No / In recent years, different genes and proteins have been highlighted as potential biomarkers for psoriasis, one of the most common inflammatory skin diseases worldwide. However, most of these markers are not only psoriasis-specific but also found in other inflammatory disorders. We performed an unsupervised cluster analysis of gene expression profiles in 150 psoriasis patients and other inflammatory skin diseases (atopic dermatitis, lichen planus, contact eczema, and healthy controls). We identified a cluster of IL-17/tumor necrosis factor-α (TNFα)-associated genes specifically expressed in psoriasis, among which IL-36γ was the most outstanding marker. In subsequent immunohistological analyses, IL-36γ was confirmed to be expressed in psoriasis lesions only. IL-36γ peripheral blood serum levels were found to be closely associated with disease activity, and they decreased after anti-TNFα-treatment. Furthermore, IL-36γ immunohistochemistry was found to be a helpful marker in the histological differential diagnosis between psoriasis and eczema in diagnostically challenging cases. These features highlight IL-36γ as a valuable biomarker in psoriasis patients, both for diagnostic purposes and measurement of disease activity during the clinical course. Furthermore, IL-36γ might also provide a future drug target, because of its potential amplifier role in TNFα- and IL-17 pathways in psoriatic skin inflammation.

No / The remote robotic exploration of extraterrestrial scenarios for evidence of biological colonization in 'search for life' missions using Raman spectroscopy is critically dependent on two major factors: firstly, the Raman spectral recognition of characteristic biochemical spectral signatures in the presence of mineral matrix features; and secondly, the positive unambiguous identification of molecular biomaterials which are indicative of extinct or extant life. Both of these factors are considered here: the most important criterion is the clear definition of which biochemicals truly represent biomarkers, whose presence in the planetary geological record from an analytical astrobiological standpoint will unambiguously be indicative of life as recognized from its remote instrumental interrogation. Also discussed in this paper are chemical compounds which are associated with living systems, including biominerals, which may not in themselves be definitive signatures of life processes and origins but whose presence provides an indicator of potential life-bearing matrices.

Yes / Personalized medicine through Pharmacogenomics: choosing the right drug, and the right dose, for the right patients based on patient’s genetic makeup-is gradually being realised in Western countries. Yet, the practice of pharmacogenomics in Asian countries lags behind that of the West, but the medical needs for pharmacogenomics are expected to surge as better patient care is demanded in Asia. As next-generation sequencing technology advances quickly, previous technical challenges for performing pharmacogenomic studies or practices in Asia have been mostly resolved. What is lacking in Asia is an effective model of community-wide pharmacogenomics. On the delivery front, pharmacists, the drug and dosing professionals, can potentially be the main healthcare providers for pharmacogenomic services in Asia. The first large “Genomics for Precision Drug Therapy in the Community Pharmacy” in Canada, which is close to its completion, has successfully identified community pharmacists as key contact professionals for smooth facilitation and implementation of pharmacogenomics for personalized medication. It is anticipated that Asian pharmacists, with appropriate training, can have the capacity to provide expert pharmacogenomic supports for both physicians and patients in Asia. / The School of Biomedical Sciences Start-up Fund, the Chinese University of Hong Kong, the General Research Fund (#17114814; #17121616), the Theme-based Research Scheme (T12-401/13-R), Research Grant Council, Hong Kong, as well as the Hong Kong Cancer Fund, Hong Kong.

Yes / Background: This study investigated predictors of response to topical diphenylyclopropenone (DPCP) immunotherapy in patients with alopecia areata (AA). Objective: To identify predictors of response, or resistance, to treatment for AA through clinical observations and serum tests. Methods: Eighty four AA patients were treated with DPCP. Serum cytokine levels were measured in 33 AA patients pre- and post-treatment, and in 18 healthy controls, using ELISA assays. Results: Of patients, 56.1% responded to DPCP with satisfactory hair regrowth; the response rate was negatively correlated with hair loss extent. Before DPCP treatment, higher serum IFN-γ and IL-12 cytokine levels were observed in AA patients compared to healthy controls. Non-responders to DPCP had significantly elevated serum IL-4 pre-treatment (3.07 fold higher) and lower IL-12 levels compared with responders. After DPCP treatment, non-responders had persistently high IL-4, increased IL-12, negligible decrease in IFN-γ and decreased IL-10. Post-treatment DPCP responders exhibited significantly decreased IFN-γ and IL-12, and increased IL-4 and IL-10. Development of adverse side-effects was significantly associated with higher pre-treatment serum IgE levels. Limitations: A small number of subjects were evaluated. Conclusions: Potentially, elevated pre-treatment serum levels of IL-4 and IL-12 can be used as unfavorable and favorable predictors of DPCP therapeutic effect, respectively. In addition, pre-treatment elevated serum total IgE may predict increased risk for severe adverse side-effects to DPCP application. Whether serum cytokine expression levels can be used as predictors of response to other forms of treatment is unknown, but it may warrant investigation in the development of personalized treatments for AA. / This work is supported by the National Natural Science Foundation of China (81573066) and Natural Science Foundation of Guangdong Province (2014A030313098) to Xingqi Zhang.

Despite improvements in therapy, the prognosis for non-small cell lung cancer (NSCLC) patients remains poor, and cure is only possible in localized tumors after surgical resection. A new generation of targeted cancer drugs has led to the expectation that lung cancer therapy can be significantly improved, but these drugs are today only an option in a small subset of NSCLC patients, and their effect is temporary. Therefore, the aim of this thesis was to characterize NSCLC in order to find new treatment targets and to evaluate biomarkers that further optimize therapy selection. In Paper I, the expression of the potential treatment targets claudin 6 and claudin 18.2 were evaluated based on immunohistochemical- and gene expression analysis. High ectopic protein and gene expression were demonstrated for both claudins in small subgroups of NSCLC. Clinical trials using humanized monoclonal antibodies against both proteins are ongoing in other cancer forms and may be extended to NSCLC. In Paper II, the prognostic impact of the inflammatory mediator cyclooxygenase 2 (COX-2) was evaluated. No prognostic significance was found in a meta-analysis incorporating gene expression data of 1337 NSCLC patients. Likewise, COX-2 protein expression in tumor cells was not associated with survival in two independent NSCLC cohorts. However, in one of the analyzed cohorts, higher COX-2 expression in the tumor stroma was associated with longer survival and may therefore be a subject for further investigation. In Paper III, tumor and stromal COX-2 protein expression was examined in patients treated with the COX-2 inhibitor celecoxib in order to evaluate if COX-2 expression is a predictive biomarker for benefit of celecoxib therapy. Celecoxib did not prolong overall survival neither in the whole cohort nor in patients stratified according to COX-2 expression in tumor or stromal cells. Noteworthy, a tendency towards longer survival was again demonstrated in patients with high COX-2 stromal expression. In Paper IV, the diagnostic methods for identification of ALK rearrangements were assessed in a large representative Swedish NSCLC population. Fluorescence in situ hybridization (FISH), as the diagnostic standard, was compared to two immunohistochemical assays. ALK gene expression levels were incorporated to supplement the molecular data. The frequency of ALK rearrangements was lower than previously reported. The different methods to detect the ALK fusion demonstrated overlapping results. However, the overlap was poor, so the methods cannot be regarded as interchangeable and should thereby be interpreted with caution when used in clinical diagnostics. In summary, this thesis applied an integrative translational approach to characterize potential new treatment targets and to evaluate the detection of existing predictive biomarkers in NSCLC.

There is an unmet need for novel biomarker discovery in Inflammatory Bowel Diseases (IBD) to aid clinical management in several clinical settings including diagnosis and prognosis. With an ever-advancing repertoire of biological therapies on the horizon, it is important to personalise treatments at an early stage. The aim of this thesis is to explore the clinical utility of novel blood-based biomarkers in diagnosis, disease classification and prognosis in 2 cohorts: newly diagnosed IBD and acute severe colitis. Investigating the circulating methylome, 290 probes exhibited Holm significant IBD-associated methylation differences, including VMP1/MIR21 (p=7.5×10-14) and RPS6KA2 (1.1×10-19) and were consistent within the European cohort. 11 Differentially methylated positions (DMPs) predicted treatment escalation after Holm adjustment (top probe p=0.003). A panel of 6 probes identified 2 patient subgroups that have significantly different disease courses (Hazard Ratio (HR) 10.5, 95%CI: 4.3-25.6; logrank p=1.5×10-24). The 6 probe marker outperformed conventional biomarkers in predicting treatment escalation (hsCRP > 4mg/L, HR 3.2(1.7-5.8), logrank p=0.0004 and Alb < 36g/L, HR 2.9(1.5-5.6), p=0.0001). Within the same cohort, a novel proximity extension assay (PEA) was then utilised to identify novel diagnostic and prognostic protein markers. 61 proteins were significantly associated with IBD including MMP12 (Holm-adjusted p=4.1×10-26). A total of 9 proteins predicted disease course in this cohort. Using a panel of 7 randomly selected top prognostic probes, 2 patient groups were identified that had significantly different disease courses: logrank p=2.2×10-10, HR 5.6(2.0-15.6), outperforming conventional biomarkers in predicting treatment escalation (hsCRP > 4mg/L, HR 3.2(1.7- 5.8), logrank p=0.0003 and Alb < 36g/L, HR 2.7(1.4-5.2), p=0.0004). In a subcohort, serum calprotectin (SC) and conventional blood markers were investigated for their utility in diagnosis and prognosis in IBD. SC performed at par with CRP at differentiating IBD from controls with an area under the curve (AUC) of 0.87 (CI 0.81-0.92). For prognostication, both albumin and SC remained significant predictors of treatment escalation in IBD (logrank test p=5.1×10-5). MicroRNAs (miRNA) are small non-coding nucleic acids that have the capacity to modulate gene expression. Using small RNA sequencing in acute severe colitis (ASUC) and healthy controls (HC), 10 serum-based miRNA markers were significantly associated with acute severe colitis, including miR-30a-5p. Validating the findings using qPCR, miR-30a-5p was downregulated in ASUC (p=0.003). Furthermore, miR30a-5p remained a significant predictor of eventual colectomy in acute colitis (logrank test p=0.0014). These data highlight the translational potential for methylation, miRNA and proteomic biomarkers in diagnosing and prognosticating in IBD.

Magister Chirurgiae Dentium (MChD) / Water-pipe tobacco smoking (WTS) is a form of tobacco use with different names. There is a misconception that passing tobacco smoke through water reduces its harmful effects to increase its popularity. One million individuals smoke water-pipe daily, resulting in approximately five million deaths per annum globally. The toxic effects of WTS are related to the several components of the tobacco mixture. WTS contains 100 times more tar, four-fold more nicotine, eleven-fold more Carbon Monoxide (CO), and two to five-fold more polycyclic aromatic hydrocarbons than cigarettes.

Artificial insemination (AI) using cryopreserved sperm has an important positive impact on cattle production. Fertility is the most critical trait controlling livestock production; however, molecular, cellular, and physiological determinants of bull fertility and sperm freezability are not well understood. Better understanding of molecular, cellular, and physiological underpinnings of bull fertility may increase the success rate of AI. The objective of this study was to test the hypothesis that expression dynamics of sperm nuclear proteins, post-translational modifications (PTM) of sperm Histone 4 (H4), and seminal plasma metabolome are associated with bull fertility and sperm freezability (P = 0.043). Flow cytometry experiments were conducted to quantify H4 and acetylated histone 4 (H4ac) in sperm from high and low fertility Holstein bulls. The analysis of flow cytometry experiments clarified that retained levels of H4ac in bull sperm are associated with bull fertility. In addition, gas chromatography-mass spectrometry (GC-MS) was applied to ascertain the amino acid concentration of seminal plasma from bull semen with various freezability. A total of 21 amino acids and isomers were identified, and phenylalanine was positively associated with sperm post-thaw viability (r = 0.57, P-value = 0.043). Lastly, a quantitative western blotting experiment was utilized to ascertain relative quantification of sperm nuclear proteins including protamine 1 (PRM1), protamine 2 (PRM2), Histone 3 (H3), and H4. Also, sperm functional parameters including acrosome reaction, DNA fragmentation index, PAWP expression were analyzed using flow cytometry. In addition, immunocytochemistry experiments were applied to analyze sperm chromatin decondensation ability. The analyses of western blotting experiments revealed that the relative abundance of PRM2 in poor freezability sperm (PF) was greater than those in good freezability sperm (GF) (P = 0.0259). The relative abundance of retained H3 was greater in PF bulls than in GF bulls (1.02 ± 0.005 and 0.969 ± 0.021, respectively; P = 0.0272). There was a positive correlation between the abundance of retained H4 and sperm decondensation state (r = 0.71, P = 0.05). These results are important because they can help advance fundamental andrology and the assisted reproductive technologies both for cattle and other mammals, including humans and endangered species.

Brown algae have been the focus of metal ecotoxicology research for over 60 years, mainly because of their high metal accumulation capacity and reputed resistance. Now that Ectocarpus siliculosus has been positioned as a model for the study of brown algae, and that the genome has been recently sequenced and annotated, new lines of research have been made possible on these ecologically and economically important organisms, including the field of ecotoxicology. Several strains of E. siliculosus have been collected and isolated from locations around the world, thus providing the opportunity to study inter-population differences in their responses to environmental stress. This investigation can be split into three main sections. In the first part Cu exposure experiments were carried out under laboratory conditions using three strains of E. siliculosus: Es524 from a Cu polluted location in Chile, REP10-11 from a metal polluted (including Cu) location in England and LIA4A from a pristine site in Scotland. Strains were exposed for 10 d to concentrations ranging between 0 and 2.4 μM Cu. We measured different parameters: relative growth rates; metal accumulation (extracellular and intracellular); phytochelatins and the expression of related enzymes; oxidative stress responses as manifested in lipid peroxidation and levels of H2O2, and levels of pigments; levels of antioxidants glutathione and ascorbate (in reduced and oxidised forms), and phenolic compounds; and the activity of the antioxidant enzymes superoxide dismutase, catalase, and ascorbate peroxidise. Strain Es524 was the most efficient in counteracting the effects of Cu stress as manifested by a combination of Cu exclusion production of metal chelators, upregulation of oxidative enzymes, and strong antioxidant metabolism. REP10-11 also showed effective Cu defences, especially related to glutathione-ascorbate interactions. LIA4A was the least tolerant strain, with metabolic defences significantly less effective against Cu exposure. In part two a novel transplantation experiment was developed to compare responses in the field with those obtained in the laboratory. The study was carried out at a metal polluted and a low-impacted site in central Chile using strain Es524 (as in the laboratory experiments) and Es147, isolated from a low metal-polluted site in Chile. From the biomass, we conducted similar measurements of Reactive Oxygen Metabolism (ROM) as for the laboratory experiments described in the first part. In agreement with the laboratory experiments, strain Es524 displayed a higher resistance to metal stress. Because they behaved similarly between strains, the best suggested biomarker candidates for future assessments are metal accumulation, glutathione and ascorbate in reduced and oxidised forms, phenolic compounds, and the activity of superoxide dismutase. The method is simple, widely applicable in temperate environments, cost-effective, and provides a reliable representation of metal bioavailability in the environment. In the final part of the study a novel technique for the co-extraction of RNA and DNA, using a high pH Tris-HCl buffer, from small amounts of biomass of different strains of E. siliculosus was successfully developed. The extraction of nucleic acids from brown algae is considered to be difficult and the product is of poor quality due to the high concentrations of interfering secondary metabolites such as phenolics and polysaccharides. The protocol devised here provided high yields of pure RNA and DNA that are suitable for molecular analyses. This investigation provides new insights on metal stress metabolism in brown algae, and demonstrates that metal resistance is dependent on inherited defences developed over a long history of exposure. Furthermore, the good agreement between the results obtained in the laboratory with those from the field study confirms that the responses expressed under controlled laboratory conditions are representative of stress metabolism of E. siliculosus under natural conditions.

Low levels of survival motor neuron (SMN) protein cause the autosomal recessive neurodegenerative disease spinal muscular atrophy (SMA), through mechanisms that are poorly defined. SMN protein is ubiquitously expressed, however the major pathological hallmarks of SMA are focused on the neuromuscular system, including a loss of lower motor neurons in the ventral horn of the spinal cord and atrophy of skeletal muscle. At present there is no cure for SMA. Most research to date has focused on examining how low levels of SMN lead to pathological changes in motor neurons, therefore the contribution of other tissues, for example muscle, remains unclear. In this thesis I have used proteomic techniques to identify intrinsic molecular changes in muscle of SMA mice that contribute to neuromuscular pathology in SMA. I demonstrate significant disruption to the molecular composition of skeletal muscle in pre-symptomatic SMA mice, in the absence of any detectable degenerative changes in lower motor neurons and with a molecular profile distinct from that of denervated muscle. Functional cluster analysis of proteomics data and phospho-histone H2AX labelling of DNA damage revealed increased activity of cell death pathways in SMA muscle. In addition robust up-regulation of VDAC2 and down-regulation of parvalbumin was confirmed in two mouse models of SMA as well as in patient muscle biopsies. Thus intrinsic pathology of skeletal muscle is an important event in SMA. I then used proteomics to identify individual proteins in skeletal muscle of SMA that report directly on disease status. Two proteins, GRP75 and calreticulin, showed increased expression levels over time in different muscles as well as in skin samples, a more accessible tissue for biopsies in patients. Preliminary results suggest that GRP75 and calreticulin can be detected and measured in SMA patient muscle biopsies. These results show that proteomics provides a powerful platform for biomarker identification in SMA, revealing GRP75 and calreticulin as peripherally accessible potential protein biomarkers capable of reporting on disease progression in muscle as well as in skin samples. Finally I identified a role for ubiquitin-dependent pathways in regulating neuromuscular pathology in SMA. Levels of ubiquitin-like modifier activating enzyme 1 (UBA1) were reduced in spinal cord and skeletal muscle tissue of SMA mice. Dysregulation of UBA1 and subsequently the ubiquitination pathways led to the accumulation of β-catenin. I show here that pharmacological inhibition of β-catenin robustly ameliorates neuromuscular pathology in animal models of SMA. Interestingly, downstream disruption of β-catenin was restricted to the neuromuscular system in SMA mice. Pharmacological inhibition of β-catenin failed to prevent systemic pathology in organs. Thus disruption of ubiquitin homeostasis, with downstream consequences for β-catenin signalling, contributes to the pathogenesis of SMA, thereby highlighting novel therapeutic targets for this disease.