Submitted by Irene Nascimento (irene.kessia@ufpe.br) on 2016-10-06T17:20:04Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Denise Maria do Nascimento Costa.pdf: 2568349 bytes, checksum: 97687424c47175731885cd254c815ad4 (MD5) / Made available in DSpace on 2016-10-06T17:20:04Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Denise Maria do Nascimento Costa.pdf: 2568349 bytes, checksum: 97687424c47175731885cd254c815ad4 (MD5) Previous issue date: 2016-07-21 / Introdução: Glomerulopatia membranosa (GM) é uma causa de síndrome nefrótica cuja etiologia pode ser primária (GMP) ou secundária, dentre estas é frequente o Lúpus eritematoso sistêmico (LES). Trata-se de uma doença imunologicamente mediada, caracterizada pela deposição de imunocomplexos no espaço subepitelial glomerular. A maioria dos antígenos envolvidos identificados são alvos da imunoglobulina G4 (IgG4), subclasse predominante em imunofluorescências renais na GMP, em contraste com a GM secundária a LES (GMS) na qual IgG1, IgG2 e IgG3 prevalecem. Apesar da IgG4 ser um subtipo de imunoglobulina com baixa capacidade de ativação do complemento, há várias evidências deste envolvimento na GMP. Esses dados, em conjunto com achados de depósitos glomerulares de lectina ligadora de manose (MBL), um dos principais componentes da via das lectinas do complemento, podem sugerir que tanto a via da lectina como a IgG4 estão envolvidas nesta patologia. Sabe-se ainda que o desenvolvimento de GMP também está associado a alterações genéticas. Entretanto, a etiopatogenia da GMP ainda não é totalmente conhecida e estudos para avaliação gênica do MBL2 e dosagem sérica de IgG em GM são escassos. Assim, foi realizado este estudo com o objetivo de avaliar a frequência de polimorfismos do gene MBL2 em portadores de GM, comparados a indivíduos saudáveis. Um segundo objetivo foi comparar pacientes com GMP e GMS quanto a diferenças do percentual de IgG4 sérico em relação a IgG (%IgG4) e da frequência de polimorfismos do MBL2. Métodos: Estudo realizado entre 2014 e 2015, em Pernambuco - Brasil. A amostra incluiu 60 pacientes adultos com diagnóstico histopatológico de GMP ou GMS. Outras causas de GM secundárias foram excluídas. Foram avaliados 35 pacientes com GMP e 24 com GMS, e um grupo controle (GC), formado por 101 indivíduos saudáveis. Resultados: O alelo mutante O do gene MBL2 foi mais frequente no grupo com GM comparados aos GC (42% x 22%; p < 0,001). A heterozigose A/O, em relação ao genótipo A/A, predominou entre os pacientes comparados ao GC, associando-se a GM com OR = 11,16 (95% IC = 4,77 - 28,41). À análise comparativa entre os pacientes com GMP e GMS, não houve diferença das frequências dos polimorfismos genéticos entre os grupos. O grupo GMP apresentou menor mediana de IgG sérica total (p = 0,008) e maior %IgG4 (p = 0,016), comparado ao grupo GMS. Nível sérico de IgG4 não diferiu significativamente entre os grupos GMP e GMS (p = 0,289). Conclusão: O polimorfismo do éxon 1 do gene MBL2 associou-se à GM, comparado a indivíduos saudáveis, porém sem diferença entre as etiologias avaliadas. Já o %IgG4 sérico foi maior na GMP em relação a GMS. Estes resultados sugerem que esta mutação genética possa conferir maior vulnerabilidade a GMP e que o %IgG4 sérico possa ser utilizado como marcador adicional para diagnóstico diferencial entre as duas etiologias da GM. / Introduction: Membranous glomerulopathy (MG) is a cause of nephrotic syndrome whose etiology may be primary (PMG) or secondary, wich is frequent systemic lupus erythematosus (SLE). It is an immune-mediated disease characterized by the deposition of immune complexes in the glomerular subepithelial space. Most of the identified antigens are targets to immunoglobulin IgG4, most common subclass in renal immunofluorescence in GMP, in contrast to the SLE secondary MG (SMG) in which IgG1, IgG2 and IgG3 prevail. Although IgG4 is a immunoglobulin subtype with low complement activation capacity, there is abundant evidence of this involvement in PMG. These data, together with glomerular deposits of mannose-binding lectin (MBL), a major component of the lectin pathway of complement, may suggest that both the lectin pathway and IgG4 are involved in this pathology. It is also known that the development of PMG is associated with genetic alterations. As the pathogenesis of PMG is not yet fully known, and studies for genetic evaluation of MBL2 and serum IgG in MG are scarce, this study was conducted to evaluate the frequency of MBL2 gene polymorphisms in patients with MG, compared to healthy subjects. A second objective was to compare patients with PMG and SMG with respect to the percentage of serum IgG4 (IgG4%) and frequency MBL2 polymorphisms. Methods: This study was conducted between 2014 and 2015 in Pernambuco - Brazil. The sample included 60 adult patients with histopathologic diagnosis of PMG or SMG. Other causes of secondary MG were excluded. Thity five patients with PMG and 24 with SMG were evaluated, compared to a control group (CG) of 101 healthy subjects. Results: The mutant allele O was more frequent in the MG population compared to CG (42% vs. 22%; p <0.001). The heterozygous A/O, compared to genotype A/A, predominated among patients compared to the control group, and was associated with MG (OR = 11.16; 95% CI = 4.77 to 28.41). In the comparative analysis between patients with PMG and SMG, there was no difference in the frequency of genetic polymorphisms between groups. The PMG group had lower median total serum IgG (p = 0.008) and higher IgG4% (p = 0.016) compared to the SMG group. Serum IgG4 did not differ significantly between the groups PMG and SMG (p = 0,289). Conclusion: The polymorphism of exon 1 MBL2 gene was associated with MG, compared to healthy subjects, but no difference between the assessed etiologies. Serum IgG4% was higher in PMG relative to SMG. These results suggest that this gene mutation can confer increased vulnerability to PMG and the serum IgG4% may be used as an additional marker for the differential diagnosis between the two etiologies MG.

Malária é uma das principais causas de doença e morte no mundo, principalmente de crianças. É considerada a força de seleção evolucionária mais forte que se conhece na história recente do genoma humano. Além dos fatores ambientais e do próprio parasito, fatores genéticos do hospedeiro têm um papel fundamental tanto na suscetibilidade como na evolução clínica da infecção. O sistema imune inato reconhece os plasmódios através de um número limitado de receptores de reconhecimento padrão (PRRs) e inicia vários mecanismos de defesa que resultam no desenvolvimento de inflamação e resistência do hospedeiro à infecção. Mas, a eliminação completa do parasito requer respostas imunes adaptativas que são amplificadas pela ativação do sistema imune inato. As manifestações clínicas de malária são dependentes dos níveis de citocinas próinflamatórias circulantes produzidas, as quais em níveis altos contribuem para a imunopatologia da doença. O balanço entre respostas pró e antiinflamatórias dirigidas contra o parasito é considerado crítico para a proteção clínica, assim a resposta imune inata pode contribuir tanto para proteção da malária como para modular a resposta imune adaptativa. Neste estudo, nós investigamos polimorfismos de um único nucleotídeo (SNP) dos genes de três PRRs: TLR, MBL e CR1 de indivíduos infectados por Plasmodium e residentes em áreas endêmicas de malária no Brasil. Os SNPs TLR1 (I602S), TLR4 (D229G), TLR6 (S249P), TLR9 (T-1237C/ -1486C), MBL [exon 1 nos códons 52, 54, e 57 (MBL2*A ou D, A ou B e A ou C, respectivamente); na região do promotor na posição -221 (*X ou *Y); e na posição +4 da região não traduzida (*P ou *Q)] e CR-1(C5507G) foram determinados por PCR-RFLP. Nós observamos associações entre os polimorfismos TLR1 I602S, TLR6 S249P e da região não traduzida +4 (*Q) e manifestações clínicas de malária e entre os polimorfismos TLR9 T-1486C, TLR T-1237C, MBL*D (códon 52) e do diplótipo de produção insuficiente de MBL (XA+O/O) e parasitemias mais altas. Nenhuma associação foi observada entre o polimorfismo CR-1 C5507G e manifestações clínicas de malária ou com parasitemia. Ao analisarmos juntos os polimorfismos de MBL e TLR, observamos que indivíduos com diplótipo de produção suficiente de MBL (YA/YA+YA/XA+YA/O+XA/XA) TLR1 I602S tinham menos manifestações clínicas de malária e indivíduos com diplótipo de produção suficiente de MBL e não carreadores do alelo TLR9 -1486C tinham parasitemias mais baixas do que os indivíduos com diplótipo de produção insuficiente de MBL e carreadores dos alelos variantes de TLR1 I602S e TLR9 -1486C, respectivamente. Juntos, nossos dados indicam que polimorfismos do promotor de TLR-9 e os diplótipos de produção insuficiente de MBL (XA+O/O) devem de algum modo controlar o nível de parasitemia por plasmódios enquanto a deficiência de TLR1 parece predispor para a presença de manifestações clínicas de malária. Também, podemos sugerir que existe uma cooperação entre TLR1, TLR9 e MBL na ativação da resposta imune inata na malária. Estes achados genéticos devem contribuir para o entendimento da patogênese da malária e levantar uma questão potencialmente interessante que é digna de investigações posteriores em outras populações a fim de validar a contribuição genética destes loci na patogênese da malária / Malaria is one of the major causes of disease and death worldwide, mainly of children. It is also the strongest known force for evolutionary selection in the recent history of the human genome. Besides environmental and parasite factors, host genetic factors play a major role in determining both susceptibility to malaria and the course of infection. Innate immune mechanisms directed against Plasmodium parasites both contribute to protection from malaria and modulate adaptive immune responses. The innate immune system recognizes Plasmodium via a limited number of pattern-recognition receptors (PRRs) and initiates a broad spectrum of defense mechanisms that result in the development of inflammation and host resistance to infection. But, the complete control of the infection requires adaptive immune responses; and the innate immune system is also very efficient in instructing the cellular mediators of adaptive immunity to lead a powerful additional strike force against the parasite. Clinical malaria is characterized by high levels of circulating proinflammatory cytokines, which are thought to contribute to the immunopathology of the disease. The balance between pro- and anti-inflammatory responses toward the parasite is considered critical for clinical protection. The innate immune system initiates and thus sets the threshold of immune responses. In this study, we investigated single nucleotide polymorphisms (SNP) in the genes of three PRRs: TLR, MBL and CR1 in Plasmodium-infected individuals living in endemic areas of Brazil. The SNPs TLR1 (I602S), TLR4 (D229G), TLR6 (S249P), TLR9 (T-1237C/ -1486C), MBL [in the coding sequence of exon 1 at codons 52, 54, and 57 (MBL2*A or D, A or B, and A or C, respectively); in the promoter region at position -221 (*X or *Y); and in the untranslated sequence at position +4 (*P or *Q)] and CR-1(C5507G) were determined by PCR-RFLP. We observed associations of the TLR1 I602S, TLR6 S249P and untranslated sequence at position +4 MBL (*Q) variants with clinical manifestations of malaria and of the TLR9 T-1486C, TLR9 T-1237C, MBL2*D and MBL-insufficient diplotype (XA+O/O) with higher parasitemias. No association was observed to the CR-1 C5507G ) and clinical manifestations of malaria or parasitemia. Also, we observed that individuals with MBLsufficient haplotype (YA/YA+YA/XA+YA/O+XA/XA) and not bearing the allele TLR1 I602S had less clinical manifestations of malaria and individuals with MBL-sufficient haplotype and not bearing TLR9 -1486C had lower parasitemias when compared to individuals with MBL-insufficient diplotype and bearing the variant alleles TLR1 I602S and TLR9 -1486C, respectively. Altogether, our data indicate that TLR-9 promoter and MBL-insufficient haplotype (XA+O/O) polymorphisms to some extent may control the level of Plasmodium parasitemia while TLR1 deficiency seems to predispose to mild malaria. Also, they could suggest cooperation among TLR1, TLR9 and MBL in the immune response against malaria. These genetic findings may contribute to the understanding of the pathogenesis of malaria and raise a potentially interesting issue that is worthy of further investigation in other population in order to validate the genetics contribution of these loci to the pathogenesis of malaria

Carbon nanotubes possess various unique and interesting properties. They have very high thermal and electrical conductivities, high stiffness, mechanical strength, and optical properties. Due to these properties, CNTs are widely used materials in a variety of fields. It is used for biotechnological and biomedical applications, as chemical and biosensor, in energy storage and field emission transistor. Experimentally synthesized CNTs are generally found in bundle form due to the strong vander Waals (vdW) at-traction between the individual tubes. To use CNTs in real life applications, we often require specific nanotubes with particular characteristics. The nanotube bundle is a mixture of various chirality, diameters and electronic properties (metallic and semiconducting). Only thermal energy is not sufficient to disperse nanotubes from the bundle geometry overcoming the strong vdW attraction between nanotubes. The hydrophobic and insoluble nature of CNTs in the aqueous medium makes the dispersion of CNTs even more difficult. So, it is a big challenge to get single pristine nanotube from the bundle geometry. Many experimental and theoretical studies have addressed the problem of nanotube dispersion from the bundle geometry. Ultrasonic dispersing method is a widely used technique for this purpose where ultrasonic sound is applied to agitate particles in a system. Other methods include using different organic and inorganic solutions, various surfactant molecules, different polymers as dispersing agents. In this study we extend our e orts to develop some better methods and improved dispersing agents. In this thesis, we address the problem of CNT dispersion. To address this issue, we rst give a quantitative estimation of the effective interaction between nanotubes. Next, we introduce different polymers (ssDNA and dendrimers) as external agents and show that they help to overcome the strong adhesive interaction between CNTs and make nanotube dispersion possible from the bundle geometry. For all of the works presented in this thesis, we have used fully atomistic MD simulation and DFT level calculations. We study ssDNA-CNT complex using all-atom MD simulation and calculate various structural quantities to show the stability of ssDNA-CNT complex in aqueous medium. The adsorption of ssDNA bases on CNT surface is driven by - interaction between nucleic bases and CNT. Using the potential of mean forces (PMF) calculation, we study the binding strength of the polynucleotide ssDNA for poly A, T, G, and C with CNT of chirality (6,5). From the PMF calculation, we show the binding sequence to be A > T > C > G. Except for poly G, our result is in good agreement with earlier reported single molecule force spectroscopy results where the sequence of binding interaction was reported to be A > G > T > C. To explore how the interaction between two CNTs mod-i ed in presence of ssDNA between them, we perform PMF calculation between the two ssDNA-wrapped CNTs. The PMF shows the sequence of interaction strength between two ssDNA-wrapped CNTs for different nucleic bases to be T > A > C > G. Thus, from PMF calculations we show the poly T to have the highest dispersion efficiency, which is consistent with earlier reported experimental study. Our PMF calculation shows that poly C and poly G reduce the attraction between two CNTs drastically, whereas poly A and poly T make the interaction fully repulsive in nature. We also present microscopic pictures of the various binding conformations for ssDNA adsorbed on CNT surface. We also study the dendrimer-CNT complex for both the PAMAM and PETIM dendrimers of different generations at various protonation states and present microscopic pictures of the complex. We calculate PMF between two dendrimer wrapped CNTs and show that protonated and higher generations (G3, G4, and so forth) non-protonated PAMAM dendrimers can be used as e ective agents to disperse CNTs from bundle geometry. We also study the chirality dependence of PMF respectively. Finally, we study the interaction of mannose dendrimer with CNTs and show that the wrapping of mannose dendrimer can drive a metal to semiconducting transition in a metallic CNT. We attribute the carbon-carbon bond length assymetry in CNT due to the wrapping of mannose dendrimer as the reason for this band gap opening which leads to metal-semiconductor transition in CNT. Thus, the wrapping of mannose dendrimer on CNT can change its electronic properties and can be used in the band gap engineering of CNT in future nanotechnology. Thus, the works carried out here in this dissertation will help to address the problem of nanotube dispersion from the bundle geometry which will in turn help to use CNT for various applications in diverse fields.

Abstract The aims of this work were to study the carriage of respiratory bacteria and to identify risk factors affecting pharyngeal colonisation by these pathogens among young Finnish men during military service, and also to investigate the role of mannose-binding lectin (MBL) concentrations and MBL2 gene polymorphisms in the carriage of respiratory bacteria. A total of 892 military recruits entering the Kainuu Brigade, including 224 men with asthma, were followed up prospectively to the end of their military service. Carriage of Streptococcus pneumoniae, Neisseria meningitidis and beta-haemolytic streptococci appeared to be higher during and at the end of military service than at the beginning. Smoking was found to be a significant risk factor for colonisation by these bacteria. S.pneumoniae was more common in the asthmatic than military conscripts in the non-asthmatic ones at the beginning of military service. A low MBL level increased the risk of carrying N. meningitidis and beta-haemolytic streptococci during military service among non-smokers but not among smokers. Low MBL levels producing MBL2 haplotypes seemed to be associated with the carriage of N. meningitidis and S. pneumoniae. Characterisation of all the oropharyngeal N.meningitidis isolates obtained (n=215) by phenotypic and genotypic methods showed that most of them belonged to the carriage-associated ST-60 clonal complex. Clonal complexes ST-41/44, ST-32, and ST-23, which have previously been associated with disease, also accounted for a third of the carriage strains. Furthermore, a significant association was indicated between an acute upper respiratory infection and oropharyngeal carriage of the virulent meningococcal ST-23 clone. In conclusion, the results reported here show a significant increase in bacterial carriage during military service and provide new information on the association between MBL and carriage of respiratory bacteria. These findings also highlight the importance of smoking cessation, especially among military conscripts, who have been found to be a risk group for invasive bacterial diseases, and they also point to the importance of meningococcal vaccination for military recruits and the need for an efficacious vaccine against serogroup B meningococci. / Tiivistelmä Hengitystieinfektiot ovat yleisiä varusmiespalvelun aikana. Myös oireeton bakteerien nielukantajuus on lisääntynyt. Useimmiten infektiot ovat lieviä virusinfektioita, mutta bakteerien nielukantajuus voi johtaa myös vaikeisiin bakteeritulehduksiin. Tämän väitöskirjatyön tarkoituksena oli tutkia bakteerien nielukantajuutta varusmiespalveluksen alkaessa ja päättyessä sekä mahdollisten hengitystieinfektioiden aikana ja näin saada uutta tietoa bakteerien nielukantajuuteen vaikuttavista tekijöistä. Lisäksi tavoitteena oli selvittää mannoosia sitovan lektiinin (MBL) sekä MBL2-geenin polymorfismien yhteyttä bakteerien nielukantajuuteen. Työn tarkoituksena oli myös feno- ja genotyypittää varusmiehiltä palveluksen aikana eristetyt meningokokkikannat ja verrata niitä vastaavana ajankohtana invasiivista tautia sairastaneista henkilöistä eristettyihin meningokokkikantoihin. Tutkimuksessa seurattiin prospektiivisesti 892 varusmiestä, jotka suorittivat asepalveluksen Kainuun Prikaatissa vuosina 2004–2006. Tutkimukseen osallistuneista varusmiehistä 224:llä oli astma. Tutkimuksessa havaittiin, että oireeton bakteerien nielukantajuus lisääntyy merkitsevästi varusmiespalveluksen aikana. Lisäksi havaittiin, että tupakointi oli merkittävä itsenäinen riskitekijä pneumokokin, meningokokin sekä beta-hemolyyttisten streptokokkien nielukantajuudelle varusmiespalveluksen aikana. Astmaatikkojen pneumokokin nielukantajuus varusmiespalveluksen alussa oli yleisempi kuin terveiden varusmiesten. Tutkimuksessa osoitettiin myös pienen seerumin MBL-pitoisuuden sekä MBL2-geenin polymorfismin eksoni 1:n alueella ja geenin säätelyalueella olevan riskitekijöitä meningokokin, pneumokokin sekä beta-hemolyyttisten streptokokkien nielukantajuudelle tupakoimattomilla varusmiehillä. Meningokokin nielukannoista jopa kolmasosa kuului genotyyppiryhmään, jonka on aiemmissa tutkimuksissa havaittu liittyvän invasiiviseen tautiin. Tutkimuksessa osoitettiin myös tietyn hyperinvasiivisen meningokokin genotyypin (ST-23) liittyvän hengitystieinfektioepisodeihin. Tässä väitöskirjatyössä osoitettiin, että bakteerien nielukantajuus lisääntyy merkitsevästi varusmiespalveluksen aikana ja että oireettomilla varusmiehillä tavataan myös hyperinvasiivisia meningokokkikantoja. Tutkimus antoi myös uutta tietoa hyperinvasiivisten meningokokin genotyyppien liittymisestä hengitystieinfektioihin sekä MBL:n vaikutuksesta bakteerien nielukantajuuteen. Tutkimushavainnot tukevat tupakoimattomuuden edistämisen tärkeyttä myös varusmiespalveluksen aikana.

Cystic fibrosis (CF) is a single gene Mendelian disorder characterized by pulmonary disease and pancreatic insufficiency. Pulmonary disease is the major cause of death in CF patients. Although some cystic fibrosis transmembrane conductance regulator (CFTR) genotypes are associated with less severe disease, patients possessing the same genotype show great variation in pulmonary disease severity and progression. Genes involved in modulating the inflammatory response and genes increasing susceptibility to infection are proposed as modifiers of pulmonary disease severity. Polymorphisms selected for based on evidence that they affect the function of the gene and prevalence of the putative risk allele: 1) antiprotease gene alpha-1-antitrypsin (alpha-1-AT), 2) innate immunity genes: mannose binding lectin (MBL2) (promoter [G→C] at -221 and codon 52 (Arg52Cys, D allele), 54 (Gly54Asp, B allele), and 57 (Gly57Glu, C allele), and pulmonary surfactant genes SPA-1 (Arg219Trp), SPA-2 (Thr9Asn, Lys223Gln) and SPD (Thr11Met), 3) antioxidant genes GSTM1 and T1 (gene deletion polymorphisms), GSTP1 (Ile105Val) and GCLC repeats, 4) mucin genes (MUC2 and MUC5B). Pulmonary disease progression and survival in patients with chronic Burkholderia cepacia complex (BCC) infection were also investigated controlling for genomovar and RAPD type of the organism. BCC infection was associated with more severe pulmonary disease progression and worse survival. Alpha-1-AT genotype was not a major contributor to variability of pulmonary disease severity, but the results suggest that alpha-1-AT plasma levels during pulmonary infections may be affected by poor nutritional status. We showed similar pulmonary disease progression and MBL2 genotype. Contrary to the previous literature, wild-type MBL2 genotype was associated with steeper decline in pulmonary disease over time following chronic infection with BCC, but genotype was not associated with increased susceptibility to BCC infection. We showed inconsistant results for the pulmonary surfactant gene polymorphisms, GSTM1, T1 and GSTP1 polymorphisms, and number of repeats for GCLC and MUC5B depending on the phenotype investigated. We conclude that some of the variability in pulmonary disease severity and progression in CF is explained by polymorphisms in secondary genes. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

Nanodiamanty (250 nm) boli podrobené oxidácií, a tiež separované podľa veľkosti. Následne bola na povrch nanodiamantov adsorbovaná rozvetvená vrstva polyglycerolu. Táto vrstva vzniká kyslo katalyzovanou ,,ring opening“ polymeráciou glycidolu s hydroxylovými a karboxylovými skupinami povrchu nanodiamantov. Vzniknutá polyglycerolová vrstva zvyšuje stabilitu a biokompatibilitu nanodiamantov. Po vytvorení polyglycerolovej vrstvy bol do reakcie pridaný glycidyl propargyl éter, čím došlo k terminácií polymerácie, v dôsledku zavedenia propargylu do reakcie. Prítomná trojná väzba umožňuje nové modifikácie povrchu, napr. prostredníctvom CuAAC reakcie. S využitím CuAAC reakcie je možné na povrch fluorescenčných nanodiamantov pripojiť manózu (so spojkou obsahujúcou azidoskupinu). Pripravené nanočastice boli charakterizované použitím fyzikálno chemických metód. Distribúcia veľkosti častíc bola meraná pomocou dynamického rozptylu svetla (DLS), a taktiež bola potvrdená obrazovou analýzou mikrografov z elektrónového transmisného mikroskopu (TEM). Vyhodnotené bolo aj správanie sa nanodiamantov v biologických podmienkach (stabilitné testy v 1xPBS, 10xPBS a 100 % FBS) a bol meraný aj zeta potenciál. Nakoniec boli pripravené fluorescenčné diamanty (250 nm) s naviazanou manózou. Vzhľadom k výsledkom termogravimetrie, meraniam DLS a pozorovania pomocou TEM môžeme povedať, pripravené nanodiamanty sú pokryté tenkou vrstvou polyglycerolu. Prítomnosť manózy bola zistená na základe FTIR spektier. Nami pripravené fluorescenčné nanodiamanty obalené vrstvou polyglycerolu sú vhodné pre ďalšie testovanie in vitro a in vivo vďaka ich stabilite v médiách s vysokým obsahom solí ako napr. fosfátový pufor (PBS) a nízkemu viazaniu proteínov vo fetalnom bovinnom sere (FBS).

Orchideoid mycorrhizal symbiosis (OM) can be found in nearly one tenth of higher plant species. This symbiosis is absolutely critical for orchids as they are unable to grow from seeds without external energy which is in nature provided by symbiotic fungi. The mechanism of transport between symbionts remains unknown. It is supposed that trehalose is one of the substances transported from fungi to plants as the source of energy. This thesis mainly aims to find out which other fungal saccharides could contribute to the process. The ability to utilize selected compounds was tested on protocorms of the common marsh orchid, Dactylorhiza majalis. The results showed that arabitol, erythritol, mannitol and sucralose are not utilized, while xylitol, sorbitol, glycerol and mannose are. Glutamin, an amino acid also suspected of participation in the OM transport, does not suffice as a source of energy. In orchids there were identified three groups of sequences coding for manitol dehydrogenase and two groups of sequences coding for sorbitol dehydrogenase. Key words: orchideoid mycorrhizal symbiosis, sugar alcohols, mannose, glutamine, carbon flow, energy flow, sorbitol dehydrogenase, in vitro

Chronic granulomatous disease (CGD) is associated with defective function of the NADPH-oxidase system in conjunction with phagocytic defects which leads to granuloma formation and serious infectious complications. This is often associated with significant morbidity and mortality. The association of defective phagocyte function with other coincidental immune defects is unknown. Defects in innate pathways seen with CGD, including complement systems, and toll-like and dectin receptor pathways, have not been described before. We present the case of a 2-year old male patient hospitalized with recurrent pneumonia, a non-healing skin ulcer, necrotizing lung granulomas, and epididymo-orchitis. Defective neutrophil chemiluminescence was detected by dihydrorhodamine (DHR) testing. Further evaluation demonstrated characteristic molecular mutations of CYBB consistent with CGD. Immune evaluation demonstrated polyclonal hyperglobulinemia, but a greatly reduced mannose binding lectin (MBL) level. Six biallelic polymorphisms in MBL gene and its promoter were analyzed using Light Cycler™ Real-time PCR assay. The LXPA/LYPB haplotype of MBL was detected in our patient; the latter is the defective haplotype associated with low MBL levels. Due to the implications for innate immunity and the protection against bacterial, viral, and fungal infections provided by MBL, a deficiency of this protein may have disastrous consequences on the long term outcomes of CGD. MBL deficiency can also complicate other disorders affecting the immune system, significantly increasing the risk of infection in such patients. Further studies looking at the frequency and implications of MBL deficiency in CGD are needed.

37.3 million Americans (11.2% of the US population) currently have Type 2 diabetes mellitus (T2DM) with over 1.5 million new cases being diagnosed each year. The multifactorial etiology of the patient having neuropathy, overweight/obesity, foot deformities, ischemia, and infection leads to a condition called diabetic foot ulcer (DFU). One in six patients with a DFU will require amputation with infected DFUs have a 155-fold increased risk of amputation. Staphylococcus aureus is the most common bacteria isolated from severe DFU infections that require amputation. Interestingly, diabetics are more heavily colonized with S. aureus compared to non-diabetics suggesting a unique advantageous adaptation to diabetes. The specifics of the underlying molecular mechanisms and triggers by which S. aureus adapts and thrives in the T2DM patient that increase its pathogenicity and colonization compared to non-diabetics with skin ulcer infections are not fully elucidated. Thus, our studies aimed to identify the key virulence components in the pathogenesis of S. aureus infected DFUs and using that information to develop therapeutics aimed at disrupting these components to increase the success rate of conservative treatment and prevent non-traumatic lower extremity amputations in T2DM patients. Our studies found that several different elevated sugars in T2DM patients can trigger virulence factor production in S. aureus. We also found by comparing several different clinical DFU S. aureus isolates that there are clear differences in the ability of each isolate to cause necrotic infections. And lastly, we identified a possible therapeutic, the amino acid L-arginine, that can help prevent/treat S. aureus infections in the Tallyho diabetic mouse model. In conclusion, we have increased the understanding of the pathogenesis of S. aureus infected DFU and have proposed a possible therapeutic to add to the conservative treatment regimen.

La vaccination est l’un des moyens les plus efficaces de la médecine moderne dans le combat contre les maladies infectieuses. L’amélioration de l’efficacité des vaccins requiert la mise au point d’adjuvants permettant d’accroître la qualité de la réponse immunitaire. À titre d’exemple, les nanoparticules (NP) de poly(acide lactique) (PLA) constituent un système efficace pour la délivrance d’antigènes. Afin de renforcer leur potentiel vaccinal, ce travail de recherche a eu pour objectif d’élaborer des NP de PLA décorées en surface par des molécules immunostimulantes, le D-mannose ou un peptide dérivé de l’interleukine-Beta, et au cœur, par l’imiquimod. Notre stratégie repose sur l’utilisation d’un tensioactif macromoléculaire composé d’un bloc de PLA et d’un bloc de poly(N-acryloxysuccinimide-co-N-vinylpyrrolidone) (P(NAS-co-NVP)), dont les fonctions ester de N-succinimidyle (NS) permettent le couplage de biomolécules. Ce copolymère a été synthétisé par combinaison de la polymérisation par ouverture de cycle et de la polymérisation radicalaire contrôlée par les nitroxydes (NMP). Après l’étude de la copolymérisation du NAS et de la NVP par NMP à partir d’une alcoxyamine modèle (MAMA-SG1), leur copolymérisation a été réalisée à partir de la macro-alcoxyamine PLA-SG1, conduisant au copolymère PLA-b-P(NAS-co-NVP) désiré. Des NP de PLA ont alors été préparées par nanoprécipitation et diafiltration en présence du copolymère, conduisant à des tailles respectives de 150 et 500 nm. Des études de potentiel zêta et de spectrométrie UV ont démontré la présence des esters de NS à la surface des NP (2.4 fonctions.nm-2), disponibles pour le couplage des biomolécules. Des micelles de copolymère ont été également préparées, après substitution des esters de NS par des sucres, et permettent une encapsulation efficace de l’imiquimod, contrairement aux NP de PLA. Ces systèmes constituent une plateforme flexible d’adjuvants potentiels comme alternative aux adjuvants non biodégradables actuellement utilisés. / Vaccination represents one of the most powerful tools of medicine for the fight against infectious diseases. The improvement of vaccine efficiency needs the development of adjuvants able to increase the quality of the immune response. Poly(lactic acid) (PLA) nanoparticles (NPs) represent an efficient system for antigen delivery. In order to improve their vaccine potential, the goal of this research work was to elaborate PLA NPs decorated at the surface with immunostimulatory molecules, D-mannose or peptide derived from interleukine-Beta, and into the core with imiquimod. Our strategy relies on the use of a macromolecular surfactant composed of a PLA block and a poly(N- acryloxysuccinimide-co-N-vinylpyrrolidone) (P(NAS-co-NVP)) block, whose N-succinimidyl (NS) activated esters allow the coupling of biomolecules. This diblock copolymer was synthesized by the combination of ring opening polymerization and nitroxide mediated polymerization (NMP). After the study of the copolymerization of NAS and NVP by NMP from the MAMA-SG1 model alkoxyamine, their copolymerization was performed from the macro-alkoxyamine PLA-SG1, leading to the desired copolymer PLA-b-P(NAS-co-NVP). PLA NPs were then prepared by nanoprecipitation and diafiltration, in the presence of the copolymer, leading to 150 nm and 500 nm sized particles, respectively. Studies of zeta potential and UV spectrometry demonstrated the presence of NS-activated esters at the NP surface (2.4 functions.nm-2), available for the coupling of biomolecules. Micelles from copolymer were also prepared, after substitution of esters with carbohydrates, and allowed an efficient encapsulation of imiquimod, contrary to PLA NPs. These systems represent a flexible platform of potential adjuvants as an alternative to non-biodegradable adjuvants currently used.