The pathological hallmark of Alzheimer's disease (AD) is the accumulation of amyloid β (Aβ) plaques, present in the majority of clinically diagnosed cases. Treatment options, such as the FDA-approved Lecanemab, target early-stage AD by promoting Aβ clearance via microglial (MG) phagocytosis, but no effective therapy exists for late-stage AD. Hyperhomocysteinemia (HHcy), prevalent in the elderly, is an established risk factor for AD, with previous studies linking it to various pathologies but not to Aβ dynamics or MG function. This study examined the hypothesis that HHcy-induced epigenetic changes impair MG clearance of Aβ, potentially exacerbating AD. It also investigated the role of IGF-1 (Insulin-like growth factor 1) in MG phagocytosis, considering the established influence of hypomethylation on both IGF-1 expression and AD progression. In this study, we probed the interplay among HHcy, Aβ accumulation, and MG phagocytosis in AD using Cbs-/- mice as a model. Our approach delineated an HHcy-induced hypomethylation status in both plasma and brain cortex. HHcy increased Aβ deposition without a corresponding rise in Aβ production in the 5xFAD x Cbs-/- mouse brains. This was characterized by elevated levels of soluble Aβ40 and insoluble Aβ42, alongside unaltered the β-secretase and s-APPβ expression. Single-nucleus multiomic profiling unveiled five distinct MG subclusters (MG_0 to MG_4) in the hippocampi of both Cbs-/- and control mice. MG_0, specific to control mice, involved activated MG phagocytosis. Subclusters MG_2 and MG_3, predominantly identified in Cbs-/- mice, exhibited decreased migration and phagocytosis. Subcluster MG_4, unique to Cbs-/- mice, displayed impaired cytoskeleton organization and dendritic development. HHcy suppressed MG Aβ phagocytosis activity in the mouse MG cell line SIM-A9 (ex vivo phagocytosis), freshly isolated MG from HHcy mice with Cbs-/- or a high-methionine (HM) diet (ex vivo phagocytosis), and in 3xTg-AD mice with an HM diet (in vivo phagocytosis). Through meta-analysis over transcriptomes of Cbs-/- mouse MG, human and mouse AD MG, Aβ phagocytosis model, and human AD methylome, we identified 5 differentially expressed genes (DEGs) (Flt1, Calponin 3, Igf1, Cacna2d4, and Celsr) in HHcy-suppressed phagocytic AD MG. All of them have been reported to regulate the migration and Aβ phagocytosis of MG and macrophages (MΦ). IGF-1 expression changes are the most significant between liver-specific Ahcy-/- (model of hypomethylation) and HHcy-altered phagocytic AD MG. In both AD and non-AD mice, the HHcy group exhibited significantly lower IGF-1 levels in the brain, plasma, and MG than the non-HHcy group. Additionally, a negative correlation was observed between Igf1 levels and MG Aβ phagocytosis. Our study underscored the critical role of HHcy in AD progression, particularly through its detrimental impact on MG Aβ phagocytosis and altered MG subclusters, leading to reduced Aβ clearance. We identified key genes, such as IGF-1, essential for MG function. These findings indicated that HHcy may exacerbate AD and potentially diminish the efficacy of treatments like Lecanemab, highlighting the importance of HHcy-targeted strategies in AD therapy. / Biomedical Sciences

Chez les mammifères, l'ADN des cellules interphasiques s'organise en une fibre chromatinienne confinée à l'intérieur de « territoires chromosomiques ». Ce confinement autorise l'établissement d'interactions à longue distance permettant une régulation fine des fonctions génomiques. Toutefois, l'organisation et la dynamique de la chromatine à l'échelle dite supranucléosomale (10 à 500 kb) reste méconnue. Afin d'étudier la chromatine à cette échelle, nous avons utilisé la méthode dite de 3C-qPCR qui permet de mesurer les fréquences d'interactions entre deux portions génomiques. Dans un premier temps, nous avons analysé les collisions aléatoires afin de déterminer l'organisation intrinsèque de la chromatine à l'échelle supranucléosomale. Nos résultats indiquent que, en l'absence d'interactions spécifiques, les collisions aléatoires dans les régions riches en gènes présentent une modulation d'une périodicité d'environ 90kb. Cette modulation semble être sous-jacente à de nombreuses interactions spécifiques et avoir des répercutions sur leur positionnement génomique contribuant ainsi à l'évolution des génomes. Des modèles, dérivés de la physique des polymères, suggèrent que la chromatine s'organise dans ces régions en une hélice statistique. Dans un second temps, nous avons abordé l'organisation tridimensionnelle du locus murin Igf2/H19 soumis à l'empreinte génomique parentale. Les interactions spécifiques identifiées entre des « enhancers » endodermiques et certaines portions du locus ont confirmé l'existence d'une hiérarchie des interactions et ont permis la découverte d'un nouveau locus soumis à l'empreinte (PIHit). Ce locus produit un ARN non codant que nous avons caractérisé mais dont la fonction exacte reste à déterminer.Finalement, mes travaux de thèse ont aussi conduit à la mise au point d'une nouvelle technologie (HRS-SEQ) qui permettra d'aborder l'organisation génomique globale par le biais des séquences récupérées à haut-sel (HRS). / In mammal, the DNA of interphasic cells is organized into the chromatin fiber which is itself confined inside “chromosome territories”. This compact organization allows the establishment of long-range interactions involved in the fine regulation of genomic processes. However, the organization and the dynamic of the chromatin at the so-called supranucleosomal scale (10 to 500kb) remain unclear. In order to study the chromatin at this scale, we used the 3C-qPCR method that allows to measure interaction frequencies between two genomic regions. Firstly, we have analyzed random collisions in order to determine the intrinsic organization of the chromatin at the supranucleosomal scale. Our data indicates that, in the absence of specific interactions, random collisions in gene-rich regions show a periodic modulation of about 90kb. This modulation seems to be underlying numerous locus-specific interactions and have repercussions on their genomic location, thus contributing to genome evolution. Models, derived from polymers physics, suggest that, in these regions, the chromatin is shaped in a statistical helix. Secondly, we have investigated the tridimensional organization of the Igf2/H19 mouse locus which is subject to genomic imprinting. Specific interactions identified between endodermic enhancers and some regions of the locus have confirmed the existence of a hierarchy of interactions and allowed the discovery of a new imprinted locus (PIHit). This locus produces a non-coding RNA that we have characterized but for which the function remains to be determined.Finally, my work also led to the development of a new technology (HRS-SEQ) that allows to study global genome organization through mapping of high-salt recovered sequences (HRS).

INTRODUÇÃO: A crescente incidência de câncer de mama, que cada vez mais acomete mulheres jovens, tem despertado muito interesse no diagnóstico precoce e na busca do tratamento mais eficaz e minimamente agressivo. Da mesma forma, vem aumentando a parcela da população com alto risco para câncer de mama, para a qual as atenções têm se voltado no sentido de se encontrar um caminho que permita prevenir o surgimento da neoplasia. Inspirados por Peyrat, que primeiro associou o câncer de mama com a presença de fator de crescimento insulina like tipo 1 (IGF-1), vários autores se lançaram nesta procura e, apesar de controversa, a literatura internacional tende a mostrar uma relação direta entre IGF-1 e câncer de mama na pré-menopausa. A proteína carreadora de IGF do tipo 3, ou IGFBP-3, também foi adicionada ao rol de sustâncias com possibilidade de promover câncer, porém, não tem demonstrado clara regularidade em seu mecanismo de ação. Tendo como alvo a população com alto risco para câncer de mama, quer histopatológico, quer familiar, e direcionada pelos cálculos matemáticos de Gail e Tyrer-Cuzick, essa tese objetivou avaliar a relação entre IGF-1 e IGFBP-3 em mulheres brasileiras atendidas pelo Setor de Mastologia da Disciplina de Ginecologia do Departamento de Obstetrícia e Ginecologia da Faculdade de Medicina da Universidade de São Paulo. MÉTODOS: estudo transversal em que foram comparados os níveis séricos de IGF-1 e IGFBP-3, dosados pelo método imunométrico quimioluminescente, em 3 diferentes grupos: mulheres com câncer de mama não tratado (n= 51), mulheres com risco populacional (n= 66) e mulheres com alto risco para câncer de mama (n= 108). Foram considerados fatores de comparação: idade, estado menopausal e índice de massa corpórea. RESULTADOS: 1) não houve diferenças entre os grupos com respeito ao IMC; 2) foi estatisticamente significativa a diferença das médias de idades dos grupos, sendo as mulheres com alto risco para câncer de mama mais jovens (p<0,001); 3) quanto ao estado menopausal, houve significativa diferença entre os grupos, com predomínio de mulheres pós-menopausa no grupo de pacientes com câncer de mama (58,8%), com p <0,001; 4) não foi encontrada relação estatisticamente significante entre percentual de IGF-1 em relação à mediana, percentual de IGFBP-3 em relação à mediana e razão dos percentuais de IGF-1 e IGFPB-3 em relação à mediana para os 3 grupos estudados;4) não houve variações nas dosagens de acordo com o tipo de situação determinante do alto risco para câncer de mama (familiar ou histopatológico); 5) não houve alteração estatisticamente significativa das variáveis estudadas, mesmo após subdivisão do grupo de alto risco (risco vitalício entre 20 e 29% X risco vitalício superior a 29%). CONCLUSÃO: Não foi observada variação dos níveis séricos de IGF-I e IGFBP-3 em mulheres com câncer de mama, com alto risco para câncer de mama ou com risco populacional / INTRODUCTION: The rising incidence of breast cancer, a disease that is increasingly affecting young women, has aroused a great deal of interest in early diagnosis and the search for a more efficient and minimally aggressive treatment. Likewise, the population at high risk for breast cancer has been growing, and it is now the focus of research efforts in the struggle to prevent neoplasia. Inspired by Peyrat, who was the first to associate breast cancer with insulin-like growth factor type 1 (IGF-1), several authors have taken on the challenge, and the international literature now leans towards a direct relationship, albeit still controversial, between IGF-1 and breast cancer in premenopause. The IGF binding protein 3 (IGFBP-3) has also been added to the list of cancer-causing agents, but its mechanism of action has not shown to be clearly regular. Targeting a population at high risk for breast cancer for histopathologic or familial reasons, and guided by the mathematical calculations of Gail and Tyrer-Cuzick, this thesis aimed at evaluating the relationship between IGF-1 and IGFB-3 in Brazilian female outpatients at the Mastology Sector of the Gynecology Discipline of the Department of Obstetrics and Gynecology of the School of Medicine of the University of São Paulo. METHODS: Transverse study to compare the serum levels of IGF-1 and IGFBP- 3 measured by the chemiluminescent immunometric method. The patients were divided into 3 different groups: women with untreated breast cancer (n=51), women with a population-based risk of breast cancer (n=66), and women at high risk for breast cancer (n=108). The comparison factors were age, menopausal state, and body mass index. RESULTS: 1) there were no differences among the groups with respect to BMI; 2) but there were statistically significant differences among the groups regarding mean age, and the younger women were those at a higher risk for breast cancer (p<0.001); 3) as for menopausal state, the groups were significantly different, and the postmenopausal women were prevalent among the patients with breast cancer (58.8%; p<0.001); 4) no statistically significant relationship was found between the IGF-1 percentage and the median, the IGFBP-3 percentage and the median, or the IGF-1 and IGFBP-3 ratio and the median in any of the groups; 5) measurements did not vary according to the determinant reason for high breast cancer risk (familial or histopathologic); 6) statistically significant changes in the variables under consideration did not take place, even after subdivision of the high-risk group (lifelong risk between 20% and 29% X lifelong risk over 29%). CONCLUSION: The serum levels of IGF-I and IGFBP-3 showed no variation among women with breast cancer, at high risk for cancer, or with populationbased risk

INTRODUÇÃO: A crescente incidência de câncer de mama, que cada vez mais acomete mulheres jovens, tem despertado muito interesse no diagnóstico precoce e na busca do tratamento mais eficaz e minimamente agressivo. Da mesma forma, vem aumentando a parcela da população com alto risco para câncer de mama, para a qual as atenções têm se voltado no sentido de se encontrar um caminho que permita prevenir o surgimento da neoplasia. Inspirados por Peyrat, que primeiro associou o câncer de mama com a presença de fator de crescimento insulina like tipo 1 (IGF-1), vários autores se lançaram nesta procura e, apesar de controversa, a literatura internacional tende a mostrar uma relação direta entre IGF-1 e câncer de mama na pré-menopausa. A proteína carreadora de IGF do tipo 3, ou IGFBP-3, também foi adicionada ao rol de sustâncias com possibilidade de promover câncer, porém, não tem demonstrado clara regularidade em seu mecanismo de ação. Tendo como alvo a população com alto risco para câncer de mama, quer histopatológico, quer familiar, e direcionada pelos cálculos matemáticos de Gail e Tyrer-Cuzick, essa tese objetivou avaliar a relação entre IGF-1 e IGFBP-3 em mulheres brasileiras atendidas pelo Setor de Mastologia da Disciplina de Ginecologia do Departamento de Obstetrícia e Ginecologia da Faculdade de Medicina da Universidade de São Paulo. MÉTODOS: estudo transversal em que foram comparados os níveis séricos de IGF-1 e IGFBP-3, dosados pelo método imunométrico quimioluminescente, em 3 diferentes grupos: mulheres com câncer de mama não tratado (n= 51), mulheres com risco populacional (n= 66) e mulheres com alto risco para câncer de mama (n= 108). Foram considerados fatores de comparação: idade, estado menopausal e índice de massa corpórea. RESULTADOS: 1) não houve diferenças entre os grupos com respeito ao IMC; 2) foi estatisticamente significativa a diferença das médias de idades dos grupos, sendo as mulheres com alto risco para câncer de mama mais jovens (p<0,001); 3) quanto ao estado menopausal, houve significativa diferença entre os grupos, com predomínio de mulheres pós-menopausa no grupo de pacientes com câncer de mama (58,8%), com p <0,001; 4) não foi encontrada relação estatisticamente significante entre percentual de IGF-1 em relação à mediana, percentual de IGFBP-3 em relação à mediana e razão dos percentuais de IGF-1 e IGFPB-3 em relação à mediana para os 3 grupos estudados;4) não houve variações nas dosagens de acordo com o tipo de situação determinante do alto risco para câncer de mama (familiar ou histopatológico); 5) não houve alteração estatisticamente significativa das variáveis estudadas, mesmo após subdivisão do grupo de alto risco (risco vitalício entre 20 e 29% X risco vitalício superior a 29%). CONCLUSÃO: Não foi observada variação dos níveis séricos de IGF-I e IGFBP-3 em mulheres com câncer de mama, com alto risco para câncer de mama ou com risco populacional / INTRODUCTION: The rising incidence of breast cancer, a disease that is increasingly affecting young women, has aroused a great deal of interest in early diagnosis and the search for a more efficient and minimally aggressive treatment. Likewise, the population at high risk for breast cancer has been growing, and it is now the focus of research efforts in the struggle to prevent neoplasia. Inspired by Peyrat, who was the first to associate breast cancer with insulin-like growth factor type 1 (IGF-1), several authors have taken on the challenge, and the international literature now leans towards a direct relationship, albeit still controversial, between IGF-1 and breast cancer in premenopause. The IGF binding protein 3 (IGFBP-3) has also been added to the list of cancer-causing agents, but its mechanism of action has not shown to be clearly regular. Targeting a population at high risk for breast cancer for histopathologic or familial reasons, and guided by the mathematical calculations of Gail and Tyrer-Cuzick, this thesis aimed at evaluating the relationship between IGF-1 and IGFB-3 in Brazilian female outpatients at the Mastology Sector of the Gynecology Discipline of the Department of Obstetrics and Gynecology of the School of Medicine of the University of São Paulo. METHODS: Transverse study to compare the serum levels of IGF-1 and IGFBP- 3 measured by the chemiluminescent immunometric method. The patients were divided into 3 different groups: women with untreated breast cancer (n=51), women with a population-based risk of breast cancer (n=66), and women at high risk for breast cancer (n=108). The comparison factors were age, menopausal state, and body mass index. RESULTS: 1) there were no differences among the groups with respect to BMI; 2) but there were statistically significant differences among the groups regarding mean age, and the younger women were those at a higher risk for breast cancer (p<0.001); 3) as for menopausal state, the groups were significantly different, and the postmenopausal women were prevalent among the patients with breast cancer (58.8%; p<0.001); 4) no statistically significant relationship was found between the IGF-1 percentage and the median, the IGFBP-3 percentage and the median, or the IGF-1 and IGFBP-3 ratio and the median in any of the groups; 5) measurements did not vary according to the determinant reason for high breast cancer risk (familial or histopathologic); 6) statistically significant changes in the variables under consideration did not take place, even after subdivision of the high-risk group (lifelong risk between 20% and 29% X lifelong risk over 29%). CONCLUSION: The serum levels of IGF-I and IGFBP-3 showed no variation among women with breast cancer, at high risk for cancer, or with populationbased risk

The main purpose of this thesis was to examine the relationship of pre-diagnostic circulating levels of sex-steroids (androgens and estrogens), sex hormone binding globuline (SHBG), insulin-like growth factor-I (IGF-I), IGF binding proteins (BP) and C-peptide (as a marker of pancreatic insulin secretion) with risk of ovarian and endometrial cancer. Additionally, the interrelationships of body mass index (BMI), sex-steroids, IGF-I and IGFBP-3 were examined. Two case-control studies were nested within 3 prospective cohort studies centered in New York (USA), Umeå (Sweden) and Milan (Italy). The ovarian study included 132 cancer cases. The endometrial study included 166 cancer cases in the IGF-I and C-peptide component and 124 postmenopausal cases in the sex-steroids component. For each case, two controls matching the case for cohort, age, menopausal status and date at recruitment were selected. In total 286 and 315 controls were included in the ovarian and endometrial cancer studies, respectively. Odds ratios (OR) and their 95% confidence intervals (CI) for cancer risk associated with increasing hormone concentrations were estimated by conditional logistic regression. The cross-sectional analysis was based on anthropometric and hormonal data from 620 controls selected for the two nested case-control studies. There was no association of prediagnostic androstenedione, testosterone, DHEAS, SHBG or estrone with ovarian cancer risk in the whole study population or in women who were pre- or postmenopausal at blood donation. In the premenopausal group, risk appeared to increase with increasing androstenedione (OR (95% CI) for the highest tertile: 2.35 (0.81-6.82), p=0.12). There was no association of IGF-I, IGFBP-1, 2, 3 or C-peptide concentrations with risk of ovarian cancer risk in the study group as a whole. In analyses restricted to subjects who had developed ovarian cancer at an early age (&lt;55), circulating IGF-I was directly and strongly associated with risk (OR (95% CI): 4.74 (1.20-18.7), p&lt;0.05 for the highest IGF-I tertile). In the endometrial study, previous observations were confimed that elevated circulating estrogens and androgens and decreased SHBG increase risk of developing endometrial malignancy after menopause. Multivariate ORs (95% CI) for endometrial cancer for quartiles with the highest hormone levels were: 4.13 (1.76-9.72), p&lt;0.001 for estradiol; 3.67 (1.71-7.88), p=0.001 for estrone; 2.15 (1.05-4.40), p&lt;0.04 for androstenedione; 1.74 (0.88-3.46), p=0.06 for testosterone; 2.90 (1.42-5.90), p&lt;0.01 for DHEAS and 0.46 (0.20-1.05), p&lt;0.01 for SHBG. Prediagnostic IGF-I, IGFBP-1, -2 and –3 were not related to risk of endometrial cancer in the whole study population. In postmenopausal women, levels of IGFBP-1 were inversely related to risk with an OR for the highest quartile of 0.36 (0.13-0.95), p&lt;0.05. Endometrial cancer risk increased with increasing levels of C-peptide (p&lt;0.01), up to an OR of 4.40 (1.65-11.7) for the highest quintile after adjustment for BMI and other confounders. The cross-sectional analyses showed that in both pre- and postmenopausal women SHBG decreased with increasing BMI. In the postmenopausal group, estrogens, testosterone and androstenedione increased with BMI, while the association with IGF-I was non-linear, the highest mean IGF-I concentration being observed in women with BMI between 24 and 25. In postmenopausal women, IGF-I was positively related to androgens, inversely correlated with SHBG, and was not correlated with estrogens. In conclusion, elevated pre-diagnostic sex-steroids, IGF-I or C-peptide increase risk of developing ovarian and endometrial cancer. BMI influences the circulating levels of these hormones, especially after menopause.

Einfluss des Wachstumsfaktors Insulin-like growth factor-I (IGF-I) auf das Follikelwachstum beim Weißbüschelaffen (Callithrix jacchus) Aus dem Veterinär-Physiologisch-Chemischen Institut der Veterinärmedizinischen Fakultät der Universität Leipzig Eingereicht im September 2010 (86 S., 16 Abb., 9 Tab., 225 Lit., 4 S. Anhang) Schlüsselwörter: Insulin-like Growth Factor-I (IGF-I), Granulosazellen, Steroidhormone (Östradiol, Progesteron), Gonadotropine (FSH, hCG) In der vorliegenden Studie wurde die Rolle von IGF-I und das Zusammenwirken mit den Gonadotropinen (FSH, hCG) auf die Sekretion der Steroidhormone (Progesteron, Östradiol) kultivierter Granulosazellen von 13 Weißbüschelaffen untersucht, um zu prüfen, ob und wie weit IGF-I die Sekretion und Reifung der Granulosazellen beeinflusst. Für die in vitro-Experimente wurden Zellkulturen mit Granulosazellen kleiner ( 0,5 - 1 mm) und präovulatorischer Follikel ( > 2 mm) von Ovarien am 7. Tag der Follikelphase verwendet. Vor jedem Versuch wurde das Wachstum der Follikel durch zwei Ultraschalluntersuchungen kontrolliert. Während der Kultur wurden drei Inkubationsintervalle von je 48 h durchgeführt. Die Zellen wurden mit IGF-I allein oder in Kombination mit FSH bzw. hCG stimuliert. Zum Teil wurden die Gonadotropine auch zur Prästimulation verwendet. Das Signifikanzniveau der Hormoneffekte lag bei p<0,05. Bei den Granulosazellen kleiner Follikel lässt sich durch die alleinige Gabe von IGF-I nur am Ende der Kultur (144 h) eine signifikante Erhöhung der Progesteronsekretion feststellen. Bei einer Kombinationsgabe von IGF-I und FSH findet sich schon am Anfang (48 h) ein signifikanter Einfluss auf die Sekretion von Progesteron und Östradiol. Bei der Progesteronsekretion ist der Effekt der Kombination signifikant höher als bei Einzelgabe beider Hormone. Dagegen ist bei der Östradiolsekretion der Effekt der Kombination zwar nicht höher als bei einer alleinigen Gabe von FSH, aber die Zellen reagieren wesentlich schneller auf IGF-I, wenn sie zusammen mit FSH stimuliert werden. Keine signifikante Wirkung in der Steroidhormonsekretion ruft die Hormonkombination IGF-I und hCG im Vergleich zur alleinigen Gabe der beiden Hormone hervor. Bei dem Vergleich beider Gonadotropine ist eine signifikante Erhöhung der Steroidhormonsekretion nur bei alleiniger Gabe von FSH zu beobachten. Bei den Experimenten mit Prästimulationen (FSH oder hCG) lässt sich nur bei der FSH-Prästimulation mit einer nachfolgenden Kombinationsgabe von hCG und IGF-I eine signifikante Erhöhung der Steroidhormonsekretion feststellen. Dies bedeutet, dass FSH die kleinen Granulosazellen auf die Wirkung von hCG sensibilisiert, wobei IGF-I diesen Vorgang unterstützt. Im Gegensatz zu den kleinen Follikeln lässt sich bei den Granulosazellen der präovulatorischen Follikel ein signifikanter Effekt von verschiedenen Hormonstimulationen schon früh beobachten. Durch alleinige IGF-I-Gabe lässt sich bereits am Anfang der Kultur (48 h) eine signifikante Erhöhung der Steroidhormonsekretion feststellen. Eine Kombinationsgabe von IGF-I und der Gonadotropine (FSH oder hCG) zeigt, dass die Kombination mit FSH zu einer signifikanten Erhöhung beider Steroide im Vergleich zur Kontrolle führt. Dagegen zeigt sich bei einer Kombination von IGF-I und hCG eine signifikante Erhöhung der Steroidhormonsekretion schon ab 48 h sowohl im Vergleich zur Kontrolle als auch zur alleinigen Gabe dieser Hormone. Bei der Untersuchung des Effekts beider Gonadotropine (FSH oder hCG) ist schon ab 48 h ein signifikanter Effekt auf beide Steroidhormone zu erkennen. Beide Gonadotropinprästimulationen (FSH oder hCG) mit nachfolgender Hormonkombination (hCG und IGF-I) führen bei den Granulosazellen der präovulatorischen Follikel zu einer signifikant geringeren Steroidhormonsekretion im Vergleich zur Gabe von hCG und IGF-I ohne Prästimulation. Die Zellen reagieren offenbar in dieser Art und Weise, um eine mögliche übermäßige Steroidgenese, und somit eine pathologische Situation, zu verhindern. Die vorliegende Arbeit zeigt, dass IGF-I bei den kleinen und präovulatorischen Follikeln unterschiedliche Wirkungen hervorruft. Es scheint, dass IGF-I die Sekretion von Progesteron und Östradiol auf unterschiedliche Art und Weise beeinflusst, und dass die Granulosazellen der Weißbüschelaffen erst während der Follikelentwicklung die Fähigkeit erwerben, auf IGFI entsprechend zu reagieren. Die Ergebnisse zeigen weiterhin, dass IGF-I bei den Granulosazellen der kleinen Follikel eine eher unterstützende Rolle für die Gonadotropine spielt, und dass IGF-I mit den Gonadotropinen bei der Reifung und der Differenzierung der Follikel mitwirkt. Möglicherweise spielt IGF-I auch während der Entwicklung und des Wachstums des präovulatorischen Follikels sowie bei der Regulierung der Progesteronsekretion eine Rolle. Die vorliegenden Ergebnisse unterstützen die Hypothese, dass IGF-I zusammen mit hCG die Zelldifferenzierung bei den Granulosazellen der präovulatorischen Follikel fördert. Außerdem kann vermutet werden, dass bei den Granulosazellen der präovulatorischen Follikel IGF-I zusammen mit FSH in unabhängiger Weise wirkt. Abschließend kann gesagt werden, dass ein Zusammenwirken zwischen den Gonadotropinen und IGF-I in Bezug auf die Bildung des präovulatorischen Follikels und die darauffolgende Ovulation existiert, dies gilt es auch bei pathologischen Situationen der Follikelreifung und Ovulation zu berücksichtigen.

Insulin like Growth Factor Binding Proteins (IGFBPs) 1 to 6 have important physiological functions of regulating half life and bioavailability of Insulin like Growth Factors (IGFs). Consequently, these have been known to play important roles in embryonic development, postnatal growth and disease conditions like cancer. However, the physiological roles of IGFBPs are diverse and not restricted only to the IGF regulation. These molecules are found to be tumor suppressors or promoters depending on the physiological contexts. IGFBP-2 has been established as a tumor promoter and found to be unregulated in several cancers including breast, ovarian, prostate cancer and glioblastoma (GBM). Various in vitro and in vivo studies have convincingly demonstrated the role of IGFBP-2 in inducing tumor cell proliferation, migration, invasion and chemoresistance. Increased plasma and tissue levels of IGFBP-2 have been associated with poor clinical outcome with respect to patients’ response to the therapy, relapse and overall survival. Various studies so far have demonstrated the role of IGFBP-2 in promoting glioma cell proliferation, migration, invasion, chemoresistance and determining stamens of GICs (Glioma Initiating Cells). However, the exact mechanisms underlying these functions remain unknown. Apart from being a diagnostic and prognostic indicator, IGFBP-2 has also been proposed as a therapeutic target. Therefore it is essential to understand mechanistic insights into pro-tumorigenic functions of IGFBP-2. Apart from the conventional function of regulating IGFs, IGFBP-2 has been shown to have several IGF independent functions. In a previous study, we reported IGFBP-2 as an upstream regulator of β-catenin signaling pathway in breast cancer. Interestingly, this study linked the association of higher expression of IGFBP-2 and β-catenin with the lymph node metastasis status of breast cancer. β-catenin signaling has been considered as one of the most important pro-tumorigenic pathways in several cancers including glioblastoma. Considering the importance of IGFBP-2 and β-catenin signaling pathways in glioblastoma, it becomes important to evaluate regulation of β-catenin activity by IGFBP-2 in glioma and address its clinical relevance. With this aim, the objectives of this study are,  To study mechanism of IGFBP-2 mediated regulation of β-catenin signaling in glioma cells and prognostic significance of IGFBP-2 and β-catenin expression in GBM tissues.  Isolation of human single chain variable fragment (scFv) against IGFBP-2 and its characterization as an inhibitor for IGFBP-2 pro-tumorigenic functions. Towards this, we established stable IGFBP-2 knockdown U251 cell line and IGFBP-2 over expressing LN229 and U87 cell lines. IGFBP-2 modulation in these glioma cell lines did not alter the rate of proliferation but there was a significant effect on cellular migration and invasion. In case of U251 cell line, there was a significant decrease in the intracellular levels of β-catenin while in IGFBP-2 over expressing cell lines there was a marked increase in intracellular β-catenin suggesting that IGFBP-2 is involved in the regulation of β-catenin in these cells. It was observed that this regulation of β-catenin was not because of its transcriptional regulation or regulation of canonical Wnt ligands Wnt1, Wnt2 and Wnt3a. To further delineate the pathway and understand the mechanism behind regulation of β-catenin, upstream regulators of β-catenin were analyzed. GSK3β is an important negative regulator of β-catenin which primes it for ubiquitination and proteasomal degradation. Phosphorylation of GSK3β at Ser9 position renders this enzyme inactive. In our study, it was observed that there was a significant downregulation of p-GSK3β in U251 cells with IGFBP-2 knockdown and upregulation in IGFBP-2 over expressing cell lines. Overexpression of IGFBP-2 in LN229 and U87 cell lines resulted in considerable decrease in the GSK3β mediated phosphorylation of β-catenin. This study unequivocally established that regulation of β-catenin by IGFBP-2 is via inactivation of GSK3β. Furthermore, regulation of GSK3β was found to be due to action of FAK following binding of IGFBP-2 to integrins. The expression pattern of IGFBP-2 and β-catenin protein in the tumor tissues of 112 GBM patients was studied and its correlation with patient survival was analysed. In this analysis it was observed that co-expression of IGFBP-2 and β-catenin is a strong predictor of patient prognosis. These results further implied the importance of understanding IGFBP-2 and β-catenin association in GBM pathology. One of the interesting observations in our study is that, not only full length IGFBP-2 protein but also C-terminal domain of IGFBP-2 was sufficient to regulate β-catenin and other IGFBP-2 mediated functions. This strongly asserts the importance of C-terminal region of IGFBP-2 as a tumor promoter. Towards an attempt to develop an inhibitor for IGFBP-2 actions, we screened a human single chain variable fragment (scFv) library using phage display technique. From this screening, one scFv (B7J) was identified which was a binder of full length IGFBP-2 as well as C-terminal domain of IGFBP-2. This scFv showed inhibition of IGFBP-2-cell surface interaction and also efficiently inhibited IGFBP-2-induced signaling pathways like ERK, FAK and GSK3β/β-catenin. B7J treatment also neutralized regulation of IGFBP-2 transcriptional targets like MMP2 and CD24. Gelatin zymography indicated the ability of B7J to decrease matrix metalloprotease activity in the conditioned medium of glioma cells. These effects ultimately reflected on the IGFBP-2-induced cellular migratory and invasive behaviour which was largely abrogated by B7J scFv treatment. Considering the therapeutic importance of scFvs because of their small size, better tumor penetration and tumor retention capacity than full length antibody molecules, such kind of strategy could be of great importance in the management of GBM. Altogether, this study provides a mechanistic insight of IGFBP-2 mediated actions involving integrin/FAK/GSK3β/β-catenin pathways and the possible role of this crosstalk in the aggressiveness of glioblastoma. This study also provides a proof of principle that an inhibitor like anti IGFBP-2 scFv could be of importance for controlling invasive glioblastoma.

Einfluss des Wachstumsfaktors Insulin-like growth factor-I (IGF-I) auf das Follikelwachstum beim Weißbüschelaffen (Callithrix jacchus) Aus dem Veterinär-Physiologisch-Chemischen Institut der Veterinärmedizinischen Fakultät der Universität Leipzig Eingereicht im September 2010 (86 S., 16 Abb., 9 Tab., 225 Lit., 4 S. Anhang) Schlüsselwörter: Insulin-like Growth Factor-I (IGF-I), Granulosazellen, Steroidhormone (Östradiol, Progesteron), Gonadotropine (FSH, hCG) In der vorliegenden Studie wurde die Rolle von IGF-I und das Zusammenwirken mit den Gonadotropinen (FSH, hCG) auf die Sekretion der Steroidhormone (Progesteron, Östradiol) kultivierter Granulosazellen von 13 Weißbüschelaffen untersucht, um zu prüfen, ob und wie weit IGF-I die Sekretion und Reifung der Granulosazellen beeinflusst. Für die in vitro-Experimente wurden Zellkulturen mit Granulosazellen kleiner ( 0,5 - 1 mm) und präovulatorischer Follikel ( > 2 mm) von Ovarien am 7. Tag der Follikelphase verwendet. Vor jedem Versuch wurde das Wachstum der Follikel durch zwei Ultraschalluntersuchungen kontrolliert. Während der Kultur wurden drei Inkubationsintervalle von je 48 h durchgeführt. Die Zellen wurden mit IGF-I allein oder in Kombination mit FSH bzw. hCG stimuliert. Zum Teil wurden die Gonadotropine auch zur Prästimulation verwendet. Das Signifikanzniveau der Hormoneffekte lag bei p<0,05. Bei den Granulosazellen kleiner Follikel lässt sich durch die alleinige Gabe von IGF-I nur am Ende der Kultur (144 h) eine signifikante Erhöhung der Progesteronsekretion feststellen. Bei einer Kombinationsgabe von IGF-I und FSH findet sich schon am Anfang (48 h) ein signifikanter Einfluss auf die Sekretion von Progesteron und Östradiol. Bei der Progesteronsekretion ist der Effekt der Kombination signifikant höher als bei Einzelgabe beider Hormone. Dagegen ist bei der Östradiolsekretion der Effekt der Kombination zwar nicht höher als bei einer alleinigen Gabe von FSH, aber die Zellen reagieren wesentlich schneller auf IGF-I, wenn sie zusammen mit FSH stimuliert werden. Keine signifikante Wirkung in der Steroidhormonsekretion ruft die Hormonkombination IGF-I und hCG im Vergleich zur alleinigen Gabe der beiden Hormone hervor. Bei dem Vergleich beider Gonadotropine ist eine signifikante Erhöhung der Steroidhormonsekretion nur bei alleiniger Gabe von FSH zu beobachten. Bei den Experimenten mit Prästimulationen (FSH oder hCG) lässt sich nur bei der FSH-Prästimulation mit einer nachfolgenden Kombinationsgabe von hCG und IGF-I eine signifikante Erhöhung der Steroidhormonsekretion feststellen. Dies bedeutet, dass FSH die kleinen Granulosazellen auf die Wirkung von hCG sensibilisiert, wobei IGF-I diesen Vorgang unterstützt. Im Gegensatz zu den kleinen Follikeln lässt sich bei den Granulosazellen der präovulatorischen Follikel ein signifikanter Effekt von verschiedenen Hormonstimulationen schon früh beobachten. Durch alleinige IGF-I-Gabe lässt sich bereits am Anfang der Kultur (48 h) eine signifikante Erhöhung der Steroidhormonsekretion feststellen. Eine Kombinationsgabe von IGF-I und der Gonadotropine (FSH oder hCG) zeigt, dass die Kombination mit FSH zu einer signifikanten Erhöhung beider Steroide im Vergleich zur Kontrolle führt. Dagegen zeigt sich bei einer Kombination von IGF-I und hCG eine signifikante Erhöhung der Steroidhormonsekretion schon ab 48 h sowohl im Vergleich zur Kontrolle als auch zur alleinigen Gabe dieser Hormone. Bei der Untersuchung des Effekts beider Gonadotropine (FSH oder hCG) ist schon ab 48 h ein signifikanter Effekt auf beide Steroidhormone zu erkennen. Beide Gonadotropinprästimulationen (FSH oder hCG) mit nachfolgender Hormonkombination (hCG und IGF-I) führen bei den Granulosazellen der präovulatorischen Follikel zu einer signifikant geringeren Steroidhormonsekretion im Vergleich zur Gabe von hCG und IGF-I ohne Prästimulation. Die Zellen reagieren offenbar in dieser Art und Weise, um eine mögliche übermäßige Steroidgenese, und somit eine pathologische Situation, zu verhindern. Die vorliegende Arbeit zeigt, dass IGF-I bei den kleinen und präovulatorischen Follikeln unterschiedliche Wirkungen hervorruft. Es scheint, dass IGF-I die Sekretion von Progesteron und Östradiol auf unterschiedliche Art und Weise beeinflusst, und dass die Granulosazellen der Weißbüschelaffen erst während der Follikelentwicklung die Fähigkeit erwerben, auf IGFI entsprechend zu reagieren. Die Ergebnisse zeigen weiterhin, dass IGF-I bei den Granulosazellen der kleinen Follikel eine eher unterstützende Rolle für die Gonadotropine spielt, und dass IGF-I mit den Gonadotropinen bei der Reifung und der Differenzierung der Follikel mitwirkt. Möglicherweise spielt IGF-I auch während der Entwicklung und des Wachstums des präovulatorischen Follikels sowie bei der Regulierung der Progesteronsekretion eine Rolle. Die vorliegenden Ergebnisse unterstützen die Hypothese, dass IGF-I zusammen mit hCG die Zelldifferenzierung bei den Granulosazellen der präovulatorischen Follikel fördert. Außerdem kann vermutet werden, dass bei den Granulosazellen der präovulatorischen Follikel IGF-I zusammen mit FSH in unabhängiger Weise wirkt. Abschließend kann gesagt werden, dass ein Zusammenwirken zwischen den Gonadotropinen und IGF-I in Bezug auf die Bildung des präovulatorischen Follikels und die darauffolgende Ovulation existiert, dies gilt es auch bei pathologischen Situationen der Follikelreifung und Ovulation zu berücksichtigen.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Leishmaniose visceral (LV) é uma doença causada pelo protozoário Leishmania (Leishmania) infantum nas Américas que acomete células do sistema fagocítico mononuclear. Na doença ativa, ocorre redução nos níveis de lipoproteínas de alta densidade (HDL) e aumento nos níveis de colesterol total e triglicérides, sendo que a progressão da doença pode estar relacionada com essas alterações no nível de lipoproteínas. Desta forma, neste trabalho avaliamos: o efeito das lipoproteínas de muita baixa densidade (VLDL) e HDL no parasitismo de células de linhagem monocítica humana (THP-1) por L. (L.) infantum, a atividade da arginase e a expressão do mRNA do fator de crescimento insulina símile-I (\"insulin-like growth factor-I\" = IGF-I) e seu receptor (\"insulin-like growth factor-I receptor\" = IGF-IR). Células THP-1 foram infectadas por 6 h com promastigotas de L. (L.) infantum, na presença de 0,5% de soro com baixa concentração lipídica (infranadante) e na presença ou ausência das frações lipoprotéicas em diferentes concentrações. As células foram lavadas e mantidas depois em meio de cultura acrescido com infranadante por 24, 48 e 72 h. Quando as frações de VLDL e HDL foram adicionadas separadamente durante a incubação inicial o parasitismo aumentou em relação ao controle. Quando as frações de VLDL e HDL foram adicionadas concomitantemente houve diminuição do parasitismo em relação ao controle, mas aumento inesperado da atividade da arginase, nos períodos de 24 e 48 h. A expressão do mRNA de IGF-I e IGF-IR mostrou uma diminuição nas células infectadas na presença e ausência das frações em relação às células não infectadas. Os resultados obtidos sugerem um papel importante de VLDL e HDL na infecção de células THP-1 por L. (L.) infantum. / Visceral leishmaniasis (VL) is a disease caused by the protozoan Leishmania (Leishmania) infantum in Americas that affects cells of the mononuclear phagocytic system. During active disease reduction in high density lipoprotein (HDL) and increase in total cholesterol and triglyceride levels are observed. Thus in this study we evaluated the effect of very low density lipoprotein (VLDL) and HDL on the parasitism of cells of human monocytic line (THP-1) by L. (L.) infantum, the arginase activity and insulin-like growth factor-I (IGF-I) and insulin-like growth factor-I receptor (IGF-IR) mRNA expressions. THP-1 cells were infected for 6 h with L. (L.) infantum promastigotes in the presence of 0.5% low lipid concentration serum (infranatant) in the presence or absence of lipoprotein fractions at different concentrations. The cells were washed and then maintained in medium with infranatant for 24, 48 and 72 h. When VLDL and HDL were added separately the parasitism increased when compared with the control. When VLDL and HDL were added concomitantly the parasitism decreased in relation to the control but with unexpected increase in arginase activity. The evaluation of IGF-I and IGF-IR mRNA expression showed a decrease in the cells infected in the presence and absence of the fractions comparing with the uninfected cells. The results suggest an important effect of VLDL and HDL in the infection of THP-1 cells by L. (L.) infantum.