Estima-se que a prevalência global da população mundial com hepatite C é de 3%. Pouco se sabe sobre a resposta ao tratamento com respeito à resistência viral. Algumas mutações no fragmento de 109 aminoácidos da NS5B são associadas com resistência ao interferon (IFN) e ribavirina (RBV). Estudos moleculares e clínicos identificaram fatores associados com o hospedeiro e vírus relacionados associada com a resposta ao tratamento, tal como o gene que codifica a IL-28B. Este estudo foi dividido em duas fases, cujos objetivos foram caracterizar a frequência de mutações que conferem resistência ao HCV e avaliar a relevância das mutações em pacientes Respondedores (R) ou Não Respondedores (NR) ao tratamento e caracterizar geneticamente as populações sobre polimorfismos genéticos nos SNPs da IL-28B em relação ao prognóstico da resposta ao tratamento. As amostras dos pacientes foram submetidas a testes de genotipagem e carga viral. As sequências geradas foram comparadas no BLAST e no banco de dados Los Alamos HCV. Realizamos o alinhamento das sequências homólogas e as mutações identificadas. Com base no genótipo e carga viral determinamos a classificação dos pacientes de acordo com a resposta à terapia. O DNA genômico foi isolado a partir de sangue periférico para a realização da tipagem de SNPs de IL-28B. A metodologia utilizada foi de PCR em tempo real utilizando sondas TaqMan SNP específico. A análise dos dados foi realizada utilizando GraphPad Prism com qui-quadrado, risco relativo (RR), Odds Ratio (OR) e intervalo de confiança de 95%, com um nível de significância de P <0,05. Foi encontrado na primeira fase deste estudo uma taxa significativa mutações associadas ao tratamento nas amostras estudadas. A prevalência de mutações associadas à resistência ao IFN e RBV bem como a novos medicamentos antivirais localizados no fragmento de 109 aminoácidos da NS5B foi examinado em 69 indivíduos infectados naïve no Rio de Janeiro, Brasil. Na segunda fase, as mutações foram clinicamente relevantes. Desde então, procuramos observar as diferenças entre melhor ou pior prognóstico de acordo com a imunogenética que mostrou diferenciação entre os grupos R e NR ao tratamento em relação ao prognóstico da resposta terapêutica. Quando as diferenças entre as sequências da NS5B e a resposta ao tratamento foram consideradas verificou-se que associada a mutação R254K, estava a C316N que poderia conduzir a uma não resposta à terapia no genótipo 1b. Os nossos dados também suportaram forte associação de IL-28B rs12979860, com elevada probabilidade de resposta à terapia de IFN + RBV. Nossos dados evidenciam a presença de pacientes virgens de tratamento que abrigam mutações de resistência previamente descritas na literatura. A análise dos fatores preditores de resposta virológica mostrou que a predição de boa resposta ou não ao tratamento e ainda da progressão da doença é dependente de uma importante interação entre a genética viral e a do hospedeiro. Fato este importante para que no momento de avaliação de diagnóstico e conduta terapêutica, o médico possa tomar medidas apropriadas para o tratamento de cada paciente individualmente independentemente do genótipo do HCV em questão. / It is estimated that the overall prevalence of the average world population with hepatitis C is 3%. Little is known about the treatment response with respect to viral resistance. Some mutations in the 109-aminoacid fragment of NS5B are associated to Interferon (IFN) and Ribavirin (RBV) resistance. Molecular and clinical studies have identified factors associated with the host and related viruses associated with response to treatment, as the gene encoding IL-28B. This study was divided into two phases whose objectives were to characterize the frequency of mutations conferring resistance to HCV viral evaluating the relevance of these in Responders (R) or Non-Responders (NR) patients to treatment and to characterize genetically the populations regarding genetic polymorphisms SNPs IL-28B in relation to prognosis of response to treatment for HCV. Patient samples were subjected to tests for genotyping and viral load. The sequences generated were compared in the BLAST and the Los Alamos database HCV. We conducted the alignment of homologous sequences and mutations identified. Based on virological parameters genotype and viral load determined the classification of patients according to response to therapy. Genomic DNA was isolated from peripheral blood for carrying out the typing of SNPs of IL-28B. The methodology used was real-time PCR using TaqMan probes specific SNPs. Data analysis was performed using GraphPad Prism with chi-square, relative risk (RR), Odds Ratio (OR) and confidence interval of 95% with a significance level of P <0.05. To study these biological parameters we associated the responsive patients, non-responders, the viral load, genotype, and IL-28B polymorphism to treatment outcome. We found in the first phase of this study a significant rate of treatment-associated mutations in the samples studied. The prevalence of mutations associated to resistance to interferon and ribavirin (IFN/RBV) as well new antiviral drugs located in the 109 aminoacid fragment of NS5B was examined in 69 Hepatitis C Virus drug naïve (HCV)-infected individuals in Rio de Janeiro, Brazil. In the second phase, the mutations revealed clinically relevant from the gene in question. Since then, we seek to observe the differences between better or worse prognosis according to immunogenetic showed that differentiation between the immunogenetics of the groups R and NR to treatment in relation to prognosis of therapeutic response. When the differences between the NS5B sequences at baseline and the treatment response were considered we found that R254K associated with C316N mutations could lead to a non-response to IFN-RBV therapy in genotype 1b. Our data also strong support the association of rs12979860 IL-28B polymorphism with high probability of response to IFN + RBV therapy. Our data highlight the presence of HCV genotypes from drug naïve patients harboring resistance mutations previously described in literature. The analysis of predictors virologic response demonstrated that the prediction of better or worse therapy response and further the disease progression is dependent of a significant interaction between viral and host genetics. This fact is important for diagnosis evaluation and clinical therapeutic, the medico can take appropriate measures to treat each individual patient irrespective of the genotype of HCV in question.

A deficiência da proteína C5 do sistema complemento humano é rara com 38 casos relatados na literatura e freqüentemente associada a severas infecções provocadas por bactérias Neisseria. O objetivo do trabalho é caracterizar imunológica e geneticamente esta deficiência encontrada pela primeira vez em brasileiros. Por imunodifusão dupla obtivemos níveis expressivos de C3, C4, C6, C7, C8, C9, Fator B, Fator H e Fator I em todos os membros desta família, a proteína C5 não foi detectada no soro de três irmãos: II:9, II:4 e II:5. Por ELISA a concentração de C5 nestes indivíduos foi (0,9; 1,0; 1,3 µg/ml, 45- 190 µg/ml). Soros destes probandos não apresentam atividade hemolítica mediada pelo sistema complemento. O cDNA de C5 dos indivíduos I:1, I:2, II:4 e II:9 apresenta a deleção do éxon 30. Causada pela substituição de GAG4028 por GAA4028 no último nucleotídeo deste éxon que leva a um erro no splicing. Este defeito provavelmente produz uma proteína incompleta e destinada à degradação. / The deficiency of the C5 component of the complement system is rare with 38 described cases in the literature. This deficiency is frequently associated with severe infections, especially caused by Neisseria. Our objective is to characterize immunologically and genetically this deficiency, the first of its type described in the Brazilian population.We noted that C3, C4, C6, C7, C8, C9, Factor B, Factor H and Factor I have expressive levels in all the individuals sera of this family. C5 was absent in individuals II:4, II:5 and II:9. By ELISA a C5 concentration in this individuals were 0,9; 1,0; 1,3 µg/ml (normal: 45 - 190 µg/ml). Their serum doesn´t present hemolytic activity mediated by complement system. The C5 cDNA from individuals I:1, I:2, II:4 and II:9 has éxon 30 deleted. Caused by the substitution of GAG4028 for a GAA4028 in the last codon of exon 30. This defect was responsible for the deficiency of C5 in this family and this deletion would probably produce an unstable protein destined for degradation.

A deficiência de CD40 Ligante (CD40L) ou síndrome de Hiper-IgM ligada ao X (X-HIGM) é considerada uma imunodeficiência primária combinada de células T e B. O CD40L é expresso na superfície de linfócitos T ativados e interage com o CD40 expresso na superfície de linfócitos B, macrófagos, células dendríticas, células endoteliais e neutrófilos. A interação CD40L-CD40 transmite sinais que induzem ativação, diferenciação e proliferação celular. Nosso objetivo foi analisar as alterações genético-moleculares da molécula CD40L que acometeram indivíduos de 5 famílias brasileiras, ocasionando X-HIGM. Genotipamos 25 indivíduos, sendo 6 pacientes com X-HIGM, 13 parentes relacionados heterozigotos e 6 homozigotos sadios. Dentre os pacientes com X-HIGM dois eram de origem caucasóide e 4 eram mestiços. A idade dos pacientes variou de 2 a 20 anos e o quadro clínico de infecções de repetição teve início em média nos primeiros 4 meses de vida. As principais infecções recorrentes manifestadas pelos pacientes foram pneumonia e otite. O paciente TB apresentou blastomicose, observação original nesta imunodeficiência. A análise genético-molecular foi heterogênea. No paciente TB foi detectado um defeito de splicing levando a deleção do exon 3 (r.345_402del do gene CD40L (CD40LG) no paciente FS uma nova substituição missense g.11856 G>C (c.476 G>C, pW140C), no paciente KC uma substituição nonsense g.11855 G>A (c.475G>A, p. W140X), e nos pacientes CH, FE e VIC uma deleção g. 3074_3077delTAGA, levando a alteração no processamento do RNA. A fenotipagem dos leucócitos demonstrou que a contagem de linfócitos T auxiliares (CD3+CD4+), linfócitos citotóxicos (CD3+CD8+), linfócitos B (CD19+CD40+) e linfócitos T ativados (CD3+CD69+) dos pacientes foram similares aos controles sadios. Contudo, foi observada uma redução significativa nos níveis de expressão de CD40L na superfície de linfócitos CD3+ e CD4+ dos pacientes. A análise dos linfócitos T por microscopia confocal revelou que as células dos homozigotos com expressão residual do CD40L em sua superfície também apresentam redução na densidade da expressão da molécula CD3, sugerindo a necessidade da integridade molecular do CD40L para a expressão normal do CD3. Concluímos que mutações no CD40L que levam à síndrome de X-HIGM são heterogêneas e a análise genético-molecular permitiu um diagnóstico preciso tornando possível o aconselhamento genético e a triagem dos recém-nascidos das famílias avaliadas. / CD40-Ligand (CD40L) deficiency or X linked Hyper-IgM syndrome (X-HIGM) is considered a T and B cell combined primary immunodeficiency. CD40L is expressed on the cell surface of activated T lymphocytes and interacts with CD40, expressed on the surface of B lymphocytes, macrophages, dendritic cells, endothelial cells, and neutrophils. The CD40L-CD40 interaction induces activation, differentiation, and cell proliferation. Our aim was to analyze the molecular-genetic alterations of CD40L molecule affecting individuals of 5 brazilian families, leading to X-HIGM. We genotyped 25 individuals, whom 6 were X-HIGM patients, 13 were heterozygote related patients, and 6 were healthy homozygotes. Within the patients with X-HIGM, two of them were of caucasoid origin and four were mestiços. The patients age ranged from 2 to 20 years and their recurrent infections started in average during their first 4 months of life. The main recurrent infections were pneumonia and otitis. The patient TB presented blastomycosis, a unique observation in this immunodeficiency. The molecular-genetic analysis revealed heterogeneity. TB patient presented a splicing defect causing a deletion of exon 3 (r.345_402del) of CD40L gene (CD40LG). Patient FS presented a new missense mutation g.11856 G>C (c.476 G>C, pW140C). Patient KC presented a nonsense substitution g.11855 G>A (c.475G>A, p. W140X). Patients CH, VIC, and FE presented a deletion g. 3074_3077delTAGA, causing an alteration on RNA processing. The leukocytes fenotyping demonstrated that T helper lymphocytes (CD3+CD4+), T cytotoxic lymphocytes (CD3+CD8+), B lymphocytes (CD19+CD40+), and T activated (CD3+CD69+) cell counts of patients were similar to healthy controls. However it was observed a significant reduction of CD40L expression on cell surface patients CD3+ and CD4+ lymphocytes. The T lymphocyte confocal microscopy analysis revealed that homozygotes with residual expression of CD40L in their surface also presented a reduction on the density of CD3 molecule expression, suggesting the need of molecular integrity of CD40L for normal CD3 expression. We conclude that mutations on CD40L leading to X-HIGM syndrome are heterogeneous and the molecular-genetic analysis allowed a precise diagnosis making possible the genetic counseling and newborn screening of the involved families.

HIV-1 is the causative agent for AIDS and has been a major focus of research for the past two decades. Though there is a combination therapy in place known as the “Highly Active Anti-Retroviral Therapy” (HAART), its usefulness is confounded by the generation of escape mutants, a host of side effects, and its prohibitive cost. The most useful alternative would be the prevention of infection by vaccination. Vaccine research has been focused on the use of recombinant protein sub-units of the virus or combinations thereof to elicit a neutralizing response against the virus. These approaches have mostly resulted in a failure to generate broadly cross reactive neutralizing response against primary strains of the virus. The work reported herein is aimed at designing a rigidified version of gp120/gp120 derivatives and understanding the scope of the various antigenic regions in gp120 in generating a neutralization response. Chapter one discusses some general features of the virus and the immune system. The general nature of AIDS, its spread and its immunological characteristics are also described in this chapter. Chapter two discusses the design and NMR structural analysis of gp120 bridging sheet peptide mimics in methanol and water. The structure of gp120 can be loosely divided into two domains (the outer domain and the inner domain) that are linked together by a discontinuous four stranded antiparallel beta sheet known as the bridging sheet. The bridging sheet is known to overlap with the coreceptor binding site of gp120 and hence is a suitable target for designing virus-entry inhibitors. 17b, a neutralizing antibody isolated from an infected individual, is known to bind to this region of gp120. Our aim in this part of the work was to design a four stranded antiparallel beta sheet, based on the sequence of the bridging sheet, that would contain most of the residues involved in 17b binding. NMR and CD studies confirmed that the peptide was well structured in methanol but the structure was largely lost on addition of aqueous solvent. A small population of the peptide was found to be well-folded in aqueous solution. Chapter three discusses the design and characterization of a gp120-CD4D12 single chain. It is well known that the conformation of gp120 changes upon binding CD4 to expose cryptic epitopes, known as CD4i epitopes. In this work we report the generation of a single chain gp120-CD4 construct that has the cryptic epitopes exposed. The construct bound to 17b, a conformation specific antibody against the bridging sheet of gp120, a cryptic epitope, as well as a non-covalent complex of gp120:CD4D12. There was also very insignificant secondary structural change in gp120 upon complex formation with CD4D12 as observed by CD spectroscopy. Immunological studies with DNA and protein vaccination in guinea-pigs indicated that though 17b like antibodies are generated after immunization, they did not contribute towards the neutralization of primary isolates of the virus. It was also observed that it was the anti-CD4D12 antibodies that were responsible for the neutralization by the sera. These studies indicated towards the inability of the bridging sheet to generate effective neutralization response in case of vaccination with gp120/CD4 complexes. Chapter four discusses the design of a mimic of the gp120/CD4 complex. Since it was seen from our previous work that gp120/CD4 complexes generate a large fraction of antiCD4 antibodies and hence are unsuitable for vaccination purposes, we generated a construct with the minimal binding region of CD4. The small fragment of CD4 spanning from 21st residue to 64th residue was inserted in the V1/V2 loop of gp120. The insertion site was designed based on the region of gp120 closest to this fragment and capable of tolerating insertions. This protein did not bind to 17b as well as gp120/CD4 complex but showed a higher binding compared to full length gp120. Further immunological characterization with this protein revealed that it was not capable of generating neutralizing antibodies against the virus. Chapter five discusses the design and execution of a SPR based solution phase competition experiment to find the solution phase binding constant of CD4 and CD4 analogs to gp120. A major problem during the analysis of binding data obtained by SPR is the accurate determination of Rmax, a parameter needed to obtain an accurate equilibrium dissociation constant. In this chapter we have developed a binary as well as a ternary solution phase SPR based assay to accurately determine a solution phase equilibrium binding constant. The binding constants were determined for gp120 binding to CD4D12 and other CD4 analogs. To confirm the validity of the assay, a control antigen:antibody interaction whose equilibrium dissociation constant has been determined by other methods has been used as a test case. Chapter six discusses the design and characterization of V3 peptides inserted in the loop regions of E. coli Thioredoxin (Trx). Trx has earlier been used to display random peptide libraries between the 33rd and the 34th residue. We have constructed three constructs where the peptide has been inserted between the 33rd and 34th residue, between the 74th and 75th residue and between the 84th and 85th residue. The insertion between 74th and 75th position (74V3Trx) was found to be superior to the other two and would be a suitable alternative for display of a random peptide library. The binding of these constructs to 447-52D, a V3 peptide specific antibody was characterized. These were also characterized immunologically, and 74V3Trx was found to generate weakly neutralizing activity against the MN strain of HIV-1. Competition experiments with 447-52D with these sera indicated that there were antibodies generated that could compete out 447-52D binding to gp120 but not in sufficient concentration to provide broad neutralization. Appendix 1 discusses the rational design of disulfides to stabilize proteins based on the analysis of naturally occurring disulfides. In our attempts to design a rigidified version of gp120 we had designed disulfides in gp120 based on its crystal structure. Many of these were disulfides that would span antiparallel adjacent strands. In order to improve the design principles, we analyzed naturally occurring disulfides that span antiparallel adjacent strands and characterized them in terms of their positional preference in a beta sheet. It was found that these disulfides mostly occur on edge strands and are found exclusively between non-hydrogen bonded registered pairs of adjacent antiparallel strands. Mutagenesis on Thioredoxin was performed to verify our results. It was found that disulfides designed between the non-hydrogen bonded pairs of antiparallel strands could significantly stabilize the protein whereas the ones between hydrogen bonded pairs destabilized the protein.

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

Para atestar a imunogenicidade de vacinas contra raiva, os testes de titulação e de soroneutralização de vírus rábico em células BHK-21 foram validados quanto à linearidade, precisão, exatidão e robustez. Cinco esquemas de imunização com diferentes concentrações, vias de inoculação e intervalos entre as doses foram avaliados em camundongos com vacina contra raiva em células Vero. O grupo II que recebeu um esquema de 2 doses e intervalos maiores (0 e 60 dias) apresentou títulos de anticorpos neutralizantes [ACN] mais elevados (46,1 UI/mL) do que o Grupo I (19,4 UI/mL), de 3 doses e intervalos menores (0,7 e 28 dias). O grupo III, que recebeu 2 doses, sendo a 1ª diluída 1/10, apresentou ACN semelhantes ao grupo II (39,2 UI/mL), sendo igualmente eficaz. Nenhum animal do grupo IV, imunizado por via oral (VO) apresentou ACN, indicando supressão e todos os do grupo V, imunizados VO com antígeno adsorvido/encapsulado em sílica nanoestruturada SBA-15, apresentaram títulos de ACN detectáveis, mostrando que o antígeno foi protegido da degradação no trato gastrointestinal. / To assure the reliability of the results of immunogenicity of rabies vaccines, tests of virus titration and neutralization in BHK-21 cells were validated for linearity, precision, accuracy and robustness. Five schemes of immunization with different concentrations, routes of inoculation and intervals between doses were evaluated in mice with rabies vaccine in Vero cells. Group II, immunized with 2 doses and enlarged interval (0 and 60 days) presented higher levels of neutralizing antibody (NAs) (46,1 IU/mL) than group I (19,4 IU/mL), with 3 doses and shorter intervals (0,7 and 28 days). Group III, which received 2 doses, the 1st diluted to 1/10, presented results similar to group II (39,2 IU/mL). None mouse of Group IV, immunized by oral route, presented NAs, indicating suppression and all mice of group V, immunized by oral route with vaccine adsorbed/encapsulated in nanostructured SBA-15 silica presented detectable NAs titers, showing that the SBA-15 silica prevented the antigen degradation in the gastrointestinal tract.

Environmental stress has shown to affect both the exposed individuals and the development of their offspring. Generally, it is thought that the stressed organism responds to stress by trying to adapt to it. This thesis investigates possible evolutionary consequences of cross-generational transmissions of stress, where the parent has been stressed but the offspring has not. In two studies we have exposed chicken parents of different breeds to an unpredictable circadian light rhythm, to investigate the influence of genetic background on the transmission of behaviour and patterns of genome-wide gene expression across generations. In Paper I, we can show that the domesticated chicken, by means of epigenetic factors, transmit their behaviours as well as their gene expression profiles to their offspring to a higher extent than their wild ancestor, the red junglefowl. Furthermore, in Paper II, even though the offspring never experienced the stress or had any contact with their stressed parents, they seemed to have adapted to it, which suggests that the parents might have prepared (or pre-adapted) them for living in the unpredictable environment. Additionally, eggs of stressed hens showed increased levels of estradiol that might have affected gene expression of specific immune genes, which were up-regulated in the offspring of stressed parents. It is possible that the traditional distinction between stress responses and evolutionary adaptation may be reevaluated, since our results indicate that they could be parts of the same evolutionary event.

Background: Human Leucocyte Antigens (HLA) represent the genetic loci most strongly linked to Multiple Sclerosis (MS). Apart from HLA-DR and HLA–DQ, HLA-DP alleles have been previously studied regarding their role in MS pathogenesis, but to a much lesser extent. Our objective was to investigate the risk/resistance influence of HLA-DPB1 alleles in Hellenic patients with early- and adult-onset MS (EOMS/AOMS), and possible associations with the HLA-DRB1*15:01 risk allele. Methods: One hundred MS-patients (28 EOMS, 72 AOMS) fulfilling the McDonald-2010 criteria were enrolled. HLA genotyping was performed with standard low-resolution Sequence-Specific Oligonucleotide techniques. Demographics, clinical and laboratory data were statistically processed using well-defined parametric and nonparametric methods and the SPSSv22.0 software. Results: No significant HLA-DPB1 differences were found between EOMS and AOMS patients for 23 distinct HLA-DPB1 and 12 HLA-DRB1 alleles. The HLA-DPB1*03 allele frequency was found to be significantly increased, and the HLA-DPB1*02 allele frequency significantly decreased, in AOMS patients compared to controls. The HLA-DPB1*04 allele was to be found significantly decreased in AOMS and EOMS patients compared to controls. Conclusions: Our study supports the previously reported risk susceptibility role of the HLA-DPB1*03 allele in AOMS among Caucasians. Additionally, we report for the first time a protective role of the HLA-DPB1*04 allele among Hellenic patients with both EOMS and AOMS.

Les cellules Natural Killer (NK), un sous-type de lymphocytes, sont cruciales dans l’immunité innée de par leur cytotoxicité directe envers les tumeurs, les cellules infectées et les cellules stressées. Elles contribuent également à l’orchestration de la réponse immunitaire adaptative de par leur capacité à produire des cytokines immunorégulatrices. Pour développer ces fonctions effectrices, les cellules NK requièrent l’intégration d’une multitude de signaux. De là, les cellules NK matures (mNK) sont considérées comme appartenant à quatre sous-types conséquents et concordants avec la polarisation vers la prolifération, la production de cytokines ou les propriétés cytotoxiques. Dans certaines maladies auto-immunes, tel le diabète de type 1 (T1D), un bloc dans leur maturation, et conséquemment des défauts en nombre et en fonctions, peut être observé. Ainsi, afin de découvrir la régulation génétique derrière la proportion et la maturation des cellules NK, des études de liaison génétique ont été produites sur des souris F2 (B10.BR x NOD.H2k) et F2.Rag (B6.Rag1-/- x NOD.Rag1-/-), où le fond génétique NOD est un modèle de T1D. Ces analyses ont révélé des locus potentiellement impliqués dans la régulation de la proportion, des nombres absolus ainsi que dans la maturation fonctionnelle des cellules mNK. Les chromosomes 8, 9 et 17 ont été liés à la proportion des cellules mNK tandis que les chromosomes 2, 4, 7, 10, 11 et 18 ont été liés aux sous-types fonctionnels de ces mêmes cellules. De là, nous avons validé l’influence du locus du chromosome 9 sur la proportion des cellules mNK en générant des souris sous-congéniques avec des insertions de segment génétique B10.BR dans des souris de fond génétique NOD. La proportion et le nombre absolu de cellules mNK ont ensuite été analysés par cytométrie en flux et comparés à ceux de souris contrôles. Pour la maturation fonctionnelle, nous avons retenus certains gènes candidats, et régulateurs associés, liés à un ou plusieurs sous-types de mNK, dont Tbx21, Zeb2, c-Myb, Trp53 et Pmaip1. Par association de voies de signalisation, nous sommes également allés vérifier certaines protéines associées, dont Bim et Eomesodermin. Nous avons alors validé leur implication par l’utilisation de modèles murins knock-out, qPCR, essais de prolifération et d’apoptose. Enfin, nos résultats supportent un rôle pour le locus du chromosome 9 dans la régulation de la proportion de cellules mNK ainsi qu’un rôle pour Trp53, Bim et Pmaip1 dans la maturation fonctionnelle de celles-ci. Nos études révèlent donc de nouveaux gènes candidats potentiels dans la régulation des cellules NK, dont les mécanismes pourront être approfondis dans la perspective de développement de thérapies cellulaires et génétiques dans le combat contre les cancers et les infections chroniques. / Natural Killer (NK) cells, a subset of lymphocytes, are crucial in innate immunity due to their direct cytotoxicity towards tumors, viral infected cells and stressed cells. NK cells also contribute to the orchestration of the adaptive response by their ability to produce immunoregulatory cytokines. To develop those effector functions, NK cells require the integration of multiple signals. As of now, mature NK cells (mNK) can be separated into a four-stage model of functional maturation that concords with a polarization either toward proliferation and cytokine production or cytotoxic functional properties. In autoimmune diseases, like type 1 diabetes (T1D), a block in their maturation, and consequently an impaired functionality and diminished numbers, can be observed. Thus, in order to uncover the genetic regulation behind the proportion and functional maturation of NK cells, a linkage analysis was performed by our lab on F2 (B10.BR x NOD.H2k) and F2.Rag (B6.Rag1-/- x NOD.Rag1-/- intercross) mice, where the NOD genetic background is a model of T1D. This analysis revealed loci that were potentially involved in the regulation of mNK cells proportion, absolute numbers or functional maturation. Loci on chromosomes 8, 9 and 17 were linked to the proportion of mNK cells while loci on chromosomes 2, 4, 7, 10, 11 and 18 were linked to different subsets of functional mNK cells. Hence, we validated the influence of the chromosome 9 locus on the proportion of mNK cells by generating congenic sub-strains of mice with insertion of B10.BR genetic segments and NOD genetic background. The proportion and absolute numbers of mNK cells were assessed by flow cytometry and compared to those of wild-type mice. Regarding the functional maturation of mNK cells, we considered potential candidate genes, and their upstream regulators, that were linked to one or more mNK subsets, namely Tbx21, Zeb2, Myb, Trp53 and Pmaip1. From those, we also looked further their associated pathway with proteins such as Bim and Eomesodermin. We proceeded to the in vivo validation of their implication by qPCR, proliferation and apoptosis assays and the use of knock-out mice. Indeed, our results support a role for a locus on chromosome 9 in the regulation of mNK cells proportion and for Trp53, Bim and Pmaip1 in NK cell functional maturation. As such, our study has revealed new candidate genes in NK cell regulation. Further explorations of the mechanisms by which those genes act could lead to the development of cellular and genetic therapies for cancers and chronic infections.

<p>Environmental stress has shown to affect both the exposed individuals and the development of their offspring. Generally, it is thought that the stressed organism responds to stress by trying to adapt to it. This thesis investigates possible evolutionary consequences of cross-generational transmissions of stress, where the parent has been stressed but the offspring has not. In two studies we have exposed chicken parents of different breeds to an unpredictable circadian light rhythm, to investigate the influence of genetic background on the transmission of behaviour and patterns of genome-wide gene expression across generations. In Paper I, we can show that the domesticated chicken, by means of epigenetic factors, transmit their behaviours as well as their gene expression profiles to their offspring to a higher extent than their wild ancestor, the red junglefowl. Furthermore, in Paper II, even though the offspring never experienced the stress or had any contact with their stressed parents, they seemed to have adapted to it, which suggests that the parents might have prepared (or pre-adapted) them for living in the unpredictable environment. Additionally, eggs of stressed hens showed increased levels of estradiol that might have affected gene expression of specific immune genes, which were up-regulated in the offspring of stressed parents. It is possible that the traditional distinction between stress responses and evolutionary adaptation may be reevaluated, since our results indicate that they could be parts of the same evolutionary event.</p>