lectures were presented and the change in the short-term nutrition knowledge of the FBDGs was measured at every session by means of a pre-post-test design. The median age of the caregivers was 63 years (52-78). A significant change in nutrition knowledge was found for each FBDG in terms of the mean number of questions answered correctly by the participants between the results of each pre- and post-test. In the evaluation phase, the impact of the NEP was measured in the Boipatong experimental group and compared, regarding nutrition knowledge, to a control group in the longer term (three months after completion of the intervention). Median nutrition knowledge before the intervention was 49 percent (IQR 46-57) compared to 70 percent (IQR 68-73) after the intervention – an increase of 21 percent. In contrast, the control group showed an increase of only five percent. The results showed that the eating habits of many of the caregivers still fell substantially short of internationally recognised standards (estimated energy requirement (EER) and estimated average requirement (EAR)), and this could contribute to undernutrition as well as to an increased risk of diet-related chronic disease. A median kilojoule intake of 4788 kJ (IQR 3415-6596) per day before and 4651 kJ (IQR 3369-6664) per day after the intervention was registered. Carbohydrate foods remained an important source of energy. Calcium presented a major concern, as none of the participants reached the EAR despite a slight increase in the intake of milk (volume and frequency) after the intervention, as validated by the top 20 food lists and as measured by a nutrient average requirement (NAR) of 0.1 to 0.7 before and after the intervention respectively. The mean average requirements (MAR) remained relatively stable, at 0.7 (median) before the intervention and 0.6 after the intervention. The NEP thus had an insignificant positive effect on dietary intake, except for calcium, iodine and vitamin C intake, which showed significant increases. No relationships could be found between the MAR as an indicator of dietary quality and nutrition knowledge. However, this does not mean that an NEP is not a suitable strategy. Changes in food choices and dietary intake should be measured in the longer term, since lifestyle changes are challenging to adopt when people, especially those in deprived communities, do not have the financial means to make healthy food choices. Conclusion When planning nutrition education strategies to improve the health status of communities in deprived areas, one is faced with the difficulty of households with a low socio-economic status and poor dietary intake, which increases the risk of food and nutrition insecurity. The nutrition education programme, developed, tailored and implemented as an intervention strategy to address an identified need of caregivers, was effective in improving nutrition knowledge. Four of the South African dietary guidelines were used in the nutrition education programme tailored to the specific profile that emerged from the situational analysis and the development of supportive nutrition education material. Lesson plans and the Sesotho and English booklets are available for use in other nutrition education programmes. The study contributed to the understanding of food choices of the urban community of Boipatong as well as of the nutrient inadequacies observed. Nutrition knowledge alone is not enough to bring about changes in food choices when the means to do so are lacking. Poverty and nutrition are closely linked and thus difficult to separate.

Le système immunitaire est particulièrement dépendant des mécanismes de réparation de l’ADN, en effet le développement du système immunitaire adaptatif nécessite certains mécanismes de réparation de l’ADN, lors de la recombinaison V(D)J et lors de la commutation de classe des immunoglobulines. De plus, le système hématopoïétique est par sa nature très sensible aux lésions spontanées de l’ADN. Il existe chez l’homme de nombreux déficits immunitaires directement liés à un défaut de réparation de l’ADN. L’identification du gène responsable est importante pour un conseil génétique familial approprié et pour la prise en charge médicale. Nous avons accès aujourd’hui à de puissants outils de dépistage génétique grâce au séquençage à haut débit et la liste des gènes responsables d’un déficit immunitaire s’allonge de plus en plus en rapidement. La première partie de ce travail porte sur la mise au point d’un nouvel outil de dépistage rapide des déficits de la réparation de l’ADN, en particulier dans le cas de déficit immunitaires. Ce test est fondé sur l’observation d’un biais du répertoire du TCRdes lymphocytes T circulants lorsque les thymocytes ont une durée de vie diminuée, or un défaut de réparation de l’ADN entraîne une diminution de la survie thymocytaire. Nous avons mis au point deux techniques, par biologie moléculaire et par cytométrie en flux, pour détecter un éventuel biais du répertoire du TCRα et évaluer la pertinence de ce test dans les déficits immunitaires liés à un défaut de réparation de l’ADN. Un biais a notamment été détecté dans les cas de déficit en facteur du NHEJ et en ATM. Nous avons également établi en collaboration avec le service d’Immunologie Clinique de l’hôpital Saint-Louis une cohorte de patients atteints de déficit immunitaire commun variable (DICV) dont la présentation clinique est évocatrice d’un défaut de réparation de l’ADN. Une série de test fonctionnels de dépistages de déficit de la réparation de l’ADN ainsi que des analyses génétiques (CGH array, séquençage complet de l’exome) ont été fait chez ces patients afin d’identifier de nouveaux gènes impliqués dans les DICV. Parmi les 18 patients analysés, dans 5 cas on retrouve une sensibilité cellulaire accrue aux agents génotoxiques et chez 15 patients, un gène candidat a été identifié. Ces résultats sont encore préliminaires et la caractérisation génétique et fonctionnelle des mutations identifiées sera poursuivie par notre équipe. Pour finir, nous avons entrepris l’exploration génétique et fonctionnelle de deux mutations identifiées chez une jeune patiente atteinte de déficit immunitaire combiné (CID) associé à un syndrome lymphoprolifératif et une auto-immunité, et chez qui une hypersensibilité cellulaire à la Mitomycine C, agent pontant de l’ADN, a été détectée. La première mutation a été identifiée dans le gène ELKS qui code pour un facteur impliqué dans la réparation de l’ADN. La complémentation fonctionnelle de ce gène prouve l’implication de cette mutation dans l’hypersensibilité des cellules de la patiente à la MMC. Nous avons développé un modèle murin KO conditionnel de ce gène dans les cellules hématopoïétiques qui n’a pas montré de défaut de développement du système immunitaire. La deuxième mutation identifiée se situe dans le gène BACH2 codant pour un répresseur transcriptionnel très impliqué dans le développement du système immunitaire. Les souris KO pour ce gène ont un phénotype proche du déficit immunitaire décrit chez cette patiente. Les investigations de cette mutation sont en cours chez elle et chez les membres de sa famille également porteurs de la mutation. / The immune system is particularly dependent on DNA damage response (DDR) pathways. The development of the adaptive immune system requires certain DDR mechanisms, in particular during the V(D)J recombination and during class switch recombination (CSR), furthermore, the hematopoietic system is very sensitive to spontaneous DNA lesions. Therefore, there are many immune deficiencies in human directly related to a DDR deficiency. The identification of the responsible gene is important for appropriate genetic counseling. Today, we have access to powerful genetic screening tools, in particular next generation sequencing (NGS) and the list of genes responsible for immune deficiency is growing rapidly. The first part of this work focuses on the development of a new screening tool for DDR defects, in particular in the case of immune deficiency, and evaluation of clinical interest. This test is based on the observation of a bias of the TCRα repertoire in circulating T lymphocytes when thymocytes lifespan is diminished and we know that DDR defect causes decreased thymocyte survival. We have developed two techniques, by molecular biology and by flow cytometry, to detect a potential bias of the TCRα repetoire and assess the suitability of this test in some immunodeficiencies linked to a DDR defect. A significant bias was detected in the case of ATM and NHEJ factor deficiency. Furthermore, we have established a cohort of patients suffering from common variable immunodeficiency (CVID) with a clinical presentation highly suggestive of DDR defect, in collaboration with the Clinical Immunology Service of Hôpital Saint-Louis (Paris). Functional test for DDR defect and genetic analysis (CGHarray, whole exome sequencing) were performed in these patients to identify new genes involved in CVID. Among the 18 patients analyzed until now, five cases of cellular sensitivity to genotoxic agents have been detected and a candidate gene was identified in 15 of them. These results are still preliminary and our team will pursue genetic and functional characterization of the identified mutations. Finally, we undertook genetic and functional exploration of two mutations identified in a young patient with combined immunodeficiency (CID) associated with a lymphoproliferative disease and autoimmunity, and in whom a cellular hypersensitivity to mitomycin C, a DNA crosslinking agent, was detected. The first mutation was identified in the ELKS gene, which codes for a factor involved in DNA repair. Functional complementation of this gene demonstrates the involvement of this mutation in the hypersensitivity of patient’s cells to MMC. We have developed a conditional knockout mouse model of this gene in hematopoietic cells that did not show any defect in development of the immune system. The second mutation was identified in BACH2 gene encoding a transcriptional repressor involved in the development of the immune system. Knockout mice for this gene have a similar phenotype to the immune deficiency described in this patient. Investigations on this mutation are ongoing in the patient and among family members that also carry the mutation.

A avaliação do estado nutricional das plantas de milho usualmente é feita através de análises químicas ou pela diagnose visual das folhas da planta, esta última, sujeita a erros de interpretação já que a ausência de algum nutriente na planta gera um padrão de mudança específico na superfície da folha que depende do nível de ausência do nutriente. As dificuldades que apresentam neste processo e sua importância na agricultura, criam a necessidade de pesquisar sistemas automáticos para a avaliação do estado nutricional de plantas. Desta forma, este mestrado teve como objetivo principal o desenvolvimento de um sistema de visão artificial para verificar a possibilidade de identificação de níveis dos macronutrientes Cálcio, Enxofre, Magnésio, Nitrogênio e Potássio em plantas de milho através da análise da superfície das folhas usando métodos de visão computacional. Este projeto realiza uma revisão bibliográfica do estado da arte dos métodos de extração de características de cor, textura em escala de cinza e textura colorida utilizadas em processamento de imagens. A alta similaridade entre os sintomas produzidos pelas deficiências e a pouca similaridade entre amostras de uma mesma deficiência motivou o desenvolvimento de novos métodos de extração de características que pudessem fornecer dados necessários para uma correta separação entre as classes. Os resultados obtidos demonstraram que o sistema desenvolvido possibilita a predição de deficiências nutricionais em estágios iniciais do crescimento da planta usando unicamente a textura da superfície da folha como fonte de informação / The evaluation of the nutritional status of corn plants is usually done through chemical analysis or by visual diagnosis of the plant leaves. Visual diagnosis is subject to misinterpretation as the lack of some nutrient in the plant generates a specific pattern of change in the leaf surface that depends on the degree on which the nutrient is absent on the plant. The difficulties present in this process and its importance in agriculture creates the necessity to search automated systems for the assessment of nutritional status of plants. Thus, this dissertation had as main objective the development of an artificial vision system to verify the possibility of identifying levels of macronutrients calcium, sulfur, magnesium, potassium and nitrogen in corn plants by analyzing the surface of the leaves using computer vision methods. This project performs a review of the literature of the state of the art methods for feature extraction of color, grayscale and colored texture used in image processing. The high similarity between the symptoms caused by deficiencies and low similarity between samples of the same deficiency motivated the development of new methods for extracting features that could provide the data needed for a correct separation between classes. The results showed that the system enables the prediction of nutritional deficiencies in an initial stage of plant growth using only texture of the leaf surface as a source of information

Introdução: A hipertensão pulmonar tromboembólica crônica (HPTEC) está incluída no grupo 4 da Classificação Internacional de Hipertensão Pulmonar (HP). É caracterizada pela persistência de obstrução por trombos sanguíneos na circulação pulmonar, associada à presença de HP, após três meses de anticoagulação efetiva. O tratamento de escolha é a cirurgia de tromboendarterectomia pulmonar (TEAP), mas alguns dos mecanismos fisiopatológicos envolvidos nesta forma de hipertensão ainda permanecem incertos. O redirecionamento dos fluxos sanguíneos pulmonares e a hipóxia exercem papel importante na HPTEC, como também em casos de hipertensão pulmonar residual, após a cirurgia de TEAP. Entretanto, existem poucos dados sobre as respostas das células endoteliais pulmonares a essas mudanças de fluxo e de oxigenação, surgindo a necessidade do estudo da proteostase celular nesta doença. Objetivo: (A) Caracterização morfológica das células em culturas provenientes de artéria pulmonar de pacientes com HPTEC submetidos à TEAP. (B) Avaliação da resposta das células endoteliais, a partir da análise de proteínas envolvidas na proteostase celular, quando submetidas a diferentes níveis de stress mecânico e à hipóxia. Método: Trombos extraídos por TEAP foram processados, as células retiradas foram cultivadas, marcadas com CD31 e submetidas a stress mecânico por vinte e quatro horas, constituindo o grupo HPTEC. A proteostase celular foi avaliada pela medida de proteínas expressas por essas células, tanto em culturas quanto pela análise imuno-histoquímica do tecido vascular pulmonar. Como grupo controle foram utilizadas células endoteliais pulmonares humanas de linhagem (CE) e tecido de artérias pulmonares de doadores de transplante de pulmão. As culturas de ambos os grupos também foram colocadas em hipóxia e analisada a expressão indireta de óxido nítrico (NO) por meio da medida de nitrato. Resultado: as células do grupo HPTEC com morfologia endotelial foram marcadas positivamente com CD31 e apresentaram características semelhantes às do grupo CE. Em relação ao stress mecânico, na condição estática as células HPTEC expressaram menor quantidade de óxido nítrico sintase endotelial (eNOS). Quando submetidas a stress de alta intensidade (shear stress >= 15 dynes/cm2), as reduções ficaram ainda mais evidentes, sinalizando uma disfunção endotelial. Na análise de outras proteínas, como GRP94, GRP78, HSP70, as respostas também foram menores no alto fluxo. Na avaliação imunohistoquímica da camada íntima do vaso pulmonar, a HSP70 apresentava-se diminuída, corroborando os achados das culturas. Os valores de NO foram inferiores no grupo HPTEC quando se comparam hipóxia e normóxia. Conclusão: (A) A avaliação morfológica mostrou que as culturas de células HPTEC eram endoteliais. (B) A análise funcional revelou que estas células apresentaram redução de resposta, o que caracteriza alteração da proteostase, que se tornou mais evidente quando foram submetidas a shear stress de alta magnitude. A hipóxia reduziu a produção de NO, entretanto sem diferenciar os grupos celulares estudados / Introduction: Chronic Thromboembolic Pulmonary Hypertension (CTEPH) is included in group 4 of the International Classification of Pulmonary Hypertension (PH). It is characterized by persistent obstruction by blood clots in the pulmonary circulation, associated with the presence of PH, after 3 months of effective anticoagulation. The treatment of choice is pulmonary endarterectomy (PEA). However, some of the pathophysiological mechanisms involved in this form of hypertension still remain uncertain. The redirection of pulmonary blood flow and hypoxia play an important role in CTEPH, and also, in cases of residual pulmonary hypertension after PEA surgery. Nevertheless, there is insufficient data from the pulmonary endothelial cell responses to this flow and oxygenation changes, reflecting the need to further study of cellular proteostasis in this disease. Objective: (A) Morphological characterization of cells in cultures from the pulmonary artery of CTEPH patients submitted to PEA. (B) Evaluation of the response of endothelial cells, through the analysis of proteins involved in cellular proteostasis, when submitted to different levels of mechanical stress and hypoxia. Method: Thrombus extracted by PEA were processed and the cells removed were cultured, marked with CD31 and submitted to mechanical stress for 24 hours and constituted the group CTEPH. Cellular proteostasis was measured by the quantification of the proteins expressed in cultures and in pulmonary vascular tissue by immunohistochemistry analysis. As a control group, the human pulmonary endothelial cells (EC) and pulmonary artery tissue from lung transplant donors were used. Cultures of both groups were also placed in hypoxia and the indirect expression of nitric oxide (NO) was analyzed by nitrate measurement. Results: The cells with endothelial morphology from the CTEPH group were positively marked with CD31 and presented similar characteristics as the EC group. Regarding mechanical stress, in the static condition, the CTEPH cells expressed a lesser amount of endothelial nitric oxide synthase (eNOS). When submitted to high flow (shear stress > 15 dynes / cm2) the reductions became even more evident, signaling an endothelial dysfunction. In the analysis of other proteins, such as GRP94, GRP78, HSP70, responses were also lower in high shear stress. In the immunohistochemistry analysis of the intimal layer of the pulmonary vessel HSP70 was diminished, corroborating with the findings of the cultures. The NO values were lower in the CTEPH group when compared hypoxia and normoxia. Conclusion: (A) Morphological evaluation showed that cultures of CTEPH cells were endothelial. (B) Functional analysis revealed that these cells had reduced response, which characterizes proteostasis alterations, which became more evident when they underwent shear stress of high magnitude. Hypoxia reduced NO production, however without differentiating the cell groups studied

Le système immunitaire est particulièrement dépendant des mécanismes de réparation de l’ADN, en effet le développement du système immunitaire adaptatif nécessite certains mécanismes de réparation de l’ADN, lors de la recombinaison V(D)J et lors de la commutation de classe des immunoglobulines. De plus, le système hématopoïétique est par sa nature très sensible aux lésions spontanées de l’ADN. Il existe chez l’homme de nombreux déficits immunitaires directement liés à un défaut de réparation de l’ADN. L’identification du gène responsable est importante pour un conseil génétique familial approprié et pour la prise en charge médicale. Nous avons accès aujourd’hui à de puissants outils de dépistage génétique grâce au séquençage à haut débit et la liste des gènes responsables d’un déficit immunitaire s’allonge de plus en plus en rapidement. La première partie de ce travail porte sur la mise au point d’un nouvel outil de dépistage rapide des déficits de la réparation de l’ADN, en particulier dans le cas de déficit immunitaires. Ce test est fondé sur l’observation d’un biais du répertoire du TCRdes lymphocytes T circulants lorsque les thymocytes ont une durée de vie diminuée, or un défaut de réparation de l’ADN entraîne une diminution de la survie thymocytaire. Nous avons mis au point deux techniques, par biologie moléculaire et par cytométrie en flux, pour détecter un éventuel biais du répertoire du TCRα et évaluer la pertinence de ce test dans les déficits immunitaires liés à un défaut de réparation de l’ADN. Un biais a notamment été détecté dans les cas de déficit en facteur du NHEJ et en ATM. Nous avons également établi en collaboration avec le service d’Immunologie Clinique de l’hôpital Saint-Louis une cohorte de patients atteints de déficit immunitaire commun variable (DICV) dont la présentation clinique est évocatrice d’un défaut de réparation de l’ADN. Une série de test fonctionnels de dépistages de déficit de la réparation de l’ADN ainsi que des analyses génétiques (CGH array, séquençage complet de l’exome) ont été fait chez ces patients afin d’identifier de nouveaux gènes impliqués dans les DICV. Parmi les 18 patients analysés, dans 5 cas on retrouve une sensibilité cellulaire accrue aux agents génotoxiques et chez 15 patients, un gène candidat a été identifié. Ces résultats sont encore préliminaires et la caractérisation génétique et fonctionnelle des mutations identifiées sera poursuivie par notre équipe. Pour finir, nous avons entrepris l’exploration génétique et fonctionnelle de deux mutations identifiées chez une jeune patiente atteinte de déficit immunitaire combiné (CID) associé à un syndrome lymphoprolifératif et une auto-immunité, et chez qui une hypersensibilité cellulaire à la Mitomycine C, agent pontant de l’ADN, a été détectée. La première mutation a été identifiée dans le gène ELKS qui code pour un facteur impliqué dans la réparation de l’ADN. La complémentation fonctionnelle de ce gène prouve l’implication de cette mutation dans l’hypersensibilité des cellules de la patiente à la MMC. Nous avons développé un modèle murin KO conditionnel de ce gène dans les cellules hématopoïétiques qui n’a pas montré de défaut de développement du système immunitaire. La deuxième mutation identifiée se situe dans le gène BACH2 codant pour un répresseur transcriptionnel très impliqué dans le développement du système immunitaire. Les souris KO pour ce gène ont un phénotype proche du déficit immunitaire décrit chez cette patiente. Les investigations de cette mutation sont en cours chez elle et chez les membres de sa famille également porteurs de la mutation. / The immune system is particularly dependent on DNA damage response (DDR) pathways. The development of the adaptive immune system requires certain DDR mechanisms, in particular during the V(D)J recombination and during class switch recombination (CSR), furthermore, the hematopoietic system is very sensitive to spontaneous DNA lesions. Therefore, there are many immune deficiencies in human directly related to a DDR deficiency. The identification of the responsible gene is important for appropriate genetic counseling. Today, we have access to powerful genetic screening tools, in particular next generation sequencing (NGS) and the list of genes responsible for immune deficiency is growing rapidly. The first part of this work focuses on the development of a new screening tool for DDR defects, in particular in the case of immune deficiency, and evaluation of clinical interest. This test is based on the observation of a bias of the TCRα repertoire in circulating T lymphocytes when thymocytes lifespan is diminished and we know that DDR defect causes decreased thymocyte survival. We have developed two techniques, by molecular biology and by flow cytometry, to detect a potential bias of the TCRα repetoire and assess the suitability of this test in some immunodeficiencies linked to a DDR defect. A significant bias was detected in the case of ATM and NHEJ factor deficiency. Furthermore, we have established a cohort of patients suffering from common variable immunodeficiency (CVID) with a clinical presentation highly suggestive of DDR defect, in collaboration with the Clinical Immunology Service of Hôpital Saint-Louis (Paris). Functional test for DDR defect and genetic analysis (CGHarray, whole exome sequencing) were performed in these patients to identify new genes involved in CVID. Among the 18 patients analyzed until now, five cases of cellular sensitivity to genotoxic agents have been detected and a candidate gene was identified in 15 of them. These results are still preliminary and our team will pursue genetic and functional characterization of the identified mutations. Finally, we undertook genetic and functional exploration of two mutations identified in a young patient with combined immunodeficiency (CID) associated with a lymphoproliferative disease and autoimmunity, and in whom a cellular hypersensitivity to mitomycin C, a DNA crosslinking agent, was detected. The first mutation was identified in the ELKS gene, which codes for a factor involved in DNA repair. Functional complementation of this gene demonstrates the involvement of this mutation in the hypersensitivity of patient’s cells to MMC. We have developed a conditional knockout mouse model of this gene in hematopoietic cells that did not show any defect in development of the immune system. The second mutation was identified in BACH2 gene encoding a transcriptional repressor involved in the development of the immune system. Knockout mice for this gene have a similar phenotype to the immune deficiency described in this patient. Investigations on this mutation are ongoing in the patient and among family members that also carry the mutation.

The diploma thesis deals with the reading level of pupils in the third year, who in the first year learned to read by different methods. The thesis is divided into theoretical and research parts. The theoretical part deals with the characteristics of pupils at the younger school age, the development of reading skills, the teaching of early reading by the analytical-synthetic, genetic and Sfumato methods. Furthermore, it defines reading literacy, and refers to international research surveys, types and qualities of reading, diagnosis of reading, reading defects and their correction. The research part focuses on the diagnosis of the reading performance of pupils in the 3rd grade of primary school. The reading results are evaluated according to the criteria based on the theoretical part of the thesis.