Orientador: Maria Helena Stangler Kraemer / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-14T09:37:26Z (GMT). No. of bitstreams: 1 Magalhaes_RenataFerreira_D.pdf: 45600840 bytes, checksum: c3be46b9fbfab6e5433e2e91db336bb3 (MD5) Previous issue date: 2009 / Resumo: Psoríase é uma dermatose inflamatória crônica, determinada por desregulação do sistema imune e associada a várias comorbidades. O marcador genético mais associado à psoríase em todas as populações é o HLA-CW06. Polimorfismos na região promotora do TNF-a, especialmente a troca de uma guanina por uma adenina nas posições -238 e -308 estão relacionados à alta produção de TNF-a e risco aumentado para psoríase nas populações caucasóides e não em asiáticos. Com o objetivo de determinar se polimorfismos destes genes podem ser fatores de risco para susceptibilidade ou gravidade da doença em pacientes brasileiros, foi realizado um estudo caso-controle com 69 pacientes com psoríase de início até 40 anos, com acompanhamento por dez anos para caracterização de sua evolução clínica em doença leve (grupo I) e grave (grupo II), e 70 indivíduos sadios. Foi feita a identificação dos alelos HLA classe I e II e SNPs da região promotora do TNF-a -238 e -308. Coletaram-se 10 mL de sangue periférico dos indivíduos e se realizou a extração do DNA através do método salting out. O DNA foi amplificado pela reação em cadeia da polimerase (PCR), com primers sequência-específica. As freqüências alélicas e gênicas foram estimadas por contagem direta e a comparação entre as frequências dos grupos foi efetuada por Teste de Fisher (programa GraphPad InStat 3.05). Dois métodos computacionais foram usados para determinar os haplótipos dos indivíduo: (1) o algoritmo ELB implementado pelo software Arlequin 3.1 e (2) um método de base coalescente implementado pelo software PHASE v2, e as freqüências de cada haplótipo foram comparadas por Teste de Fisher. No grupo II, observou-se maior associação com fatores desencadeantes como estresse, início na adolescência e predominância do sexo masculino. Pode-se sugerir que os alelos HLA-B*37, -Cw*06, -Cw*12 e -DRB1*07 foram associados ao curso mais grave da doença, enquanto - B*57 à doença mais leve. O aielo DRBT04 teve tendência a associação negativa. Ao se comparar o grupo I com o grupo II, o alelo HLA-B*37 pode ser interpretado como fator de mau prognóstico. Não houve diferença estatística entre polimorfismos da região promotora do TNF-a entre pacientes e controles. Este estudo apontou uma alta frequência do genótipo TNF-a -238 G/G {OFt 3,21; Cl:1,06-9,71; p=0,04), assim como do alelo -238 G, no grupo com doença mais grave e, ao contrário, o genótipo -238 G/A com frequência maior no grupo de boa evolução. O haplótipo -238A-308G mostrou frequência reduzida conferindo um efeito protetor. Estes dados não correspondem ao reportado para as populações caucasianas, considerando que a população brasileira é miscigenada. Polimorfismos dos SNPs do TNF-a não parecem ser um fator de susceptibilidade genética mais importante do que o já conhecido HLA-Cw*06 em pacientes brasileiros, mas podem ter relação com as manifestações e evolução da doença. / Abstract: Psoriasis is an erythematous, scaly inflammatory dermatosis with a complex immunologic basis. The strongest genetic marker for psoriasis is HLA-Cw*06. Polymorphisms in the TNF-a promoter region, especially replacement of guanine with adenine in positions -238 and -308 are related to higher TNF-a production and higher risk for psoriasis in Caucasoid populations, not found in Asians. We did a case-control study of 69 patients with psoriasis type I and 70 controls, characterized clinical progression along 10-years of follow-up in mild or severe disease and determined HLA class I, II and TNF-a SNPs -238 and -308 polymorphisms to demonstrate whether these polymorphisms may be genetic risk for susceptibility to psoriasis or severity of the disease in Brazilians. Peripheral blood (10 ml) was collected. Genomic DNA from both psoriasis patients and controls was isolated using a salting out procedure. Polymorphisms were identified by PCR/SSP. Alleles and genotypes frequencies were compared by Fisher's test (GraphPad InStat 3.05 software). Two computational methods were used to determine the haplotypes of each subject, without taking into account any prior information: (1) the ELB algorithm implemented by the ARLEQUIN 3.1 software and (2) a coalescence based method as implemented by the PHASE v2 software. The haplotype frequencies were compared between group pairs by Fisher's test. Severe disease was found more frequently in male patients, associated with environmental factors and onset at adolescence. It may be suggested that alleles HLA- B*37, -Cw*06, -Cw*12 and -DRB1*07 were associated with severe disease course, while -B"57 with mild disease. No statistical difference was found between the patients and controls regarding polymorphisms frequencies in TNF SNPs. This study pointed to a higher TNF-238 G/G genotype frequency (OR 3,21; Cl:1,06-8,71; p=0,04) in the group with severe disease and -238A-308G haplotype was found in reduced frequencies revealing a protective effect. These data do not correspond to those reported for the Caucasian population, considering that Brazilian population is admixed, and this is the first consideration about TNF-a SNPs in psoriasis in this population. Polymorphisms in the TNF-a SNPs do not seem to be a more important genetic risk factor for psoriasis than the already known Cw*06 in Brazilian patients, but these markers may be related to clinical manifestations. / Doutorado / Clinica Medica / Doutor em Clínica Médica

Eine sichere Bindung zwischen Mutter und Kind in den ersten Lebensjahren ist prägend für die Entwicklung eines Kindes. Die Qualität dieser Bindung ist ein wichtiger Prädiktor für langfristige physische und psychische Gesundheit. Für den Aufbau einer starken Bindung ist die Investition von Ressourcen seitens der Fürsorgeperson auf zeitlicher, physischer und emotionaler Ebene notwendig. Multimodale biologische Hinweisreize seitens des Kindes fördern dieses Engagement. Zunächst dienen solche Signale der Identifikation des eigenen Nachwuchses (kin recognition), um nachfolgend gezielt Ressourcen zu investieren. Darüber hinaus können infantile Stimuli affektive Reaktionen vermitteln, die den Bindungsaufbau erleichtern. In diesem Zusammenhang sind auch olfaktorische Signale, z. B. Körpergerüche, wirksam, bislang gibt es jedoch nur wenig systematische Forschung zu ihrem Einfluss auf die Eltern-Kind-Beziehung. Einzelne Studien zeigen, dass Mütter ihre Kinder am Geruch erkennen können und dass kindliche Körpergerüche auch auf neuronaler Ebene positive Reaktionen vermitteln, wobei jedoch unklar ist, wie spezifisch die neuronale Aktivität für den Geruch des eigenen Kindes ist. In der vorliegenden Arbeit soll der Einfluss von kindlichen Körpergerüchen in der Mutter-Kind- Beziehung über die kindliche Entwicklungsspanne unter Berücksichtigung genetischer, hormoneller und neurobiologischer Faktoren untersucht werden. In Veröffentlichung 1 wurde geprüft, ob Mütter ihre Kinder am Geruch identifizieren können, ob sie diesen präferieren und wie beides mit genetischen und hormonellen Faktoren sowie dem kindlichen Entwicklungsstatus interagiert. Dafür wurden N = 164 Müttern mit ihren biologischen Kindern (N = 226 Kinder zwischen 0 und 18 Jahren) in die Studie eingeschlossen. Die Mütter bewerteten die Körpergerüche des eigenen und fremder Kinder, die sich im Entwicklungsstatus sowie der genetischen Ähnlichkeit unterschieden. Die genetische Ähnlichkeit wurde über das Humane Leukozytenantigen(HLA)-Profil abgebildet, der Entwicklungsstatus wurde anhand der Steroidhormonkonzentration (Testosteron, Estradiol) und einer standardisierten Einschätzung des pubertären Status erfasst. Es zeigte sich, dass die Mütter den Geruch ihres eigenen Kindes über dem Zufallsniveau identifizieren konnten und diesen Geruch präferierten. Dies galt für alle Altersgruppen, mit Ausnahme der frühen Pubertät. In diesem Alter (9-13 Jahre) konnten die Mütter den Geruch ihres Kindes weder identifizieren, noch bevorzugten sie ihn im Vergleich zu fremden Körpergerüchen. Bei den eigenen Söhnen war die Abnahme der Präferenz mit dem Anstieg des Testosteronlevels assoziiert. Mit zunehmendem Alter des Kindes (14-18 Jahre) ähnelte das Bewertungsverhalten der Mütter wieder dem vor Pubertätsbeginn, was vermuten lässt, dass die Mütter sich in diesem Zeitraum an den veränderten Geruch des Kindes gewöhnen und somit die Vertrautheit des Geruchs eine wichtige Rolle für die Wahrnehmung spielt. Zusätzlich legen die Ergebnisse nahe, dass genetische Ähnlichkeit über Körpergerüche transportiert wird: Der Geruch des eigenen Kindes wurde zwar global bevorzugt, im paarweisen Vergleich zeigte sich jedoch, dass sich die Bewertung für den Geruch des eigenen Kindes nicht signifikant von der Bewertung des gleichaltrigen und HLA-ähnlichen Kindes unterschied. Dies lässt darauf schließen, dass sich genetische Ähnlichkeit positiv auf die Geruchsbewertung im Kontext der Eltern-Kind-Bindung auswirkt. Für die zweite Veröffentlichung wurde anhand derselben Stichprobe getestet, ob Mütter den Entwicklungsstatus des Kindes anhand von Körpergerüchen klassifizieren können und welche Prädiktoren für die Klassifikation entscheidend sind. Dafür wurden sie gebeten, die jeweilige Altersgruppe des Kindes einzuschätzen, von dem der Geruch stammte. Die Ergebnisse demonstrieren, dass Mütter den kindlichen Entwicklungsstatus (prä- bzw. postpubertär) mit einer Genauigkeit von 64 % detektieren können und insgesamt dazu tendieren, kindliche Körpergerüche als präpubertär zu klassifizieren. Die mütterliche Klassifikationsleistung war besser, wenn die Probandinnen Geruchsproben aus der gleichen Altersgruppe wie der des eigenen Kindes beurteilten. Die subjektive Bewertung der Proben hinsichtlich Angenehmheit und Intensität sowie die Einschätzung des pubertären Status waren signifikante Prädiktoren für die entwicklungsbedingte Klassifikation eines Geruchs, während sich der Steroidhormonstatus des Kindes nicht auf die mütterliche Einschätzung auswirkte. Die dritte Veröffentlichung dieser Doktorarbeit diente als methodische Pilotstudie für die spezifische Untersuchung des Einflusses von Babygerüchen auf die neuronale Verarbeitung im mütterlichen Gehirn. Aus anderen Modalitäten ist bekannt, dass kindliche Stimuli Niedlichkeit vermitteln, welche mit belohnungsspezifischer neuronaler Aktivität einhergeht. Dies ist für Babygerüche bisher jedoch kaum erforscht. Die Präsentation von Körpergerüchen zur Ableitung neuronaler Korrelate im Rahmen von funktioneller Magnetresonanztomographie (fMRT) ist aufgrund von Stimuluseigenschaften sowie methodischen Schwierigkeiten herausfordernd. Bislang existieren nur wenige Studien zur neuronalen Verarbeitung von Körpergerüchen ohne einheitliche Konvention über eine geeignete Stimuluspräsentation. Im Rahmen dieser Doktorarbeit sollte daher ein effizientes Design entwickelt werden, welches neuronale Aktivität in Reaktion auf Babygerüche optimal abbildet. Dafür wurden zwei Stimuluspräsentationen verglichen, die sich in Art, Dauer und Frequenz unterschieden. Die kurze, kontinuierliche Reizdarbietung rief im Vergleich zu einer langen, gepulsten Präsentation global stärkere Aktivierungen hervor, weshalb diese als Design empfehlenswert ist, um robuste neuronale Korrelate zu erhalten. Allerdings zeigten sich differentielle Effekte in Abhängigkeit der Hirnregionen, weshalb je nach interessierendem Areal spezifisch zwischen Länge, Dauer und Art der Stimuluspräsentation abgewogen werden sollte. Das kurze Präsentationsdesign wurde im Rahmen der weiterführenden fMRT-Studie verwendet. Diese veranschaulichte, dass Babygerüche Belohnungsareale sowie Netzwerke aktivieren, die Angehmheit, Niedlichkeit und Motivation zur Fürsorge (Pleasure-Netzwerk) kodieren. Die Aktivierungsstärke des Netzwerks sagte dabei vorher, wie angenehm die Mütter den Geruch des eigenen Babys bewerteten. Im Gegensatz zu den Verhaltensdaten aus Veröffentlichung 1, in denen sich eine klare Präferenz für das eigene Kind zeigte, konnte kein Unterschied zwischen der neuronalen Reaktion auf den Geruch des eigenen im Vergleich zu einem fremden Baby gefunden werden. Daher gilt es, die Universalität des Babygeruchs als einen Stimulus, der Niedlichkeit vermittelt, in nachfolgenden Studien systematisch zu überprüfen. Zusammenfassend stellt diese Arbeit dar, dass kindliche Körpergerüche als Chemosignale in der Mutter-Kind-Beziehung wirken und sowohl zur Identifikation des eigenen Kindes beitragen als auch affektive Komponenten vermitteln. Außerdem wurde herausgefunden, dass Körpergerüche Informationen über genetische Ähnlichkeit und den Entwicklungsstatus des Kindes transportieren. Es bleibt offen, welche Faktoren auf molekularer Ebene tatsächlich die Veränderung des Körpergeruchs ausmachen. Chemosensorische Profilanalysen können in zukünftigen Untersuchungen Aufschluss darüber geben. Darüber hinaus sind Langzeitstudien notwendig, um die hier dargestellten assoziativen Zusammenhänge auch über den individuellen Entwicklungsverlauf abzubilden und somit Mechanismen der olfaktorisch vermittelten Eltern-Kind-Beziehung ableiten zu können. Langfristig sollen diese Informationen dazu beitragen, Strategien zur Förderung der Eltern-Kind-Beziehung zu generieren und bisher bestehende Interventionen (wie z. B. Neurofeedbacktraining) auf olfaktorische Stimuli auszuweiten.:Inhaltsverzeichnis Danksagung 4 1 Zusammenfassung 6 2 Summary 9 3 Einführung in die Thematik 12 4 Studienziele: Abgeleitete Forschungsfragen und Hypothesen 21 5 Methodik der Untersuchungen 23 6 Zusammenfassung der Ergebnisse 26 7 Diskussion und Ausblick 29 8 Literaturverzeichnis 40 Anhang I. Verzeichnis der wissenschaftlichen Veröffentlichungen, Konferenzbeiträge und andere Leistungen A Veröffentlichungen der Dissertation und dazugehörige Angaben B Weitere Veröffentlichungen während der Promotionsphase C Konferenzbeiträge und andere Leistungen während der Promotionsphase II. Letters of Acceptance III. Erklärung zur Eröffnung des Promotionsverfahrens IV. Bestätigung der Einhaltung der folgenden aktuellen gesetzlichen Vorgaben / A secure bond between mother and child in the first years of life is crucial for the development of a child. The quality of this bond is an important predictor of long-term physical and mental health. To create such a bond, the caregiving person has to invest resources at a temporal, physical and emotional level. Multimodal biological infantile cues facilitate this commitment. Initially, such signals serve to identify one's own offspring (kin recognition) in order to invest resources in a targeted manner. In addition, infantile stimuli can mediate affective reactions that support bonding. In this context, olfactory signals, e.g. body odors, are also effective, but so far there is little systematic research on their influence on the parent-child relationship. Individual studies show that mothers can recognize their children by their body odor and that infantile body odors also mediate positive reactions at the neural level, although it is unclear how specific they are for their own child. The present study investigates the influence of children ́s body odors in the mother-child relationship over the developmental span, integrating genetic, hormonal and neurobiological factors. Publication 1 addressed the question of whether mothers can identify their children by body odor, whether they prefer this odor and how it interacts with genetic, hormonal factors and the child's developmental status. For this purpose, N = 164 mothers with their biological children (N = 226 children between 0 and 18 years) were included in the study and evaluated the body odors of their own and unfamiliar children, which differed in their developmental stage and genetic similarity. Genetic similarity was mapped via the human leukocyte antigen (HLA) profile, the developmental status was determined on the basis of the steroid hormone concentration (testosterone, estradiol) and a standardized assessment of the pubertal status. The results showed that the mothers were able to identify their own child's odor above chance level and preferred this odor. This was true for all age groups with the exception of early puberty. At this age (9-13 years), mothers could neither identify the odor of their child nor preferred it to unfamiliar body odors. For the body odor ratings of their own sons, the decrease in preference was associated with an increase in testosterone level. In older children (14-18 years), maternal ratings resembled those before puberty suggesting that the mothers get used to the altered body odor of their child during this period and thus, the familiarity of the odor plays an important role for perception. In addition, the results demonstrated that genetic similarity is transported via body odors: Although the preference for the odor of one's own child was globally observed, pairwise comparisons showed that the ratings for the own child ́s odor did not differ significantly from the evaluation of a same-aged and HLA-similar child. This suggests that genetic similarity has a positive effect on odor assessment in the context of parent-child bonding. In the second publication, in the same sample it was examined whether mothers are able to classify the child's developmental status on the basis of body odors and which predictors are decisive for the classification. Therefore, the mothers were asked to assess the age group of the child who was the odor donor. The results revealed that mothers are able to detect the developmental status (pre- vs. postpubertal) with an accuracy of 64% and tend to classify body odors as prepubertal. The maternal classification performance was better when they rated odor samples from the same age group as their own child. The perceptual evaluation of the samples (pleasantness, intensity) as well as the assessed pubertal status predicted the development-related classification of an odor, while the child ́s steroid hormone concentration had no effect on it. The third publication of this doctoral thesis served as a methodical pilot study for the specific examination of the influence of baby odors on neural processing in the maternal brain. From other modalities, it is known that infantile stimuli transport cuteness leading to reward-related neural correlates. However, this has scarcely been investigated for baby odors so far. Body odor presentation in functional magnetic resonance imaging (fMRI) is challenging due to stimulus properties and methodological difficulties. To date, only a few studies exist on the neural processing of body odors without a uniform convention on a suitable stimulus presentation. The aim of this thesis was to develop an efficient design that optimally maps neural activity in response to baby body odors. For that reason, two stimulus presentations were compared which differed in presentation mode, duration and frequency. The short, continuous stimulus presentation revealed stronger global activations compared to a long, pulsed presentation, thus it is recommended as a design to obtain robust neuronal correlates. However, differential effects were observed depending on the brain regions, which is why the design should be specifically adapted to the regions of interest, and length, duration and type of stimulus presentation should be considered carefully. The short presentation design was used in the follow-up fMRI study. This illustrated that baby body odors activate reward areas and a network encoding cuteness and motivation to care (pleasure network). The recruitment of this network predicted how pleasantly mothers rated their own baby's odor. In contrast to the behavioral data from publication 1, which showed a clear preference for one's own child, the neural responses did not differ between one's own or an unfamiliar baby ́s odor. Therefore, the universality of baby odor as a stimulus conveying cuteness must be systematically examined in subsequent studies. In summary, this doctoral thesis reveals that children ́s body odors function as chemosignals in the mother-child relationship and mediate both the identification of the own child and affective components. In addition, it was observed that information about genetic similarity and the child's developmental status are transcribed in body odors. It remains to be explored which factors at the molecular level actually determine changes in body odor. Future investigations using chemosensory profile analyses may clarify this question. Beyond that, longitudinal studies are necessary in order to depict the associations presented here over the course of individual development and thus enabling the derivation of mechanisms of the olfactory mediated parent-child relationship. In the long term, this information should help to generate strategies for promoting the parent-child relationship and to extend existing interventions (such as neurofeedback training) to olfactory stimuli.:Inhaltsverzeichnis Danksagung 4 1 Zusammenfassung 6 2 Summary 9 3 Einführung in die Thematik 12 4 Studienziele: Abgeleitete Forschungsfragen und Hypothesen 21 5 Methodik der Untersuchungen 23 6 Zusammenfassung der Ergebnisse 26 7 Diskussion und Ausblick 29 8 Literaturverzeichnis 40 Anhang I. Verzeichnis der wissenschaftlichen Veröffentlichungen, Konferenzbeiträge und andere Leistungen A Veröffentlichungen der Dissertation und dazugehörige Angaben B Weitere Veröffentlichungen während der Promotionsphase C Konferenzbeiträge und andere Leistungen während der Promotionsphase II. Letters of Acceptance III. Erklärung zur Eröffnung des Promotionsverfahrens IV. Bestätigung der Einhaltung der folgenden aktuellen gesetzlichen Vorgaben

The HLA complex is the most polymorphic genetic system known in man. The frequency of the HLA class II antigens have been well studied in Caucasoids but little data is available concerning HLA antigen frequencies in Negroes. In this thesis the class II antigens, excluding HLA-DP, were studied in South African (SA) Negroes (Xhosa), Cape Coloureds ( a group of mixed racial origin) and SA Caucasoids using serological, cellular ( HTC typing) and Southern blot techniques. The results obtained for the SA Negroes were compared with those previously found in Nigerians and American Negroes. Marked differences in HLA distribution occurred between these groups, which in part may be explained by Khoisan admixture in the SA Negroes. In addition, striking frequency differences were observed between the three SA populations. For example, in the Xhosa the HLA-DR1, DR4, DR7, DRw8, DQw2, DQw3, Dw1 and Dw3 specificities were found at a significantly lower frequency, whereas HLA-DR3, DRw6 and Dw' RSH' were found at a significantly higher frequency compared with the SA Caucasoids. The frequency in the Cape Coloureds was intermediate between those of the Xhosa and Caucasoids. In the SA Negroes and Cape Coloureds, several new specificities were detected such as HLA-DRw18, DR2 LUM(CT), DRwl2x6, DRw8x14, Dw' RSH', Dw' JOH' and Dw' BME'. The HLA-DR and DQ haplotypes in significant linkage disequilibrium were similar in the three groups. However, several haplotypes with unusual DR and DQ combinations such as HLA-DRw17,DQw7; DR9, DQw2 and DR4, DQw5 were present in the SA Negroes and Cape Coloured families. Al though some of these unusual haplotypes could be explained in terms of recombination between the common haplotypes, none could be typed using a panel of well defined homozygous typing cells, suggesting that the response observed in mixed lymphocyte culture arises from separate molecular determinants. The data on HLA class II antigen frequencies presented in this thesis is essential for future studies on HLA and disease associations and for establishing population relationships. Knowledge of new HLA class II antigens in the various population groups is also important in renal transplantation as matching for HLA-DR antigens is known to improve graft survival.

The thesis consists of two parts. The first part introduces the topic of human chemical communication and reviews current evidence on individual variation in human body odour and its perception. This part is framed by sexual selection theory. In the first chapter, the concept of the theory of communication is introduced followed by a discussion on the specifics of chemical communication. Next, the formation of individually specific body odour signatures with reference to skin glands, their volatile products and the subsequent metabolization by skin microflora is described. The next chapters are dedicated to selected interindividual body odour cues such as sex and kin recognition, genetic compatibility in genes of Major Histocompatibility Complex, and health and reproductive status in a mate choice context. Furthermore, interactions between perfumes and body odours are discussed. Finally, methods of body odour sampling are introduced and a rationale behind presenting individual samples or body odour blends is discussed. The second part is comprised of six scientific papers, specifically three reviews and three empirical studies. Review papers summarize factors affecting human body odour quality with emphasis on diet and affective states. The first text shows that human body odours contain cues to...

Peptides generated from cellular protein degradation via the ubiquitin-proteasome pathway are presented on MHC class I as a means for the immune system to monitor polypeptides being synthesized by cells. For CD8 + T cells to prevent the spread of an incipient infection, it appears essential they should be able to sense foreign polypeptides being synthesized as soon as possible. A prompt detection of viral proteins is of great importance for the success of an adaptive immune response. Defective ribosomal products (DRiPs) have been postulated as a preferential source which would allow for a rapid presentation of peptides derived from the degradation of all newly synthesized proteins. Although this hypothesis is intellectually appealing there is lack of experimental data supporting a mechanism that would prioritize presentation from DRiPs. In this dissertation I describe a series of experiments that probe the DRiPs hypothesis by assessing the contribution to class I presentation of model epitopes derived from DRiPs or from functional proteins. The results show that even at the early stages after mRNA synthesis DRiPs do not account for a significant fraction of the class I presented peptides. These observations suggest that the currently widespread model whereby a mechanism exists which selectively allows for DRiPs to preferentially contribute to class I antigen presentation, is incorrect. Rather, properly folded functional proteins can significantly contribute to class I antigen presentation as they are normally turned over by the ubiquitin-proteasome pathway.

Antigen presented to CD4+ T cells by major histocompatibility complex class II molecules (MHCII) plays a key role in adaptive immunity. Antigen presentation is initiated by the proteolytic cleavage of pathogenic or self proteins and loading of resultant peptides to MHCII. The loading and exchange of peptides to MHCII is catalyzed by a nonclassical MHCII molecule, HLA-DM (DM). It is well established that DM promotes peptide exchange in vitro and in vivo. However, the mechanism of DM-catalyzed peptide association and dissociation, and how this would affect epitope selection in human responses to infectious disease remain unclear. The work presented in this thesis was directed towards the understanding of mechanism of DM-mediated peptide exchange and its role in epitope selection. In Chapter II, I measured the binding affinity, intrinsic dissociation half-life and DM-mediated dissociation half-life for a large set of peptides derived from vaccinia virus and compared these properties to the peptide-specific CD4+ T cell responses. These data indicated that DM shapes the peptide repertoire during epitope selection by favoring the presentation of peptides with greater DM-mediated kinetic stability, and DM-susceptibility is a strong and independent factor governing peptide immunogenicity. In Chapter III, I computationally simulated peptide binding competition reactions and found that DM influences the IC50 (50% inhibition concentration) of peptides based on their susceptibility to DM, which was confirmed by experimental data. Therefore, I developed a novel fluorescence polarization-based method to measure DM-susceptibility, reported as a IC50 (change in IC50 in the absence and presence of DM). Traditional assays to measure DM-susceptibility based on differential peptide dissociation rates are cumbersome because each test peptide has to be individually labeled and multiple time point samples have to be collected. However, in this method developed here only single probe peptide has to be labeled and only single reading have to be done, which allows for fast and high throughput measure of DM-susceptibility for a large set of peptides. In Chapter IV, we generated a series of peptide and MHCII mutants, and investigated their interactions with DM. We found that peptides with non-optimal P1 pocket residues exhibit low MHCII affinity, low kinetic stability and high DM-susceptibility. These changes were accompanied with conformational alterations detected by surface plasmon resonance, gel filtration, dynamic light scattering, small-angle X-ray light scattering, antibody-binding, and nuclear magnetic resonance assays. Surprisingly, all these kinetic and conformational changes could be reversed by reconstitution with a more optimal P9 pocket residue. Taken together, our data demonstrated that conformation of MHCII-peptide complex constrained by interactions throughout the peptide binding groove is a key determinant of DM-susceptibility. B cells recognizing cognate antigen on the virion can internalize and process the whole virion for antigen presentation to CD4+ T cells specific for an epitope from any of the virion proteins. In turn, the epitope-specific CD4+ T cells provide intermolecular (also known as noncognate or heterotypic) help to B cells to generate antibody responses against any protein from the whole virion. This viral intermolecular help model in which CD4+ T cells provide help to B cells with different protein specificities was established in small size influenza virus, hepatitis B virus and viral particle systems. For large and complex pathogens such as vaccinia virus and bacteria, the CD4+ T cell-B cell interaction model may be complicated because B cells might not be able to internalize the large whole pathogen. Recently, a study in mice observed that CD4+ T cell help is preferentially provided to B cells with the same protein specificity to generate antibody responses against vaccinia virus. However, for larger pathogens such as vaccinia virus and bacteria the CD4+ T cell-B cell interaction model has yet to be tested in humans. In Chapter V, I measured in 90 recently vaccinated and 7 long-term vaccinia-immunized human donors the CD4+ T cell responses and antibody responses against four vaccinia viral proteins (A27L, A33R, B5R and L1R) known to be strongly targeted by cellular and humoral responses. We found that there is no direct linkage between antibody and CD4+ T cell responses against each protein. However, the presence of immune responses against these four proteins is linked together within donors. Taken together, our data indicated that individual viral proteins are not the primary recognition unit and CD4+ T cells provide intermolecular help to B cells to generate robust antibody responses against large and complicated vaccinia virus in humans.

T cell receptors (TCR) bind to peptides from various sources on MHC (Major Histocompatibility Complex) molecules. A long-standing goal in the field is to understand the mechanisms of MHC-peptide exchange and MHC-TCR interactions. Here, I present work from three uniquely different systems that address the following: HLA-DR1 conformational stability, self-tolerant mechanisms of TCRs isolated from self-reactive TCR transgenic mice, and TCR cross-reactivity mechanisms between LCMV and VV. First, I present a crystal structure of HLA-DR1 in complex with A1L9 peptide, a peptide with two amino acid substitutions from the parental peptide. The singly substituted A1 peptide, which has a pocket 1 alanine substitution, decreases intrinsic half-life between MHC-peptide and increases susceptibility to HLA-DM mediated peptide exchange. This data agrees with previous models of HLA-DM-mediated peptide exchange in which the major determinant is located at the HLA-DR1 pocket 1. However, the L9 substituted peptide, which has a pocket 9 leucine substitution, displays the opposite phenotype: increased intrinsic half-life and decreased HLA-DM susceptibility. The crystal structure presented here shows that HLA-DR1 in complex with a doubly substituted peptide, A1L9, is in the same conformation as HLA-DR1 with the wild-type peptide, demonstrating that pocket 9 residues can rescue pocket 1 residue binding deficiencies and that HLA-DR1 stability is determined by amino acids along the peptide, not only at pocket 1. Next, I present crystal structures of two self-tolerant TCRs in complex with IAb-3K pMHC. To elucidate molecular mechanism for self-reactivity and self-tolerance, the TCRs J809.B5 and 14.C6 are compared to each other and its parental self-reactive TCR, YAe-62.8. In comparison to YAe-62.8, J809.B5 interacts with the same pMHC, but utilizes more peptide specific interactions, a mechanism that may distinguish self-reactive receptors from self-tolerant receptors. Additionally, the crystal structure of 14.C6 TCR, which bears a different CDR3α sequence from J809.B5, demonstrates that CDR3 sequences can modulate interactions of germline encoded CDR1 and CDR2 loops. Together, these results highlight that in addition to CDR3 VDJ recombination, diversity is generated in the mature TCR repertoire by differential chain pairing, either of which can affect the interactions of germline encoded CDR loops. Next, I present a detailed analysis of cross-reactive TCRs between Kb-GP34 and Kb-A11R. The mature LCMV-immune repertoire was analyzed by DNA deep sequencing of TCRβ CDR3 sequences, which led to the identification of new cross-reactive sequence motifs. Cross-reactive sequence motifs varied by each Vβ gene, suggesting a role of CDR1, CDR2, and CDR3 loop interplay in cross-reactivity. Lastly, I present the crystal structures of a GP34/A11R cross-reactive TCR in complex with both Kb-GP34 and Kb-A11R. Analysis of the crystal structures revealed that the two complexes are largely the same, despite differences in peptide sequences. Surprisingly, the TCR to peptide interactions were dominated by three out of eight peptide side-chains. Cross-reactivity between these two complexes is likely due to a large amount of interactions from TCR to MHC compared to interactions of TCR to peptide. We note two unique MHC-peptide interactions that may allow Kb to be an allele prone to cross-reactivity. The first is an interaction at the C-terminus of the A11R peptide which pulls A11R P7 asparagine away from TCR interactions. The second interaction is from an arginine at position 155, which sits at the interface between TCRα and TCRβ , and contributes the most buried surface area in the interaction interface. Because Kb’s arginine 155 is a long side chain that hydrogen bonds with the peptide backbone, and is also at the center of the TCR-peptide interface, GP34 and A11R peptide sequence differences may be occluded from TCR discrimination by Kb presentation. The data presented in this dissertation demonstrate that interactions between MHC-peptide and MHC-TCR act harmoniously and coopertively, whereby proximal interactions are affected by interactions elsewhere. While previous models of HLA-DR/HLA-DM interactions demonstrate the importance of interactions at HLA-DR1 pocket 1, I showed that pocket 9 also contributes to HLA-DR stability and therefore, HLA-DM susceptibility. I also showed that TCR CDR3 loop sequences affect germline CDR1/CDR2 loop interactions and vice versa. Lastly, I showed that allele specific MHC side chain interactions with the bound peptide influence TCR ligand binding and hence, TCR cross-reactivity.

Les molécules classiques du complexe majeur d’histocompatibilité de classe II (CMHII) sont des glycoprotéines de surface spécialisées dans la présentation de peptides, principalement dérivés de pathogènes extracellulaires, aux récepteurs des lymphocytes T CD4+ afin d’initier la réponse immunitaire adaptative. Elles sont encodées, avec celles du CMH de classe I, par les gènes les plus polymorphiques identifiés jusqu’à maintenant, avec plusieurs loci et une grande diversité allélique à chacun d’eux. De plus, le polymorphisme des gènes du CMHII n’est pas limité qu’aux séquences codantes. Il est également observé dans les promoteurs où on a démontré ses effets sur le niveau d’expression des gènes. La variation de la régulation d’un gène est considérée comme un facteur important et pour laquelle des modifications morphologiques, physiologiques et comportementales sont observées chez tous les organismes. Des séquences d’ADN répétées impliquées dans cette régulation ont été identifiées dans les régions non-codantes des génomes. D’un autre côté, la sélection par les pathogènes permettrait l’évolution et le maintien du polymorphisme des gènes du CMH chez les vertébrés. À ce sujet, plusieurs études ont montré l’implication de différents allèles du CMH dans la résistance ou la susceptibilité aux maladies. Cette étude avait pour objectifs de caractériser le polymorphisme du gène MHIIb chez l’omble de fontaine (Salvelinus fontinalis) et de documenter ses effets au niveau de la survie conférée par des allèles et/ou génotypes particuliers lors d’une infection, ainsi que sur la variation du niveau d’expression du gène dans différentes conditions. Dans une première partie, nous avons identifié un total de 6 allèles du gène MHIIb, désignés Safo-DAB*0101 à Safo-DAB*0601, qui montrent une grande similarité avec les séquences codantes provenant de poissons téléostéens et de l’humain. L’analyse des séquences du domaine b1 a permis de détecter l’effet d’une pression sélective positive pour maintenir le polymorphisme dans cette région de la molécule. Quatre de ces allèles ont été testés lors d’une expérience d’infection avec le pathogène Aeromonas salmonicida afin d’évaluer l’effet qu’ils pouvaient avoir sur la survie des poissons. Nous avons trouvé que l’allèle DAB*0101 était significativement associé à la résistance à la furonculose. En plus d’avoir été identifié chez les individus homozygotes pour cet allèle, l’effet a également été remarqué au niveau de la survie les poissons de génotype DAB*0101/*0201. À l’opposé, les facteurs de risque élevé obtenus pour les génotypes DAB*0201/*0301 et DAB*0301/*0401 suggèrent plutôt une association à la susceptibilité. Étant donné la faible fréquence à laquelle l’allèle DAB*0101 a été retrouvé dans la population, le modèle de la sélection dépendante de la fréquence pourrait expliquer l’avantage conféré par ce dernier et souligne l’importance de ce mécanisme pour le maintien du polymorphisme du gène MHIIb chez l’omble de fontaine. Dans une seconde partie, nous avons rapporté la présence d’un minisatellite polymorphique formé d’un motif de 32 nucléotides dans le second intron du gène MHIIb, et pour lequel un nombre exclusif de répétitions du motif a été associé à chaque allèle (69, 27, 20, 40, 19 et 25 répétitions pour les allèles DAB*0101 à DAB*0601 respectivement). L’expression relative de quatre allèles a été évaluée dans des poissons hétérozygotes aux températures de 6 ºC et 18 ºC. Les résultats indiquent que les allèles possédant un long minisatellite montrent une réduction de l’expression du gène d’un facteur 1,67 à 2,56 par rapport aux allèles qui en contiennent un court. De même, des allèles qui incluent des minisatellites de tailles similaires n’affichent pas de différence significative au niveau de l’abondance du transcrit aux deux températures. De plus, l’effet répressif associé aux longs minisatellites est amplifié à la température de 18 ºC dans des poissons de trois génotypes différents. Nous avons finalement observé une augmentation significative par un facteur 2,08 de l’expression totale du gène MHIIb à la température de 6 ºC. Ces résultats appuient l’implication des séquences d’ADN répétées dans la régulation de l’activité transcriptionnelle d’un gène et suggèrent qu’un minisatellite sensible aux différences de températures pourrait être soumis aux forces sélectives et jouer un rôle important dans l’expression de gènes et l’évolution des organismes poïkilothermes. / Classical major histocompatibility complex class II (MHCII) molecules are cell-surface glycoproteins specialized in the presentation of peptides, mainly derived from extracellular pathogens, to the antigen receptors of CD4+ T cells in the adaptive immune system. They are encoded, with those of the MHC class I, by the most polymorphic genes known to date, with multiple loci and high allelic diversity at each one. Moreover, the polymorphism within MHCII genes is not restricted to coding sequences. It has also been observed in promoters where it was shown to affect the expression level of the genes. Variation in gene regulation is believed to be an important factor from which modification in morphology, physiology or behaviour can be observed in all organisms. Repeated DNA sequences with functional roles in this regulation have been identified within the non-coding parts of the genomes. On the other hand, pathogen-driven selection is also believed to be important in the evolution and maintenance of the polymorphism of the MHC genes in vertebrates. Studies have shown the implication of different MHC alleles in disease resistance or susceptibility. In this study, our aims were to characterize the polymorphism of the MHIIb gene in brook charr (Salvelinus fontinalis), to document its effects on the survival conferred by specific alleles and/or genotypes following an infection and on the variation of the expression level of the gene in different environmental conditions. In a first part, we identified a total of 6 MHIIb alleles, designated Safo-DAB*0101 to Safo-DAB*0601, showing a high similarity to coding sequences from teleost fish and human. Analysis of the b1 domain sequences indicates the effect of a positive selection pressure to select polymorphic mutations in that region of the molecule. Four of these alleles were tested in a challenge experiment against the pathogen Aeromonas salmonicida to evaluate their effect on fish survival. We found that one allele, DAB*0101, was significantly associated with resistance to furonculosis. In addition to homozygotes for this allele, its resistance effect was also detected in the heterozygote individuals of the DAB*0101/*0201 genotype. In contrast, other allelic combinations, namely heterozygous genotypes DAB*0201/*0301 and DAB*0301/*0401 were significantly associated with increased susceptibility. Given that its frequency was relatively low in the population, the negative frequency dependant selection hypothesis could explain the advantage associated with the allele DAB*0101 over the other alleles and highlight the importance of this mechanism to sustain variation at the MHC in brook charr. In a second part, we reported the identification of a polymorphic minisatellite formed of a 32 nucleotides motif in the second intron of MHIIb gene, and for which distinctive repeat numbers of the motif were associated to each alleles (69, 27, 20, 40, 19 and 25 repeats for the DAB*0101 to DAB*0601 alleles respectively). Relative expression levels of four alleles were determined in heterozygous fish at temperature of 18 ºC and 6 ºC. Results indicate that alleles carrying the longest minisatellite showed a 1.67 to 2.56-fold reduction in the transcript expression relatively to the shortest one. In contrast, no significant differences were seen in the expression levels between alleles with comparable minisatellite length at both temperatures. Furthermore, the repressive activity associated to the longest minisatellite was more effective at temperature of 18 ºC in fish from three different genotypes. We finally observed a significant 2.08-fold up-regulation of the total MHII transcript amount at 6 ºC. The results support the implication of repeated DNA sequences in the regulation of the gene transcriptional activity and suggest that a temperature-sensitive minisatellite could potentially be submitted to selective forces and therefore play an important role in gene expression and evolution in ectothermic organisms.

Le système de différenciation entre le « soi » et le « non-soi » des vertébrés permet la détection et le rejet de pathogènes et de cellules allogéniques. Il requiert la surveillance de petits peptides présentés à la surface cellulaire par les molécules du complexe majeur d’histocompatibilité de classe I (CMH I). Les molécules du CMH I sont des hétérodimères composés par une chaîne lourde encodée par des gènes du CMH et une chaîne légère encodée par le gène β2-microglobuline. L’ensemble des peptides est appelé l’immunopeptidome du CMH I. Nous avons utilisé des approches en biologie de systèmes pour définir la composition et l’origine cellulaire de l’immunopeptidome du CMH I présenté par des cellules B lymphoblastoïdes dérivés de deux pairs de fratries avec un CMH I identique. Nous avons découvert que l’immunopeptidome du CMH I est spécifique à l’individu et au type cellulaire, qu’il dérive préférentiellement de transcrits abondants, est enrichi en transcrits possédant d’éléments de reconnaissance par les petits ARNs, mais qu’il ne montre aucun biais ni vers les régions génétiques invariables ni vers les régions polymorphiques. Nous avons également développé une nouvelle méthode qui combine la spectrométrie de masse, le séquençage de nouvelle génération et la bioinformatique pour l’identification à grand échelle de peptides du CMH I, dont ceux résultants de polymorphismes nucléotidiques simples non-synonymes (PNS-ns), appelés antigènes mineurs d’histocompatibilité (AMHs), qui sont les cibles de réponses allo-immunitaires. La comparaison de l’origine génomique de l’immunopeptidome de soeurs avec un CMH I identique a révélé que 0,5% des PNS-ns étaient représentés dans l’immunopeptidome et que 0,3% des peptides du CMH I seraient immunogéniques envers une des deux soeurs. En résumé, nous avons découvert des nouveaux facteurs qui modèlent l’immunopeptidome du CMH I et nous présentons une nouvelle stratégie pour l’indentification de ces peptides, laquelle pourrait accélérer énormément le développement d’immunothérapies ciblant les AMHs. / The self/nonself discrimination system of vertebrates allows detection and rejection of pathogens and allogeneic cells. It requires the surveillance of short peptides presented by major histocompatibility class I (MHC I) molecules on the cell surface. MHC I molecules are heterodimers that consist of a heavy chain produced by MHC genes and a light chain encoded by the β2-microglobulin gene. The peptides presented by MHC I molecules are collectively referred to as the MHC I immunopeptidome. We employed systems biology approaches to define the composition and cellular origin of the self MHC I immunopeptidome presented by B lymphoblastoid cells derived from two pairs of MHC-identical siblings. We found that the MHC I immunopeptidome is subject- and cell-specific, derives preferentially from abundant transcripts, is enriched in transcripts bearing microRNA response elements and shows no bias toward invariant vs. polymorphic genomic sequences. We also developed a novel personalized approach combining mass-spectrometry, next-generation sequencing and bioinformatics for high-throughput identification of MHC I peptides including those caused by nonsynonymous single nucleotide polymorphisms (ns-SNPs), termed minor histocompatibility antigens (MiHAs), which are the targets of allo-immune responses. Comparison of the genomic landscape of the immunopeptidome of MHC-identical siblings revealed that 0.5% of ns-SNPs were represented in the immunopeptidome and that 0.3% of the MHC I-peptide repertoire would be immunogenic for one of the siblings. We discovered new factors that shape the self MHC I immunopeptidome and present a novel strategy for the identification of MHC I-associated peptides that could greatly accelerate the development of MiHA-targeted immunotherapy.

No description available.