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In this thesis, an analytical platform was designed and applied to various in vitro bacterial and eukaryotic cell cultures. An extraction and an analytical protocol were developed for comprehensive and simultaneous analysis of both lipid and polar metabolites for intra- and extracellular metabolomics using HILIC-LC-TOF-MS. This analytical platform was applied to four diverse research questions such as the effect of oxygen environment on growth, the interplay between gene expression and metabolism, metabolic changes that occur with age, and PAH toxicity. Specifically: (i) the effect of oxygen on the growth, physiology and metabolism of the Gram positive Streptococcus intermedius were investigated by comprehensive intra- and extracellular metabolomes and transcriptome. (ii) Metabolic insights into the role of the multipartite genome of the Gram negative bacteria Sinorhizobium meliloti and its metabolic preferences in a nutritionally complex environment. (iii) Age-associated metabolic dysregulation in murine bone marrow-derived macrophages during bacterial lipopolysaccharide-induced inflammation. (iv) Comprehensive intracellular metabolomic profiles of Sinorhizobium meliloti to sub-lethal exposure of individual or mixtures of polycyclic aromatic hydrocarbon revealed additive and dose-dependent effects. This thesis has demonstrated the versatility of the designed analytical platform and its use for diverse research in cell biology. / Thesis / Doctor of Philosophy (PhD)

Capsaicin, prostaglandin E2 (PGE2) and polydopamine (PDA) were used to target macrophage and myofibroblast activity to reduce biomaterial-induced fibrosis. The lifetime and efficacy of implantable biomedical devices are determined by the foreign body response. Immediately after implantation, proteins nonspecifically adsorb onto the material and initiate inflammation. Macrophages recruited to the site can differentiate into M1 and M2 phenotypes and upregulate inflammation and fibrosis which interferes with the intended function. M1 macrophages secrete pro-inflammatory mediators that induce chronic inflammation and promote myofibroblast differentiation while M2 macrophages are wound healing cells that suppress inflammation and regulate fibroblast activity. The fibrotic tissue is developed by myofibroblasts which produce collagen in an unregulated fashion. Collagen thickening and biomaterial encapsulation decreases efficacy and sensitive of biomedical devices. We investigated the in vitro and in vivo effects of capsaicin, PGE2 and polydopamine surface modification on macrophages and myofibroblasts. Capsaicin and PGE2 reduced poly(lactic-co-glycolic) acid (PLGA)-induced fibrosis by promoting M2 macrophage phenotype to secrete anti-inflammatory IL-10 and suppressing myofibroblast marker α-smooth muscle actin (α-SMA). Capsaicin decreased collagen by 40% and upregulated IL-10 secretion by 35% while PGE2 reduced collagen by 55% after 14 days of implantation and 40% less collagen after 42 days. PDA was used to bind an anti-fibrotic compound to the surface of a poly(dimethyl siloxane) (PDMS-PDA) to reduce fibrosis. However, PDMS-PDA controls gave an unexpected result by reducing fibrosis to the same extent as anti-fibrotic compound bound PDMS- v PDA. PDA modification reduced cellularity by 50% and significantly decreased collagen thickness by 30%. Overall, our results showed that biomaterial-induced fibrosis can be reduced by promoting M2 macrophage activity and inhibiting myofibroblast differentiation. This research demonstrates three compounds that have potential to reduce fibrosis and extend the lifetime and efficacy of implantable biomedical devices. / Thesis / Master of Applied Science (MASc) / Capsaicin, prostaglandin E2 (PGE2) and polydopamine were used to reduce scar tissue development around implanted polymers. Biomedical devices implanted in the body can undergo severe scar tissue formation, or fibrosis, and fail. Fibrosis is described by the accumulation of collagen and encapsulation of an implanted polymer. Macrophages regulate fibrosis by secreting pro-fibrotic compounds and myofibroblasts produce unregulated amounts of collagen. In this thesis, capsaicin, PGE2 and polydopamine were incorporated into implants to target macrophage and myofibroblast activity and reduce fibrosis in mice. Capsaicin and PGE2, released from a degradable polymer, altered macrophages to secrete anti-fibrotic compounds and decreased collagen by 40% and 55%, respectively. Polydopamine surface modified implants gave an unexpected result and suppressed overall cell activity to reduce fibrosis by 30%. The research conducted shows the potential of these compounds to reduce fibrosis and extend the lifetime of implantable devices.

Equine asthma (EA) is a highly relevant disease, estimated to affect up to 20% of all horses, and compares to human asthma. The pathogenesis of EA is most likely immune-mediated, yet incompletely understood. To study the immune response in the affected lower airways, mixed leukocytes were acquired through bronchoalveolar lavage (BAL) and the cell populations were analyzed on a single-cell basis by flow cytometry (FC). Samples of 38 horses grouped as respiratory healthy or affected by mild to moderate (mEA) or severe EA (sEA) according to their history, clinical signs, and BAL cytology were analyzed. Using FC, BAL cells and PBMC were comprehensively characterized by cell surface markers ex vivo. An increased percentage of DH24A+ polymorphonuclear cells, and decreased percentages of CD14+ macrophages were detected in BAL from horses with sEA compared to healthy horses or horses with mEA, while lymphocyte proportions were similar between all groups. Independently of EA, macrophages in BAL were CD14+CD16+, which contrasts the majority of CD14+CD16- classical monocytes in PBMC. Percentages of CD16-expressing BAL macrophages were reduced in BAL from horses with sEA compared to healthy horses. While PBMC lymphocytes predominantly contain CD4+ T cells, B cells and few CD8+ T cells, BAL lymphocytes comprised mainly CD8+ T cells, fewer CD4+ T cells and hardly any B cells. These lymphocyte subsets' distributions were similar between all groups. After PMA/ionomycin stimulation in vitro, lymphocyte activation (CD154 and T helper cell cytokine expression) was analyzed in BAL cells of 26 of the horses and group differences were observed (p=0.01-0.11). Compared to healthy horses' BAL, CD154+ lymphocytes from horses with mEA, and CD4+IL-17A+ lymphocytes from horses with sEA were increased in frequency. Activated CD4+ T helper cells were more frequent in asthmatics' (mEA, sEA) compared to healthy horses' PBMC lymphocytes. In summary, FC analysis of BAL cells identified increased polymorphonuclear cells frequencies in sEA as established, while macrophage percentages were mildly reduced, and lymphocyte populations remained unaffected by EA. Cytokine production differences of BAL lymphocytes from horses with sEA compared to healthy horses' cells point towards a functional difference, namely increased local type 3 responses in sEA.

Two polymorphisms in the promoter region of macrophage migration inhibitory factor (MIF)—rs755622 and rs5844572—exhibit prognostic relevance in inflammatory diseases. The aim of this study was to investigate a correlation between these MIF promoter polymorphisms and the severity of hepatitis C virus (HCV)-induced liver fibrosis. Our analysis included two independent patient cohorts with HCV-induced liver fibrosis (504 and 443 patients, respectively). The genotype of the single nucleotide polymorphism (SNP) -173 G/C and the repeat number of the microsatellite polymorphism -794 CATT5–8 were determined in DNA samples and correlated with fibrosis severity. In the first cohort, homozygous carriers of the C allele in the rs755622 had lower fibrosis stages compared to heterozygous carriers or wild types (1.25 vs. 2.0 vs. 2.0; p = 0.03). Additionally, 7 microsatellite repeats were associated with lower fibrosis stages (<F2) (p = 0.04). Comparable tendencies were observed in the second independent cohort, where fibrosis was assessed using transient elastography. However, once cirrhosis had been established, the C/C genotype and higher microsatellite repeats correlated with impaired liver function and a higher prevalence of hepatocellular carcinoma. Our study demonstrates that specific MIF polymorphisms are associated with disease severity and complications of HCV-induced fibrosis in a stage- and context-dependent manner.

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