Abstract The aims of this study were to explore different aspects of dental fear and their determinants among children at different ages. The study samples comprised 378 children from the Veneto region of Italy aged 3–13 years and 1474 children from Jyväskylä and Kuopio, Finland, aged 3, 6, 9, 12 and 15 years. In the Italian study, the child’s age, first dental visit, number of subsequent visits and dental fear, and the parent’s dental fear were asked in a questionnaire. In the Finnish study, a questionnaire was used that contained 11 dental fear-related questions and family member’s dental fear and questions on oral health habits and family characteristics. Dental status was determined clinically and information on treatment procedures from three preceding years was collected from patient records. A total of 21 to 36% of Finnish children were quite or very afraid of something in dental treatment. The prevalence of dental fear among Finnish children was not lower among older children but rather fluctuated among different ages. The characteristics of dental fear differed among children at different ages. Among Finnish children, four aspects of dental fear were revealed from the questionnaire, i.e. ‘Treatment of dental decay’, ‘Attending dentist’, ‘Peak value for dental fear’ and ‘Fear of dental treatment in general’. Correlations between the four abovementioned aspects, the differences between age groups separately for the above mentioned aspects, and the determinants of dental fear were studied. At younger ages, the dental fear was abstract, commonly related to attending dentist. At older ages, dental fear was often related to invasive treatment, such as fear of local anaesthesia and drilling. Fear of pain which was common among all age groups. Among Italian children, the first dental experiences were strong determinants of dental fear. Among Finnish children, dental fear among other members of the family was more often found among children with dental fear than among non-fearful children. 15-year-old girls were more likely to be afraid than boys of the same age but gender differences were not found at younger ages. The results indicated that dental fear is not solely based on direct conditioning but rather consists of child, family and environment related determinants. / Tiivistelmä Tutkimuksen tarkoituksena oli selvittää lasten hammashoitopelon piirteitä ja niihin liittyviä seikkoja eri-ikäisillä lapsilla. Tutkimuksessa käytettiin havaintoaineistoa, joka koostui 378:sta 3–13-vuotiaasta italialaisesta lapsesta Veneton maakunnan alueelta sekä 1474:stä 3-, 6-, 9-, 12- ja 15-vuotiaasta lapsesta Jyväskylästä ja Kuopiosta. Tutkimuksessa italialaisilla lapsilla vanhempi täytti kyselylomakkeen, joka sisälsi kysymyksiä lapsen iästä, hammashoitopelosta, ensimmäisestä hammashoitokäynnistä ja seuraavien hoitokäyntien lukumäärästä sekä vanhemman omasta hammashoitopelosta. Suomalaisilla lapsilla tutkimustiedot kerättiin kyselylomakkeella, joka sisälsi 11 kysymystä lapsen hammashoitopelosta ja kysymyksiä lapsen suun terveystottumuksista sekä perheeseen liittyvistä seikoista, kuten perheenjäsenten hammashoitopelosta. Hampaiden senhetkinen kliininen tila sekä kolmen tarkastusta edeltävän vuoden hoitokäynnit ja tuolloin tehdyt toimenpiteet otettiin mukaan tutkimukseen. Suomalaisista lapsista 21–36 % pelkäsi jonkin verran tai paljon jotain asiaa hammashoidossa. Suomalaisten lasten hammashoitopelko ei ollut alempi nuoremmilla lapsilla vaan vaihteli ikäryhmien välillä. Myös hammashoitopelon luonne vaihteli ikäluokittain. Suomalaisesta kyselystä löydettiin neljä pelon osa-aluetta: paikkaushoitoon liittyvä pelko, hammaslääkärissä käymiseen liittyvä pelko, yleinen hammashoitopelko sekä voimakkaimmaksi koettu hammashoitoon liittyvä yksittäinen pelko. Tutkimuksessa tutkittiin pelon osa-alueiden välisiä korrelaatioita ikäryhmittäin, erikseen yksittäisen pelon osa-alueen vaihtelua ikäryhmien välillä sekä hammashoitopelon liittyviä seikkoja ikäryhmittäin. Nuoremmilla lapsilla hammashoitopelko oli useammin abstraktia, yleensä hammashoidossa käymiseen liittyvää pelkoa. Vanhemmilla lapsilla hammashoitopelko oli usein hammashoitotoimenpiteisiin liittyvää pelkoa, esimerkiksi puudutuksen ja porauksen pelkoa. Kivun pelko oli yleistä kaikissa ikäryhmissä. Ensimmäiset hammashoitokokemukset olivat voimakkaita hammashoitopelon selittäjiä italialaisilla lapsilla. Suomalaisten lasten hammashoitopelko oli vahvasti yhteydessä muiden perheenjäsenten hammashoitopelkoon. 15-vuotiaat tytöt pelkäsivät hammashoitoa enemmän kuin pojat, mutta sukupuolten välisiä eroja ei havaittu nuoremmissa ikäryhmissä. Tutkimus osoittaa, että lasten hammashoitopelko ei ole ainoastaan seurausta suorasta ehdollistumisesta hammashoitokokemusten kautta vaan siihen vaikuttavat enemmänkin lapseen, perheeseen ja ympäristöön liittyvät seikat.

This thesis sets out to critically examine Guidano's model of psychopathology in eating-disordered individuals . The literature review highlights the diverse etiological approaches that have been taken in understanding eating disorders. Guidano's model is presented as an alternative to traditional approaches . It is a developmental, unitary model of psychopathology, conceptualised within a systems/process-oriented approach to organised complexity. Within a qualitative framework, case study methodology is utilised to test the viability and limitations of Guidano's model. Four in-depth case histories are presented which offer appropriate material for the testing of the model. The data is analysed using the reading guide method and presented in terms of the four features of Guidano's model: (1) dysfunctional patterns of attachment; (2) sense of self; (3) major themes on systemic coherence; (4) common coping strategies. The findings of the research support Guidano's model of psychopathology in eating-disordered individuals. The findings are: (1) attachment styles are ambiguous, "intrusive, and enmeshed; participants experience a disappointment in the preferential attachment relationship; (2) that participants' sense of self is blurred and wavering; (3) the major theme on systemic coherence is the oscillation between seeking and avoiding intimacy; (4) common coping strategies are: the seeking of supportive intimacy with minimal self-exposure; withdrawal into the self; perfectionism; the development of an eating disorder; continuous thoughts about food, eating, and weight which prevents participants from becoming aware of the real issues confronting them. An evaluation of Guidano's model in terms of its specific contribution to knowledge and research on the role of father in child and adolescent psychopathology, as well as how father effects evidence in eating disorders, demonstrates the model's value as an explanatory tool and raises implications for future treatment, theory, and research practices of eating-disordered individuals.

Ménière’s disease is the third most common inner ear disorder. The individual course of Ménière’s disease in different patients makes it difficult to diagnose on the basis of symptomatology alone. The impact of Ménière’s disease on quality of life has highlighted the importance of an additional tool to support the diagnosis of Ménière’s disease. Apart from the patient’s history, audiological data provide the most relevant information for confirming the diagnosis. The aim of this study was to analyse and describe the clinical and audiological features of a cohort of subjects diagnosed with Ménière’s disease, in order to develop understanding of the pathophysiology of the disease and to facilitate the diagnostic process. The research is based on a retrospective study of the medical records of 135 subjects with Ménière’s disease which were selected according to a non-probability sample. Descriptive statistics were used to organize, analyse and interpret the data. Sixty one percent of subjects presented with definite Ménière’s disease, 14 % with probable Ménière’s disease and 25 % with possible Ménière’s disease. The results showed a higher incidence of Ménière’s disease in females especially in the vestibular type. Three percent of subjects indicated a family history of Ménière’s disease. Bilateral Ménière’s disease presented in 39 % of subjects. The results confirmed that vertigo was the most debilitating symptom in Ménière’s disease. Correlating the clinical features of subjects with audiometric and vestibular tests highlighted the clinical value of an audiological test battery including the following tests: Pure tone audiometry, Speech discrimination, Oto-acoustic emissions, Electronystagmography and Electrocochleography. This confirms the role of the audiologist in the diagnostic and rehabilitation process in patients with Ménière’s disease. / Dissertation (Communication Pathology)--University of Pretoria, 2007. / Speech-Language Pathology and Audiology / unrestricted

The composition of Joshua 3 and 4 forms the main focus of this study. Although Joshua 3 and 4 have been the subject of many studies, there have been no satisfactory explanation of the many contradictions and incoherence in these chapters. Apart form the literary problems regarding the composition of Joshua 3 and 4, some challenging historical questions arise from the study of these chapters. Therefore, this literary study of Joshua 3 and 4 will not also involve some broader literary questions like the position of the book of Joshua in the Canon, but it will also attempt to answer historical questions about Israel`s past. My hypothesis is that that the final text of Joshua 3 and 4 is the result of several redactions. The original narrative of the Jordan crossing forms the main part of these chapters. This original deuteronomistic narrative was originally part of a Deuteronomistic History that encompassed Deuteronomy, Joshua, and some parts of 1 and 2 Samuel as well as the books of 1 and 2 Kings. The Deuteronomistic History originated in the exile, and was the subject of major editing up to the Persian era. Joshua 3 and 4 reflects the process of the formation of the Pentateuch. The original deuteronomistic narrative of the Jordan crossing was subsequently supplemented by a post-priestly narrative which enhanced the miracle of the crossing. This supplement probably took place when the priestly composition (Ex-Num) was joined to the deuteronomistic composition (Deut-2 Kon) as part of an compromise between rival priestly groups. The book of Deuteronomy was eventually incorporated in the foregoing books (Ex-Num) to form a Pentateuch. As a result of this process, the book of Joshua was cut off from Deuteronomy and became a post-Deuteronomic book. This explains not only the affinities and differences between Joshua and Deuteronomy, but also the peculiar position of the book of Joshua in the Canon. The narrative of the twelve memorial stones (Joshua 4) forms part of an etiological formula, found throughout Joshua 1-12. These etiological reference points reflects the lists of the returning exiles and the builders of the Jerusalem`s walls in the books of Ezra and Nehemiah. Furthermore, these etiological references reflects the borders of the post-exilic Israel. Joshua 4 is the post-exilic Israel`s way of interpreting the pre-exilic conquest narratives in Joshua 1-12 in order to make sense of their present situation. There are also references to the post-deuteronomistic emphasis on many sanctuaries (which probably served to legitimize the Samaritan Temple at Mount Gerizim. The book of Joshua is not to be classified as history. This study enhances the hypothesis that the book of Joshua embodies the ideology of post-exilic Judaism. In its final form, the book probably served as an attempt to bring a compromise between rival priesthoods and rival ideologies. Although the book had pre-exilic (deuteronomistic) origins, it was subsequently edited to function in a post-exilic context when the people of Israel were facing a new future with new possibilities. / Thesis (PhD)--University of Pretoria, 2010. / Old Testament Studies / unrestricted

Determinamos o comportamento destes mediadores em pacientes com insuficiência aórtica crônica importante ( IAo ). Materal e métodos:Analisamos 89 portadores de Insuficiência Aórtica crônica importante, média etária de 33,6±11,5 anos, 84,6% sexo masculino, 60% assintomáticos, todos de etiologia reumática . Os pacientes foram submetidos a avaliação clínica e ecocardiográfica. Os valores médios foram: diâmetro diastólico (DD ) do ventrículo esquerdo ( VE ) de 71,9±8,3mm e o diâmetro sistólico ( DS ) do VE de 50,4±9,3mm, e a fração de ejeção ( FE ) do VE de 0,64±0,11. Realizamos a dosagem de Fator de Necrose Tumoral ( TNF ), seus receptores solúveis tipo I e II ( sTNFRI e sTNFR II ) , Interleucina-6 ( IL-6 ), seu receptor ( IL-6R), interleucina 1-beta ( IL-1beta ) , seu antagonista ( IL1-RA ) e endotelina-1 ( ET-1 ). Comparamos com níveis séricos de controles saudáveis. Conjuntamenete analisamos o polimorfismo genético do gene do TNF, localizado a -308 pares de bases do sítio de iniciação. Resultados: Os níveis séricos de TNF forma significativamente maiores em pacientes com IAo do que em controles normais ( 92,65±110,24 pg/ml contra 1,67±1,21 em controles normais, p < 0,001 ). Tiveram comportamentos similares os níveis séricos de sTNFRI ( 894,75±348,87pg/ml vs 521,42±395.13pg/ml em controles, p=0,007 ) e IL-6 ( 7,17±7,78pg/ml vs 0,81±0,38pg/ml, p=0,0001 ). Observamos correlação significativa entre níveis de sTNFRII e DDVE ( r=-0,329, p=0,038 ) e DSVE ( r=-0,352, p=0,027). Não observamos relação de níveis séricos de citocinas com sintomas. As outras citocinas não guardaram relação com parâmetros de gometria e função ventricular. A presença do alelo 2 do polimorfismo genético do TNF associou-se a paciente assintomáticos com IAo. Conclusão: Demonstramos níveis elevados de citocinas proinflamatórias em pacientes com IAo em relação a controles normais. Os níveis de sTNFRII diminuem com o aumento dos diâmetros ventriculares. A presença do alelo 2 do polimorfismo -308 do TNF associa-se a pacientes assintomáticos com IAo / Background - Proinflamatory cytokines are implied in the phisiopatology of heart failure secoundary to ischaemic or idiophatic dilated cardiomiopathy, but there are few studies regarding these mediators in valular heart disease. We determined the behaivour of proinflamatory cytokines and their antagonists in patients with chronic severe aortic regurgitation ( IAo ) Methods - We analised 89 patients with IAo mean age 33.6±11.5 years, 84.6% male, 60% asymptomatic, all of rheumatic etiology. Patients were evaluated clinnicaly and by echocardiography. Mean values were : left ventricular ( LV ) diastolic diameter ( DD ) 71.9±8.3mm, LV systolic diameter ( SD ) 50.4±9.3mm and ejection fraction 0.64±0.11. We made the plasma dosages of tumor necrosis factor-alpha ( TNF ), its soluble receptors type I and II ( sTNFRI and sTNFR II ) , Interleukin 6 ( IL-6 ) and its receptor ( IL-6R ), Interleukin 1-beta ( IL-1beta ) , its antagonist ( IL1-RA ) and endothelin-1 ( ET-1 ). Plasma levels were compared to healthy controls. We also analysed the TNF gene polimorphism, located at - 308 base pairs from the initation site. Results - Plasma TNF levels were significantly increased in IAo patients in telation to normal controls (92.65±110.24 pg/ml vs 1.67±1.21 pg/ml in controls, p < 0.001 ). Similar behaivour was observed with IL-6 (7.17±7.78pg/ml in IAo patients vs 0,81±0,38pg/ml in controls , p=0.0001 ) and sTNFRI (894.75±348.87pg/ml vs 521.42±395.13pg/ml in controls, p=0,007 ). We observed and significant relation between sTNFRII levels and LVDD ( r=-0,329, p=0,038 ) e LVSD ( r=-0,352, p=0,027). Levels of cytokines were similar in asymptomatic and sympromatic patients and the other cytokines had no relation to ventricular diameters or function. Presence of the alele 2 of the -308 TNF polimorphism was associated to asymptomatic patients. Conclusion - We showed increased plasma levels of proinflamatory cytokines in patients with IAo in relation to normal controls. There was an decrease of sTNFRII levels with increase in ventricular diameters. The presence of the alele 2 of the -308 TNF polimorphism was associated to asymtomatic patients

Introdução: A litíase biliar e o consumo de álcool são as etiologias mais frequentes para pancreatite aguda (PA), sendo reportadas como responsáveis por cerca de 40 e 30% dos casos respectivamente. No entanto, no Rio Grande do Sul - BR observamos uma frequência de pancreatite aguda biliar (PAB) em torno de 77% dos casos e pancreatite aguda alcoólica (PAA) em apenas 8%. Além da possibilidade de diferenças próprias da nossa população, é possível que a incidência de PAB esteja aumentando. Objetivo: Estimar as frequências globais da PAB, PAA e dos casos considerados pancreatite aguda idiopática (PAI) em estudos publicados de 2006 a 18 de outubro de 2017. Comparar as frequências de PAB, PAA e PAI entre os estudos que realizaram revisão de prontuários individuais dos pacientes ou foram prospectivos e os que utilizaram apenas os códigos de alta hospitalar para o diagnóstico etiológico. Comparar as frequências de PAB, PAA, PAI de acordo com região geográfica da população dos estudos. Métodos: Uma revisão sistemática de estudos observacionais em Inglês, Espanhol e Português, de 2006 a 18 de outubro de 2017 foi realizada. Metanálise pelo modelo de efeitos randômicos foi utilizada para calcular as frequências de PAB, PAA e PAI globais e nos subgrupos (diagnóstico por código da alta hospitalar, diagnóstico por avaliação individualizada do prontuário do paciente, estudos dos EUA, estudos da América Latina, estudos da Europa e estudos da Ásia). Resultado: Foram incluídos quarenta e seis estudos representando 2.341.007 casos de PA em 36 países. A estimativa global para a pancreatite aguda biliar (PAB) foi 41,6% (IC 95% 39,2-44,1), seguido por PA alcoólica (PAA) com 20,5% (IC 95% 3 16,6-24,6) e PA idiopática (PAI) em 18,3% (IC 95% 15.1 - 21,7). Em estudos com diagnóstico etiológico por código de alta a PAI foi a mais frequente com 37,9% dos casos (IC 95% 35,1 - 40,8). Nos estudos que revisaram os prontuários dos pacientes a PAB foi a mais frequente com 46% (IC 95% 42,3 - 49,8). Nos EUA a PAI foi a mais frequente com 34,7% (IC 95% 32,3 - 37,2). Na América Latina a estimativa de PAB foi de 68,5% (IC de 95% 57,8 - 78,3). Na Europa, na Ásia e em 1 estudo Australiano, a etiologia mais frequente foi a PAB em 41,3% (IC 95% 37,9 - 44,7), 42% (IC 95% 28,8 - 55,8) e 40% (IC 95% 36,8 - 43,2), respectivamente. Na África do Sul 1 artigo apresentou frequência de 70,2% (IC de 95% 64,5 - 75,4) para PAA. Conclusão: A PAB é a etiologia mais prevalente da PA, sendo 2 vezes mais frequente que o segundo lugar. A América Latina apresenta uma frequência para PAB muito maior do que o resto do mundo. Grandes estudos populacionais que utilizam diagnósticos codificados e estudos americanos apresentam elevadas taxas de PA sem classificação. A importância do diagnóstico etiológico consiste no tratamento da causa para prevenção da recorrência. / Background: Gallstones and alcohol are the most common etiology of acute pancreatitis (AP) and is reported to account for about 40% and 30% of cases respectively. However, in Rio Grande do Sul - BR, we observed a frequency of acute biliary pancreatitis (ABP) around 77% of cases and alcoholic acute pancreatitis (AAP) in only 8%. Besides the possibility of differences of our own population, it is possible that the incidence of PAB is increasing. Objective: estimate the global frequency of ABP, AAP and the cases considered idiopathic pancreatitis (IAP) in published studies from 2006 to October 18 2017. Compare the frequencies for ABP, AAP and AIP among studies that performed review of individual records of patients or collected data prospectively and those using only the hospital discharge diagnostic codes for etiologic diagnosis. Compare the frequency of ABP, AAP and IAP by geographic region. Methods: A systematic review of observational studies in English, Spanish and Portuguese, from 2006 to October 18, 2017 was done. Random-effects metaanalysis was used to assess the frequency of biliary, alcoholic and idiopathic AP worldwide and to perform the analysis of 6 subgroups (hospital discharge coded diagnosis, individual patient chart review, studies from US, Latin America, Europe and studies from Asia). Results: Forty-six studies were included representing 2.341.007 cases of PA in 36 countries. The overall estimate for ABP was 41.6% (95% CI 39.2 to 44.1), followed by AAP with 20.5% (95% CI 16.6 to 24 6) and IAP with 18.3% (95% CI 15.1 - 21.7). In studies with hospital discharge coded diagnosis IAP was the most frequent with 37.9% (95% CI 35.1 to 40.8). In studies with individual patient chart review PAB 5 was more frequent with 46% (95% CI 42.3 to 49.8). In US studies IAP was he most frequent etiology with 34.7% (95% CI 32.3 to 37.2). In Latin America PAB was estimated 68.5% of the cases (95% CI 57.8 to 78.3). In Europe, Asia and one Australian study, the most frequent cause was the ABP in 41.3% (95% CI 37.9 to 44.7), 42% (95% CI 28.8 to 55.8) and 40% (95% CI 36.8 to 43.2) of the cases respectively. One study from South Africa had AAP in 70.2% (95% CI 64.5 to 75.4) of the cases. Conclusion: Gallstones are the main etiology of AP globally, twice as frequent as the second one. Latin America has a frequency for ABP much higher than the rest of the world. Large population studies using coded diagnoses and American studies show high rates IAP. The importance of the etiological diagnosis resides in treating the cause in order to prevent recurrence.

O câncer é caracterizado pelo crescimento anormal de células em consequência ao acúmulo de alterações no DNA. Diferentes tecidos apresentam variadas incidências de tumores. Em 2015, Tomasetti & Vogelstein demonstraram uma forte correlação positiva (r = 0.804) entre o número de divisões de células tronco e o risco de câncer em diferentes tecidos, em que tecidos com maior número de divisões de células tronco estão mais susceptíveis aos efeitos estocásticos da replicação do DNA, e, assim, mais propícios a desenvolverem tumores. Assim, esta correlação é justificada pelo número de mutações. Neste trabalho, propomos e testamos in silico um novo conceito para justificar parte desta correlação positiva entre o número de divisões de células tronco e o risco de câncer entre diferentes tecidos, o qual denominamos concentração de ancestrais (AC). Em nossa hipótese, tecidos com alta taxa de proliferação concentram mais seus ancestrais, amplificando as chances de perpetuar células ancestrais mutadas, e, por isso, estão relacionados a maiores riscos de câncer. Assim, tecidos com altos valores de divisão de células tronco apresentam um perfil de alto AC e tecidos com baixo número de divisão de células tronco apresentam um perfil de baixo AC Para comprovar nossa hipótese, simulamos a evolução tumoral através do software esi- Cancer e aplicamos diferentes valores de proliferação e morte (CTOR). Os resultados demonstraram uma correlação positiva de 0.995 entre o valor de CTOR e o perfil de AC (P = 0:002). Como esperado, demonstramos que maiores CTORs estão relacionados a maiores médias de gerações com esiTumors para valores totais de mutações por divisão iguais. Entretanto, esta relação se mantém quando aplicadas valores corrigidos conforme o número de divisões para os diferentes CTORs, a fim de o número de mutações totais ser igual. Logo, apenas variações não são suficientes para explicar a incidência observada em diferentes tecidos. Nossos resultados demonstram que tecidos com maior número de divisões de células tronco apresentam um perfil de alto AC, o qual amplifica as chances de concentrar ancestrais mutados, aumentando as chances de desenvolver tumores. Assim, justificando parte da correlação encontrada por Tomasetti & Vogelstein (2015). / Cancer is characterized by an abnormal replication of somatic cells as a result of DNA alterations. Different types of tissues present differences in cancer incidence. Tomasetti & Vogelstein (2015) have shown that lifetime cancer risk of different tissues presents a strong correlation of 0.804 with the number of stem cells divisions, in which tissues with higher number of stem cells divisions are more susceptible to stochastic effects of DNA replication and thus more likely to develop cancer. Thus, the number of mutations was used to explain this correlation. In our work, we propose and test in silico a new concept to explain this positive correlation, which we denominated ancestral concentration (AC). In our hypothesis, a tissue with high proliferation rates concentrates more their ancestral cells and increases the chance of a mutated ancestral to persist; which result in a higher risk of cancer. Tissues with a high number of stem cells divisions presents a high AC profile whereas tissues with a low number of stem cells divisions presents a low AC profile To prove our hypothesis, we simulated tumor evolution using esiCancer software and applied different initial rates of proliferation and death (CTOR). We observed a positive correlation of 0.995 between CTOR values and the AC profile (P = 0:002). Besides, higher CTOR values are associated to higher mean generations with esiTumors when equal mutation rates are applied. Nevertheless, this association still exist in simulations with mutation rates corrected by total number of divisions, whereas the total mutation rate is similar for different CTORs. This way, modifications of mutations solely are not sufficient to explain the observed cancer risks in different tissues. Our results showed that tissues with higher number of stem cells divisions present a high AC profile, which rises the probabilities of concentrate mutated ancestral cells, increasing the tumor risk. In this way, justifying partly the correlation that was founded by Tomasetti & Vogelstein (2015).

Neurodegenerative diseases are highly multifaceted. Despite their heavy burden, treatment options are limited and our understanding of their molecular triggers even less so. In this thesis, I focus on the pathogenesis of Alzheimer’s Disease (AD) due to familial, sporadic and environmental causes. Previous research shows that early AD stages are characterized by upregulated functionality of mitochondria-associated endoplasmic reticulum (ER) membranes. These “MAM” domains of the ER are dynamic contacts between the ER and mitochondria distinguished by a unique lipid composition equivalent to a lipid raft. These sites cluster a specific set of metabolic enzymes that regulate cellular lipid uptake, trafficking and turnover. We find that cleavage of the amyloid precursor protein at MAM domains is intimately involved in MAM regulation through localization of its C-terminal fragment of 99 a.a., C99, to MAM regions. C99 upregulates MAM functionality by promoting cholesterol uptake and trafficking to the ER for esterification, observable in both familial and sporadic AD samples. Here, we recapitulated these phenotypes in a mouse model of an environmental AD trigger: traumatic brain injury (TBI). Through biochemical, transcriptional and lipidomic analyses, we observed MAM functionality to be upregulated following a single brain injury. This was determined by assessment of phospholipid synthesis and cholesterol esterification. This correlated with increased deposition of C99 in MAM domains as well as cell type-specific lipidomic alterations. Specifically, cholesterol esterification was predominant in microglia, triglyceride elevations were predominant in microglia and astrocytes, and polyunsaturated phospholipid elevations were predominant in neurons. We hypothesize that, in the acute phase, MAM upregulation serves to promote lipid synthesis for tissue repair. However, if these phenotypes are sustained (such as after multiple injuries), cognitive functions dependent on neuronal functionality could become compromised. Altogether, we propose that the induction of AD pathogenesis following brain injury may arise from chronic upregulation of MAM activities. This work advances our understanding of neurodegenerative disease etiology.

The human neocortex is a highly sophisticated and organized brain structure that is thought to mediate some of the most complex cognitive functions in humans including language and abstract thought. As such, environmental and genetic insults to its normal structure or function can result in devastating neurological conditions including severe epilepsy and intellectual disability. Malformations of cortical development are an increasing collection of disorders that cause neocortical abnormalities due to impaired developmental processes. One recently identified disorder in this class is a thin lissencephaly variant (TLIS) associated with several mutations in the C-terminus death domain of the caspase-2 activation adaptor CRADD (also known as RAIDD). Beyond this, little is known about the mechanism underlying TLIS pathophysiology despite an increasing number of identified individuals suffering from it. In order to better understand this disorder, as well as the normal developmental mechanisms that are impaired in its pathogenesis, I have developed and characterized three murine models by introducing one of a number of different genetic perturbations associated with TLIS. These animal models show behavioral and biochemical abnormalities similar to those seen in human TLIS subjects. Focusing future studies on the developmental processes that underlie differences seen in these mouse models could greatly inform understanding of disease mechanism in humans and assist in the development in therapeutic interventions. My work presented in this dissertation thus effectively establishes a translationally relevant animal model of TLIS.

DNA double strand breaks (DSB) are cytotoxic lesions that can lead to genome rearrangements and genomic instability, which are hallmarks of cancer. The two main DSB repair pathways are non-homologous end joining and homologous recombination (HR). While HR is generally highly accurate, it has the potential for gross chromosomal rearrangements (GCRs) that occur directly or through intermediates generated during the repair process. Whole genome sequencing of cancers has revealed numerous types of structural rearrangement signatures that are often indicative of repair mediated by sequence homology. However, it can be challenging to delineate repair mechanisms from sequence analysis of rearrangement end products from cancer genomes, or even model systems, because the same rearrangements can be generated by different pathways. Numerous studies have provided insights into the types of spontaneous GCRs that can occur in various Saccharomyces cerevisiae mutants. However, understanding the mechanism and frequency of formation of these GCR without knowledge of the initiating lesions is limited. Here, we focus on DSB-induced repair pathways that lead to GCRs. Inverted duplications occur at a surprisingly high frequency when a DSB is formed near short inverted repeats in cells deficient for the nuclease activity of Mre11. Similar to previously proposed models, the inverted duplications occur through intra-strand foldback annealing at resected inverted repeats to form a hairpin-capped chromosome that is a precursor to dicentric chromosomes. Surprisingly, we found that DNA polymerase δ proof-reading activity but not the Rad1-Rad10 nuclease is required for inverted duplication formation, suggesting a role for Pol δ in the removal of the heterologous tails formed during foldback annealing. Contrary to previous work on spontaneous inverted duplications, we find that DSB-induced inverted duplications require the Pol δ processivity subunit Pol32 and that RPA plays little role in their inhibition, suggesting that spontaneous inverted duplications arise differently than those induced by DSBs. We show that stabilization of dicentric chromosomes after breakage involves telomere capture through a strand-invasion step mediated by repeat sequences and requires Rad51. Previous work on spontaneous inverted duplications suggested that Tel1, but not Mre11-Sae2, inhibits inverted duplications that initiate from inverted repeats separated by long spacers (> 12 bp). However, we do not find evidence for this requirement. Cells with Tel1 deletion can still resolve hairpins containing loops up to 30 nt long. Furthermore, deletion of Sae2, but not Tel1, increases the frequency of inverted duplications when a DSB is induced near an inverted repeat separated by a 20 bp-long spacer. This highlights another difference between spontaneous and DSB-induced GCRs. Finally, we find that the sequence context of a DSB affects the type of GCR outcome. Inverted repeats are required for the formation of inverted duplications, as the deletion of a DSB-proximal inverted repeat significantly reduces the incidence of this type of rearrangement. Furthermore, introduction of a DSB near short telomere-like sequence is required for chromosome truncations stabilized by de novo telomere addition. The effect of the sequence context can partly explain how repair pathways can be channeled to different mutagenic outcomes. Our results highlight the importance of considering how the initiating lesion can affect the type of resulting GCRs and the mechanisms by which they occur.