Higher eukaryotes produce a vast range of antimicrobial peptides (AMPs) that play important roles in their defence against microbial infection. Beta defensins are small (3-5 kDa), cationic peptides that display broad, potent antimicrobial activity against a range of microbes and also act as chemoattractants of important immunomodulatory cells. To generate highly pure peptides for structural and functional studies, we developed a method to prepare recombinant human beta defensin-2 (HBD2). The HBD2 gene was synthesised by recursive PCR with codons optimised for expression in Escherichia coli. HBD2 was expressed as an insoluble fusion to a His-tagged ketosteroid isomerase. After cleavage from the fusion with cyanogen bromide, 1H NMR spectroscopy and mass spectrometry confirmed that the oxidised HBD2 was folded and possessed the correct b-defensin disulfide bond topology. The recombinant HBD2 was active against E. coli, P. aeruginosa, S. aureus and C. albicans and was also a chemoattractant against HEK293 cells expressing the chemokine receptor CCR6. 15N-labelled HBD2 was also prepared and was highly suitable for future structural studies. Since defensins are thought to interact with bacterial membranes we also tested the recombinant HBD2 in biophysical studies (surface plasmon resonance, SPR, Biacore). We observed different binding to artificial model membranes containing either E. coli Kdo2-lipid A or phospholipids. Bacterial resistance to AMPs has been linked to the covalent modification of the outer membrane lipid A by 4-amino-4-deoxy-L-arabinose (L-Ara4N). This neutralises the charge of the LPS, thereby decreasing the electrostatic attraction of cationic peptides to the bacterial membrane. The pathogen Burkholderia cenocepacia displays extremely high resistance to AMPs and other antibiotics and the Ara4N pathway appears to be essential. To explore this further we expressed recombinant forms of two enzymes (ArnB and ArnG) from the B. cenocepacia Ara4N pathway. Purified ArnB is a pyridoxal 5’-phosphate (PLP)-dependent transaminase and we tested its ability to bind amino acid substrates. We investigated the binding of inhibitors L- and D-cycloserine to ArnB and tested their antibiotic activity against Burkholderia strains. We also studied the B. cenocepacia ArnG – a proposed membrane protein undecaprenyl-L-Ara4N flippase – and showed that the protein behaved as a dimer by non-denaturing gel analysis. The B. cenocepacia ArnG failed to complement E. coli knock-out strains encoding the equivalent flippase proteins ArnE and ArnF, suggesting that ArnG is a Burkholderia-specific protein.

Submitted by Caroline Falcao (caroline.rfalcao@ufpe.br) on 2017-04-06T19:27:03Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Dissertação-JeanSauvé.pdf: 1000481 bytes, checksum: ba9527b7ead65c4a43be6a94ad0aa462 (MD5) / Made available in DSpace on 2017-04-06T19:27:03Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Dissertação-JeanSauvé.pdf: 1000481 bytes, checksum: ba9527b7ead65c4a43be6a94ad0aa462 (MD5) Previous issue date: 2012 / O papilomavírus humano (HPV) é o principal agente etiológico de lesões malignas, a exemplo do câncer cervical e de suas lesões precursoras, as Neoplasias Intraepiteliais Cervicais (NICs). Investigamos se os polimorfismos de base única (SNPs) no gene da Beta-Defensina Humana 1 (DEFB1) poderiam estar associados à susceptibilidade à infecção e à progressão da lesão cervical provocadas por HPV de alto risco. Polimorfismos neste gene têm sido alvo de diversos estudos e revelaram ser de suma importância na susceptibilidade a diversas infecções. Neste estudo, os SNPs -52G/A, -44C/G e -20 G/A na região 5' UTR do gene DEFB1 foram analisados através de sequenciamento, em um grupo de 160 pacientes do sexo feminino HPV positivas e 71 indivíduos sadios. As análises estatísticas foram feitas utilizando o Teste exato de Fisher e o Teste do Qui-quadrado. Os resultados indicam que o alelo -44G e o haplótipo ACA podem estar relacionados com a susceptibilidade à infecção pelo HPV de alto risco. Os resultados não apontam diferenças significativas nas frequências alélicas e genotípicas dos outros polimorfismos entre pacientes infectados com HPV de alto risco e indivíduos sadios. Também não foram encontradas diferenças estatisticamente significativas nas frequências alélicas e genotípicas nos pacientes HPV positivos quanto ao grau de lesão cervical. / Human papillomavirus (HPV) is the main etiological agent for development of cervical cancer and its precursor lesions, the cervical intraepithelial neoplasia (CINs). We investigated whether single-nucleotide polymorphisms (SNPs) in the human Beta-Defensin 1 gene (DEFB1) could be associated with susceptibility to infection and progression of cervical lesions caused by high-risk HPV. Polymorphisms in this gene have been the subject of several studies and proved to be extremely important in susceptibility to various infections. In this study, SNPs -52 G/A, -44 C/G and -20 G/A in the 5 'UTR of DEFB1 were analyzed by direct sequencing in a group of 160 HPV-positive female patients and 71 healthy individuals. Statistical analysis was performed using Fisher's exact Test and Chisquare. The results indicate that the -44G allele and haplotype ACA may be associated with susceptibility to infection caused by high-risk HPV. The results show no significant differences in allelic and genotypic frequencies of the other polymorphisms in patients infected with high-risk HPV and healthy individuals. We also found no statistically significant differences in allelic and genotypic frequencies in HPV-positive patients related to the progression of cervical lesion.

No description available.

In etlichen Studien konnte die gesundheitsfördernde Wirkung von probiotischen Bakterien nachgewiesen werden. Heutzutage werden diese zahlreich in der Nahrungsmittel- und Kosmetikindustrie verwendet, um das Immunsystem des Menschen positiv zu beeinflussen. Probiotische Bakterien oder deren Extrakte können zum Beispiel die Produktion von Zytokinen anregen oder herabsetzen und dadurch Entzündungsreaktionen verhindern oder die körpereigene Abwehr steigern. Auch die Produktion des humanen Beta-Defensins-2 (hBD-2) kann durch probiotische Bakterien verändert werden. Dieses wirkt im menschlichen Körper wie ein körpereigenes Antibiotikum und wird von verschiedenen Zellen kontinuierlich gebildet. Gerade in entzündetem Gewebe unterliegt es einer gesteigerten Produktion. Inwieweit sich die hBD-2-Produktion in oralen Keratinozyten der Zelllinie OKF6/hTERT-2 verändert, während diese mit den Extrakten der probiotischen Bakterien Lactobacillus salivarius (L.s.) und Lactobacillus rhamnosus GG (L.GG.) inkubiert wurden, war das Ziel dieser Arbeit. Die Ergebnisse zeigen deutlich eine verringerte hBD-2-Produktion der oralen Keratinozyten nach der Stimulation mit den Bakterienextrakten zu verschiedenen Konzentrationen und Zeiten. Ein günstiger Therapieansatz durch die Gabe von probiotischen Bakterienextrakten bei entzündlichen Erkrankungen der Mundschleimhaut, wäre durch eine gesteigerte hBD-2-Produktion denkbar. Dieses bekämpft nämlich pathogene Erreger und unterstützt somit die Immunabwehr. Mit den Bakterienextrakten von L.s. und L.GG. ließ sich die Produktion von hBD-2 in den oralen Keratinozyten nicht steigern. Einen anti-inflammatorischen Effekt der Extrakte auf die Zellen ist dennoch nicht auszuschließen, da die hBD-2-Konzentration auch indirekt als Indikator einer Entzündung angesehen werden kann und hier mit zunehmender Bakterienextraktkonzentration sinkt. Jedoch sind andere Ursachen für den Konzentrationsabfall ebenso denkbar.

No description available.

Anxiety disorders are the most prevalent psychiatric conditions within primary care, affecting up to 29% of people across their lifetime. Generalized Anxiety disorder (GAD) is frequently comorbid with Major Depressive Disorder (MDD), resulting in greater functional impairment. Gut microbiota have been shown to modulate brain chemistry and function, possibly also playing a role in the genesis of anxiety. Bacteria are also able to produce, or interact with the host metabolism of neuroactive substances, including classical neurotransmitters and trace amines, like octopamine, which although found in trace concentrations in the mammalian brain, can affect CNS function. Specifically, trace amines can affect catecholamine release, reuptake and biosynthesis, and modulate dopamine and serotonin metabolism. We investigated whether microbiota from patients with GAD with no signs of immune activation can alter behaviour in gnotobiotic mice and whether this is accompanied by changes in metabolites within the gastrointestinal tract. Germ-free NIH Swiss mice (n=35) were colonized with microbiota from either a GAD patient (n=18) with severe anxiety, comorbid depression, and low serum and fecal octopamine, or an age and sex-matched healthy control (HC) (n=17). Three weeks post- colonization, mouse behaviour was assessed by standard psychometric tests. Emotionality z-scores were calculated to provide a robust integrated behavioural assessment. Microbiota profiles were assessed by 16S rRNA based Illumina, fecal β-defensin-3 level was measured by ELISA. After sacrifice, mouse brain BDNF and GDNF expression was assessed by immunofluorescence, and gene expression in the hippocampus, amygdala, and olfactory bulbs was assessed by Nanostring. Stool and cecum metabolites were measured in all colonized mice by multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). There were no differences in fecal β-defensin levels between mice colonized with GAD microbiota as compared to mice colonized with HC microbiota. However, GAD mice exhibited greater anxiety and depressive-like behavior compared to HC mice in the digging and tail suspensions tests. Behavioural z-scoring across all six standard psychometric tests showed a significant increase in group emotionality score means of GAD-colonized mice compared to HC-colonized mice. Mice colonized with microbiota from a GAD patient had distinct bacterial profiles from mice colonized with HC microbiota. Compared to HC mice, GAD mice had lower levels of dopamine, octopamine and acetylcholine in cecum contents. Furthermore, GAD mice had higher expression of BDNF in the amygdala, lower expression of BDNF in the hippocampus, and lower expression of GDNF in the midbrain. GAD mice also had lower expression of CCR2 in the hippocampus, higher Cnlp/CAMP in the amygdala and olfactory bulb, and higher Nfkb1 in the olfactory bulb compared to HC mice. Our results suggest that microbiota from a selected patient with GAD has the ability to induce anxiety and depressive-like behavior, by mechanisms independent of immune system, likely by altered production of biogenic amines and neurotransmitters. / Thesis / Master of Science (MSc)