Adverse environmental effects have a significant impact on plants' life and influ-ence the plants' metabolome. Specific stress-related responses are manifested both on plant development and its metabolites. This thesis entitled "Plant metabolome and its response to stress" outlines the composition of plant metabolome and reviews stress-related metabolites and metabolomics processes. The experimental part summarizes the results of two independent experiments. First, Hordeum vulgare response to stress was followed via targeted LC-MS metabolomics and proteome analysis. Next, a drought response was analyzed in Solanum lycopersicum via GC-MS metabolome profiling. Results highlighted a tissue-specific response to stress and pinpointed several putative abiotic stress markers

According to the World Health Organization (WHO), M. tuberculosis, the causative agent of TB, accounts for approximately 1.7 million deaths annually. Further contributing causes to the worldwide TB incidence, is the widespread unavailability and ineffectiveness of TB vaccines, time consuming diagnostic methods and unsuccessful treatment approaches. Research for better characterising mycobacteria in general, or other Mycobacterium species, may help us to better understand M. tuberculosis and TB disease mechanisms, which will in turn lower the future TB disease prevalence, as this may lead to the development of better treatments, diagnostics and vaccines. Mycobacteria use various secretion pathways, including the ESX- or type VII secretion (T7S) system, to ensure transport across the complex cell wall. The genome of M. tuberculosis has five copies of a gene cluster known as the ESX gene cluster region, which is associated with virulence and viability of mycobacteria. The ESX-3 gene cluster is thought to be essential for growth of M. tuberculosis and proposed to be involved in iron / zinc homeostasis. Mycobacteria synthesise siderophores, which are proposed to be involved in the uptake of iron over their cell wall. M. tuberculosis are known to produce two types of siderophores, namely: carboxymycobactins and mycobactins. Loots and colleagues, however illustrated, that ESX-3 knockouts, show signs of iron overload, despite the absence of the mycobactins induced by knocking out the ESX-3 gene cluster. It was hypothesised, that this overload occurs due to an increase in exochelin synthesis, another iron uptake protein not associated with ESX-3, overcompensating for the perceived iron depletion in the knockout organism. A Metabolomics research approach was subsequently used in this study, to generate new information in order to better characterise the role of iron on the metabolism of these organisms, and additionally confirm the role of ESX-3 in iron uptake. In this study, we firstly determined the most optimal extraction conditions for this metabolomics investigation. Two extraction methods were subsequently investigated and compared, considering their repeatability and their respective capacities to extract those compounds which best differentiate the M. smegmatis ESX-3 knockouts and wild-type parent strains. Considering the results generated, the total metabolome method was chosen for further analyses, for the following reasons: 1) it is simpler, 2) faster, 3) showed better repeatability, 4) extracts those compounds best differentiating the compared groups and 5) has been previously described for metabolomics analyses characterising ESX-3 gene functionality, hence potentially allowing us to compare results to that previously generated and published data. Subsequently, we used the chosen extraction method, followed by GCxGC-TOFMS analysis of the separately cultured M. smegmatis wild-type sample extracts, cultured in normal, low and high iron conditions, to determine the influence of varying iron concentrations on the metabolome of this organism, by metabolomics comparisons of these groups. Following this, an identical research approach was used to compare the metabolome of a M. smegmatis ESX-3 knock-out strain, to that of a M. smegmatis wild type parent strain, both cultured in normal / standardised iron concentrations. Considering the results generated when comparing the metabolome of a M. smegmatis ESX-3 knock-out strain to that of a M. smegmatis wild type parent strain, the altered metabolome of the M. smegmatis ESX-3 knockouts correlated well to that of the M. smegmatis wild type cultured in elevated iron growth conditions. This suggests ESX-3 is involved in iron uptake, and that knocking out the ESX-3 gene cluster of M. smegmatis does in fact result in a metabolome profile suggesting iron overload, as was proposed by Loots et al (2012), most probably due the exochelins overcompensating for the absence of mycobactins, in M. smegmatis ESX-3 knockouts. / MSc (Biochemistry) North-West University, Potchefstroom Campus 2013.

Tuberculosis (TB) is one of the deadliest infectious diseases of our time, with 1.4 million deaths globally, recorded in 2010 (3800 deaths a day) by the World Health Organization (WHO). Currently, South Africa ranks third on the 2011 list of 22 high-burden TB countries in the world and it was estimated that each active-TB person could potentially infect 10–15 people annually. The WHO additionally reported that in the year 2009, 87% of all TB patients worldwide were successfully treated, with a treatment success rate of 74% reported for South Africa. Despite this however, non-adherence to anti-TB treatment is still a major issue, due to it resulting in a global increased prevalence of drug resistant TB and subsequently TB treatment failure. Treatment failure is thought to be caused by a number of factors, however, it still remains largely misunderstood. One aspect of this, that isn't clearly addressed in the literature, is the underlying variation in each patient, resulting in his/her varying reaction to the drug regimen, and hence it’s varying efficacy from one patient to the next. Furthermore, little is known about the underlying variation of the host to the primary TB infection or response to the TB disease state, and how some patients have more effective mechanisms for eliminating the infection, or recovering from the disease. Considering this, a metabolomics research study using GC×GC-TOFMS was conducted, in order to identify potential metabolite markers which may be used to better characterise the underlining mechanisms associated with poor treatment outcomes (treatment failure). The first aim was to evaluate the accuracy and efficiency of the methodology used, as well as to determine the capability and accuracy of the analyst to perform these methods. In order to evaluate the GCxGC-TOFMS analytical repeatability, one QC sample was extracted and injected repeatedly (6 times) onto the GC×GC-TOFMS. Similarly, the analyst's repeatability for performing the organic acid extraction and analyses was also determined, using 10 identical QC samples, which were extracted and injected separately. CV values were subsequently calculated from the collected and processed data as a measure of this. Of all the compounds detected from the 6 QC sample repeats used for GCxGC-TOFMS repeatability, 95.59% fell below a 50% CV value, and 93,7% of all the compounds analysed for analyst repeatability had a CV < 50. Subsequently, using the above metabolomics approach, in addition to a wide variety of univariate and multivariate statistical methods, two patient outcome groups were compared. A sample group cured from TB after 6 months of treatment was compared vs a sample group where treatment failed after the 6 month period. Using urine collected from these two patient groups at various time points, the following metabolomics comparisons where made: 1) at time of diagnosis, before any anti-TB treatment was administrated, 2) during the course of treatment, in order to determine any variance in these groups due to a varying response to the anti-TB drugs, 3) over the duration of the entire 6 months treatment regimen, in order to determine if differences exist between the two groups over time. A clear natural differentiation between the cured and failed outcome groups were obtained at time of diagnosis, and a total of 39 metabolites markers were subsequently identified. These metabolites were classified according to their various origins, and included (1) those associated with the presence of M. tuberculosis bacteria, (2) those resulting from an altered host metabolism due to the TB infection, and (3) metabolites of various exogenous origins. The detailed interpretation of these metabolites suggests that a possible underlying RCD or some sort of mitochondrial dysfunction may be present in the treatment failure group, which may also be induced through an external stimulus, such as alcohol consumption. We hypothesise that this may possibly result in a far greater severity to M. tuberculosis infection in this group, subsequently causing a reduced capacity for a successful treatment outcome, also considering the critical role of the mitochondria in the metabolism of anti-TB drugs. Furthermore, 20 metabolite markers were identified when comparing the two outcome groups during the treatment phase of this metabolomics investigation. A vast majority of these 20 metabolites were also identified as markers for time 0 (time of diagnosis). Additionally, metabolites associated with anti-TB drug induced side effects, were also found to be comparatively increased in the treatment failure group, indicative of more pronounced liver damage, accompanied by metabolites characteristic of a MADD metabolite profile, due to a deficient electron transport flavoprotein, confirming previous experiments done in rats. These side effects have also previously been implicated as a major contributor of poor treatment compliance, and ultimately treatment failure. Lastly, 35 metabolite markers were identified by time dependent statistical analysis and represented those metabolites best describing the variation between the treatment outcome groups over the entire study duration (from diagnosis, to week 26). This time dependent statistical analysis identified markers, using an alternative statistical approach, and confirmed previous findings and added in a better characterisation of treatment failure. Considering the above, we successfully applied a metabolomics approach for identifying metabolites which could ultimately aid in the prediction and monitoring of treatment outcomes. This additionally led to a better understanding and or characterisation of the phenomenon known as treatment failure, as well as the underlying mechanisms related to this occurrence. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2013

According to the World Health Organization (WHO), M. tuberculosis, the causative agent of TB, accounts for approximately 1.7 million deaths annually. Further contributing causes to the worldwide TB incidence, is the widespread unavailability and ineffectiveness of TB vaccines, time consuming diagnostic methods and unsuccessful treatment approaches. Research for better characterising mycobacteria in general, or other Mycobacterium species, may help us to better understand M. tuberculosis and TB disease mechanisms, which will in turn lower the future TB disease prevalence, as this may lead to the development of better treatments, diagnostics and vaccines. Mycobacteria use various secretion pathways, including the ESX- or type VII secretion (T7S) system, to ensure transport across the complex cell wall. The genome of M. tuberculosis has five copies of a gene cluster known as the ESX gene cluster region, which is associated with virulence and viability of mycobacteria. The ESX-3 gene cluster is thought to be essential for growth of M. tuberculosis and proposed to be involved in iron / zinc homeostasis. Mycobacteria synthesise siderophores, which are proposed to be involved in the uptake of iron over their cell wall. M. tuberculosis are known to produce two types of siderophores, namely: carboxymycobactins and mycobactins. Loots and colleagues, however illustrated, that ESX-3 knockouts, show signs of iron overload, despite the absence of the mycobactins induced by knocking out the ESX-3 gene cluster. It was hypothesised, that this overload occurs due to an increase in exochelin synthesis, another iron uptake protein not associated with ESX-3, overcompensating for the perceived iron depletion in the knockout organism. A Metabolomics research approach was subsequently used in this study, to generate new information in order to better characterise the role of iron on the metabolism of these organisms, and additionally confirm the role of ESX-3 in iron uptake. In this study, we firstly determined the most optimal extraction conditions for this metabolomics investigation. Two extraction methods were subsequently investigated and compared, considering their repeatability and their respective capacities to extract those compounds which best differentiate the M. smegmatis ESX-3 knockouts and wild-type parent strains. Considering the results generated, the total metabolome method was chosen for further analyses, for the following reasons: 1) it is simpler, 2) faster, 3) showed better repeatability, 4) extracts those compounds best differentiating the compared groups and 5) has been previously described for metabolomics analyses characterising ESX-3 gene functionality, hence potentially allowing us to compare results to that previously generated and published data. Subsequently, we used the chosen extraction method, followed by GCxGC-TOFMS analysis of the separately cultured M. smegmatis wild-type sample extracts, cultured in normal, low and high iron conditions, to determine the influence of varying iron concentrations on the metabolome of this organism, by metabolomics comparisons of these groups. Following this, an identical research approach was used to compare the metabolome of a M. smegmatis ESX-3 knock-out strain, to that of a M. smegmatis wild type parent strain, both cultured in normal / standardised iron concentrations. Considering the results generated when comparing the metabolome of a M. smegmatis ESX-3 knock-out strain to that of a M. smegmatis wild type parent strain, the altered metabolome of the M. smegmatis ESX-3 knockouts correlated well to that of the M. smegmatis wild type cultured in elevated iron growth conditions. This suggests ESX-3 is involved in iron uptake, and that knocking out the ESX-3 gene cluster of M. smegmatis does in fact result in a metabolome profile suggesting iron overload, as was proposed by Loots et al (2012), most probably due the exochelins overcompensating for the absence of mycobactins, in M. smegmatis ESX-3 knockouts. / MSc (Biochemistry) North-West University, Potchefstroom Campus 2013.

Tuberculosis (TB) is one of the deadliest infectious diseases of our time, with 1.4 million deaths globally, recorded in 2010 (3800 deaths a day) by the World Health Organization (WHO). Currently, South Africa ranks third on the 2011 list of 22 high-burden TB countries in the world and it was estimated that each active-TB person could potentially infect 10–15 people annually. The WHO additionally reported that in the year 2009, 87% of all TB patients worldwide were successfully treated, with a treatment success rate of 74% reported for South Africa. Despite this however, non-adherence to anti-TB treatment is still a major issue, due to it resulting in a global increased prevalence of drug resistant TB and subsequently TB treatment failure. Treatment failure is thought to be caused by a number of factors, however, it still remains largely misunderstood. One aspect of this, that isn't clearly addressed in the literature, is the underlying variation in each patient, resulting in his/her varying reaction to the drug regimen, and hence it’s varying efficacy from one patient to the next. Furthermore, little is known about the underlying variation of the host to the primary TB infection or response to the TB disease state, and how some patients have more effective mechanisms for eliminating the infection, or recovering from the disease. Considering this, a metabolomics research study using GC×GC-TOFMS was conducted, in order to identify potential metabolite markers which may be used to better characterise the underlining mechanisms associated with poor treatment outcomes (treatment failure). The first aim was to evaluate the accuracy and efficiency of the methodology used, as well as to determine the capability and accuracy of the analyst to perform these methods. In order to evaluate the GCxGC-TOFMS analytical repeatability, one QC sample was extracted and injected repeatedly (6 times) onto the GC×GC-TOFMS. Similarly, the analyst's repeatability for performing the organic acid extraction and analyses was also determined, using 10 identical QC samples, which were extracted and injected separately. CV values were subsequently calculated from the collected and processed data as a measure of this. Of all the compounds detected from the 6 QC sample repeats used for GCxGC-TOFMS repeatability, 95.59% fell below a 50% CV value, and 93,7% of all the compounds analysed for analyst repeatability had a CV < 50. Subsequently, using the above metabolomics approach, in addition to a wide variety of univariate and multivariate statistical methods, two patient outcome groups were compared. A sample group cured from TB after 6 months of treatment was compared vs a sample group where treatment failed after the 6 month period. Using urine collected from these two patient groups at various time points, the following metabolomics comparisons where made: 1) at time of diagnosis, before any anti-TB treatment was administrated, 2) during the course of treatment, in order to determine any variance in these groups due to a varying response to the anti-TB drugs, 3) over the duration of the entire 6 months treatment regimen, in order to determine if differences exist between the two groups over time. A clear natural differentiation between the cured and failed outcome groups were obtained at time of diagnosis, and a total of 39 metabolites markers were subsequently identified. These metabolites were classified according to their various origins, and included (1) those associated with the presence of M. tuberculosis bacteria, (2) those resulting from an altered host metabolism due to the TB infection, and (3) metabolites of various exogenous origins. The detailed interpretation of these metabolites suggests that a possible underlying RCD or some sort of mitochondrial dysfunction may be present in the treatment failure group, which may also be induced through an external stimulus, such as alcohol consumption. We hypothesise that this may possibly result in a far greater severity to M. tuberculosis infection in this group, subsequently causing a reduced capacity for a successful treatment outcome, also considering the critical role of the mitochondria in the metabolism of anti-TB drugs. Furthermore, 20 metabolite markers were identified when comparing the two outcome groups during the treatment phase of this metabolomics investigation. A vast majority of these 20 metabolites were also identified as markers for time 0 (time of diagnosis). Additionally, metabolites associated with anti-TB drug induced side effects, were also found to be comparatively increased in the treatment failure group, indicative of more pronounced liver damage, accompanied by metabolites characteristic of a MADD metabolite profile, due to a deficient electron transport flavoprotein, confirming previous experiments done in rats. These side effects have also previously been implicated as a major contributor of poor treatment compliance, and ultimately treatment failure. Lastly, 35 metabolite markers were identified by time dependent statistical analysis and represented those metabolites best describing the variation between the treatment outcome groups over the entire study duration (from diagnosis, to week 26). This time dependent statistical analysis identified markers, using an alternative statistical approach, and confirmed previous findings and added in a better characterisation of treatment failure. Considering the above, we successfully applied a metabolomics approach for identifying metabolites which could ultimately aid in the prediction and monitoring of treatment outcomes. This additionally led to a better understanding and or characterisation of the phenomenon known as treatment failure, as well as the underlying mechanisms related to this occurrence. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2013

<p>Medonosna pčela (Apis mellifera&nbsp; Linnaeus, 1758) je &scaron;iroko rasprostranjena vrsta iz grupe socijalnih insekata, glavni opra&scaron;ivač mnogih divljih biljaka i poljoprivrednih kultura. Pored njene primarne uloge u opra&scaron;ivanju, koristi se i za dobijanje pčelinjih proizvoda, pre svega meda, voska&nbsp; i propolisa,&nbsp; čime se dodatno nagla&scaron;ava njen privredni značaj. Uzimajući u obzir značaj medonosne pčele, a sa druge strane aktuelan problem pada broja kolonija, postoji potreba za razumevanje uzroka&nbsp; i&nbsp; procenu&nbsp; rizika koji leže u osnovi ovog kompleksnog problema. Stoga, istraživanja u ovoj doktorskoj disertaciji obuhvataju praćenje biohemijskih i fiziolo&scaron;kih parametara kod medonosne pčele kao pokazatelja stanja pčelinjih zajednica tokom letnjeg i zimskog perioda&nbsp; na stani&scaron;tima sa različitim antropogenim uticajem.&nbsp; U prvoj fazi istraživanja su izvedeni akutni laboratorijski testovi izlaganja&nbsp; medonosne pčele&nbsp; subletalnim dozama pesticida, tiakloprida i klotianidina, a dobijeni rezultati su&nbsp; ukazali&nbsp; na naru&scaron;enu neurotransmisiju, na izmene u aktivnosti antioksidativnih enzima i redoks statusa u ćeliji, kao i na imunosupresiju.&nbsp; U drugoj fazi istraživanja su analizirane kolonije&nbsp; medonosne pčele sa različitih lokaliteta&nbsp; na području AP Vojvodine, sa primenom stacionarnog i migratornog tipa pčelarenja, tokom letnjeg i zimskog perioda, a dobijeni rezultati su ukazali&nbsp; na značajne razlike&nbsp; u biohemijskom odgovoru medonosne pčele u zavisnosti od starosti pčela, sezone godine i tipa pčelinjaka. Na osnovu rezultata prethodne dve faze istraživanja, izvr&scaron;en&nbsp; je&nbsp; odabir uzoraka, kao i izbor parametara za&nbsp; treću fazu&nbsp; istraživanja,&nbsp; gde&nbsp; je praćeno stanje medonosne pčele&nbsp; iz stacionalnih pčelinjih zajednica, tokom letnjeg&nbsp; aktivnog perioda, kao i početkom&nbsp; i&nbsp; krajem&nbsp; zimskog perioda&nbsp; mirovanja pčela, sa tri lokaliteta u Republici Srbiji sa različitim stepenom urbanizacije i industrijalizacije: Beograd kao urbano, Susek kao ruralno i Zajača kao industrijsko područje. Dobijeni rezultati su ukazali na&nbsp; značajne razlike u zavisnosti od sezone&nbsp; godine, kao i&nbsp; lokaliteta sa različitim antropogenim uticajem.&nbsp; Zimske pčele, krajem perioda mirovanja&nbsp; u odnosu na početak mirovanja,&nbsp; karakteri&scaron;e vi&scaron;i&nbsp; nivo antioksidativne za&scaron;tite, udružen sa&nbsp; visokim&nbsp; stepenom&nbsp; oksidativnih o&scaron;tećenja, zatim&nbsp; bolja imunokompetencija, kao i niske energetske rezerve u masnom telu.&nbsp; Sezonskim povećanjem temperatura tokom godine povećava se sadržaj zasićenih, dok se smanjuje udeo nezasićenih masnih kiselina, gde se oleinska 18:1(9) kiselina izdvaja kao dominantna komponenta u ukupnom sadržaju masnih kiselina masnog tela&nbsp; pčela&nbsp; sa sve tri lokacije, tokom sva tri perioda.&nbsp; Niži nivo ukupnih ugljovodonika je jo&scaron; jedna fiziolo&scaron;ka karakteristika zimskih pčela. Takođe, zapažene su promene&nbsp; i&nbsp; u neurotransmisiji holinergičkog tipa tokom sezone, sa vi&scaron;im aktivnostima acetilholinesteraze kod pčela krajem zime i tokom leta.&nbsp; Metabolomičkom analizom hemolimfe pčela je&nbsp; identifikovano&nbsp; 125 metabolita, uključenih u 36 metaboličkih puteva, gde se kao biomarkeri od značaja za razlikovanje letnjih i zimskih pčela izdvajaju&nbsp; metaboliti u metabolizmu glicerolipida i aminokiseline glicin, cistein, glutamin, fenilalanin, prolin i lizin.&nbsp; Poređenjem dobijenih rezultata između različitih lokaliteta,&nbsp; pčele iz Beograda se izdvajaju sa nižim nivoom oksidativnog stresa,&nbsp; nižim aktivnostimaacetilholinesteraze, kao i fenol oksidaze i glukoza oksidaze, ključnih enzima u imunom odgovoru pčela. Pčele iz Suseka su se izdvojile sa najvećom masom masnog tela, &scaron;to ukazuje na dobre nutritivne i energetske rezerve u pčelama tokom zime i leta, dok se Beograd izdvaja sa značajno vi&scaron;im udelom nezasićenih masnih kiselina&nbsp; u masnom telu pčela.&nbsp; Takođe su zabeležene razlike u relativnoj zastupljenosti&nbsp; n-alkana u pčelama sa različitih lokaliteta, te je u Beogradu najzastupljeniji nonakozan&nbsp; (C29), dok je heptakozan (C27) najzastupljeniji u uzorcima pčela iz Suseka i Zajače.&nbsp; Analizom sadržaja metala u pčelama, pergi i medu, zapažamo da svaku sredinu karakteri&scaron;e specifičan profil zagađenja,&nbsp; kao i da je u medu znatno niža koncentracija svih analiziranih metala.&nbsp; Dobijeni rezultati pružaju dobru osnovu za dalja ispitivanja biolo&scaron;kog odgovora medonosne pčele, u pogledu uticaja faktora okruženja, a time i dalje definisanje odgovarajućih mera za&scaron;tite pčelinjih zajednica.</p> / <p>The&nbsp; honey&nbsp; bee&nbsp; (Apis&nbsp; mellifera&nbsp; Linnaeus,&nbsp; 1758)&nbsp; is&nbsp; a&sbquo;widespread&nbsp; social&nbsp; insect&nbsp; species&nbsp; and&nbsp; the&nbsp; main pollinator of various wild plants and agricultural crops.In addition to its primary role in pollination, the honey bee&nbsp; is&nbsp; also&nbsp; used&nbsp; for&nbsp; obtaining&nbsp; honeybee&nbsp; products, mostly honey, wax, and propolis, with a significant role in&nbsp; the&nbsp; economy.&nbsp; Number&nbsp; of&nbsp; honey&nbsp; bee&nbsp; colonies&nbsp; is declining,&nbsp; thus&nbsp; necessitating&nbsp; the&nbsp; assessment&nbsp; of underlying&nbsp; reasons&nbsp; and&nbsp; risks.&nbsp; Therefore,&nbsp; in&nbsp; this doctoral&nbsp; dissertation,&nbsp; biochemical&nbsp; and&nbsp; physiological parameters&nbsp; in&nbsp; the&nbsp; honey&nbsp; bee&nbsp; were&nbsp; monitored&nbsp; as indicators&nbsp; of&nbsp; the&nbsp; colonies&rsquo;&nbsp; condition&nbsp; during&nbsp; the summer&nbsp; and&nbsp; winter&nbsp; period&nbsp; in&nbsp; habitats&nbsp; with&nbsp; different anthropogenic&nbsp; impacts.&nbsp; In&nbsp; the&nbsp; first&nbsp; phase&nbsp; of&nbsp; the research,&nbsp; acute laboratory tests of honey bee exposure to&nbsp; sublethal&nbsp; doses&nbsp; of&nbsp; pesticides&nbsp; (thiacloprid&nbsp; and clothianidin)&nbsp; were performed and the obtained results indicated&nbsp; impaired&nbsp; neurotransmission,&nbsp; changes&nbsp; in&nbsp; the activity of antioxidant enzymes, and redox status in the cell,&nbsp; as&nbsp; well&nbsp; as&nbsp; immunosuppression.&nbsp; In&nbsp; the&nbsp; second phase&nbsp; of&nbsp; the&nbsp; research,&nbsp; honey&nbsp; bee&nbsp; colonies&nbsp; from different&nbsp; localities&nbsp; in&nbsp; the&nbsp; area&nbsp; of&nbsp; AP&nbsp; Vojvodina&nbsp; were analyzed,&nbsp; using&nbsp; the&nbsp; stationary&nbsp; and&nbsp; migratory&nbsp; types&nbsp; of beekeeping, during the summer and winter period, and the obtained results indicated significant differences in the&nbsp; biochemical response of the honey bee depending on the age of the bees, the season of the year, and the type of apiary. Based on the results of the previous two phases&nbsp; of&nbsp; the&nbsp; research,&nbsp; the&nbsp; selection&nbsp; of&nbsp; samples&nbsp; was performed,&nbsp; as&nbsp; well&nbsp; as&nbsp; the&nbsp; selection&nbsp; of&nbsp; parameters&nbsp; for analysis within the third phase of the research, where the&nbsp; condition&nbsp; of&nbsp; honey&nbsp; bees&nbsp; from&nbsp; stationary&nbsp; bee communities was monitored during the summer active period,&nbsp; as&nbsp; well&nbsp; as&nbsp; the&nbsp; beginning&nbsp; and&nbsp; end&nbsp; of&nbsp; winter dormancy of bees, from three localities in the Republic of&nbsp; Serbia&nbsp; with&nbsp; different&nbsp; degrees&nbsp; of&nbsp; urbanization&nbsp; and industrialization: Belgrade as urban, Susek as rural and<br />Zajaca&nbsp; as&nbsp; industrial&nbsp; area.&nbsp; The&nbsp; obtained&nbsp; results indicated&nbsp; significant&nbsp; differences&nbsp; depending&nbsp; on&nbsp; the season&nbsp; of&nbsp; the&nbsp; year,&nbsp; as&nbsp; well&nbsp; as&nbsp; the&nbsp; location&nbsp; with different&nbsp; anthropogenic&nbsp; influence.&nbsp; Older&nbsp; winter&nbsp; bees are&nbsp; characterized&nbsp; by&nbsp; a&nbsp; higher&nbsp; level&nbsp; of&nbsp; antioxidant protection,&nbsp; associated&nbsp; with&nbsp; a&nbsp; high&nbsp; degree&nbsp; of&nbsp; oxidative damage,&nbsp; better&nbsp; immunocompetence,&nbsp; as&nbsp; well&nbsp; as&nbsp; lowenergy reserves in the fat body. The seasonal increase in&nbsp; the&nbsp; temperature&nbsp; during&nbsp; the&nbsp; year&nbsp; increases&nbsp; the content&nbsp; of&nbsp; saturated&nbsp; while&nbsp; decreasing&nbsp; the&nbsp; ratio&nbsp; of unsaturated&nbsp; fatty&nbsp; acids,&nbsp; where&nbsp; oleic&nbsp; 18:1(9)&nbsp; acid stands&nbsp; out&nbsp; as&nbsp; the&nbsp; dominant&nbsp; component&nbsp; in&nbsp; the&nbsp; total fatty&nbsp; acid&nbsp; content&nbsp; of&nbsp; bee&nbsp; body&nbsp; fat&nbsp; from&nbsp; all&nbsp; three locations,&nbsp; during&nbsp; all&nbsp; three&nbsp; periods.&nbsp; The&nbsp; lower&nbsp; level&nbsp; of total&nbsp; hydrocarbons&nbsp; is&nbsp; another&nbsp; physiological characteristic&nbsp; of&nbsp; winter&nbsp; bees.&nbsp; Also,&nbsp; changes&nbsp; in cholinergic&nbsp; neurotransmission&nbsp; were&nbsp; observed&nbsp; during the&nbsp; season,&nbsp; with&nbsp; higher&nbsp; acetylcholinesterase&nbsp; activities in&nbsp; bees&nbsp; in&nbsp; late&nbsp; winter&nbsp; and&nbsp; summer.&nbsp; Metabolomic analysis of bee hemolymph identified 125 metabolites,included in 36 metabolic pathways, where metabolites in&nbsp; the&nbsp; metabolism&nbsp; of&nbsp; glycerolipids&nbsp; and&nbsp; amino&nbsp; acids glycine,&nbsp; cysteine,&nbsp; glutamine,&nbsp; phenylalanine,&nbsp; proline, and&nbsp; lysine&nbsp; stand&nbsp; out&nbsp; as&nbsp; biomarkers&nbsp; of&nbsp; importance&nbsp; for distinguishing summer and winter bees. By&nbsp; comparing the obtained results between different localities, honey bees&nbsp; from&nbsp; Belgrade&nbsp; stand&nbsp; out&nbsp; with&nbsp; lower&nbsp; levels&nbsp; of oxidative&nbsp; stress,&nbsp; lower&nbsp; activities&nbsp; of acetylcholinesterase&nbsp; as&nbsp; well&nbsp; as&nbsp; phenoloxidase&nbsp; and glucose oxidase, key enzymes in the immune response of&nbsp; bees.&nbsp; The&nbsp; honey&nbsp; bees&nbsp; from&nbsp; Susek&nbsp; had&nbsp; the&nbsp; largest mass of the fat body, which indicates good nutritional and&nbsp; energy&nbsp; reserves&nbsp; in&nbsp; bees&nbsp;&nbsp; during&nbsp; winter&nbsp; and summer,&nbsp; while&nbsp; the&nbsp; honey&nbsp; bees&nbsp; from&nbsp; Belgrade&nbsp; were shown&nbsp; to&nbsp; have&nbsp; a&nbsp; significantly&nbsp; higher&nbsp; ratio&nbsp; of unsaturated&nbsp; fatty&nbsp; acids&nbsp; in&nbsp; the&nbsp; fat&nbsp; body&nbsp; of&nbsp; bees&nbsp; during the&nbsp; year.&nbsp; There&nbsp; were&nbsp; also&nbsp; differences&nbsp; in&nbsp; the&nbsp; relative prevalence&nbsp; of&nbsp; n-alkanes&nbsp; in&nbsp; honey&nbsp; bees&nbsp; from&nbsp; different localities, nonacosane (C29) being the most common in Belgrade, while heptacosane (C27) is most prevalent in honey&nbsp; bee&nbsp; samples&nbsp; from&nbsp; Susek&nbsp; and&nbsp; Zajaca.&nbsp; By analyzing the metal content in honey bees, perga, and honey,&nbsp; it&nbsp; was&nbsp; noted&nbsp; that&nbsp; each&nbsp; environment&nbsp; is characterized by a specific pollution profile, as well as that the concentration of all analyzed metals in honey is significantly lower compared to samples of bees and perga.&nbsp; The&nbsp; obtained&nbsp; results&nbsp; provide&nbsp; a&nbsp; good&nbsp; basis&nbsp; for further studies of the biological response of&nbsp; the honey bee,&nbsp; in&nbsp; terms&nbsp; of&nbsp; the&nbsp; influence&nbsp; of&nbsp; the&nbsp; environmental factors,&nbsp; enabling&nbsp; further&nbsp; defining&nbsp; of&nbsp; appropriate measures for the protection of honey bee colonies.</p>

Pancreatic cancer is a malignant disease with an unfavorable prognosis. Currently, the diagnosis of pancreatic cancer is based mainly on CT, MR and EUS imaging methods, because a reliable biomarker of pancreatic cancer is not available. It is considered a success in the current clinical practice if patients suitable for surgical resection can be selected, because in this group the overall survival is slightly better than in the group where surgery is not technically possible. In the first part, the presented dissertation focuses on testing of already established imaging diagnostic methods used in common practice, i.e. EUS and EUS FNA, as well as modern examination possibilities, such as contrast endosonography, and the comparison of the accuracy of the methods used. The dominant role of the EUS FNA was confirmed. In the next phase of the work, the methodological procedure of EUS FNA collection and the preprocessing of pancreatic tissue samples was tested with regard to the quantity and quality for epigenetic examination and further testing using the prognostic role of KRAS and miR-21. It has been suggested that cytological smears are the most suitable source of biological material for DNA and miRNA analysis, where non-tumor tissue contamination is low. Although the prognostic role of KRAS is...

Aging is associated with natural biological changes, which result in a decrease in the functional capacity of the body, which is also accompanied by an increased incidence of inflammatory processes that contribute to the development of neurodegeneration. The intestinal microbiota and its interaction with the intestine and the central nervous system play a key role in maintaining the function of biological homeostatic systems at an older age. This opens the possibility of influencing or modifying human microbiota for the development of therapeutic strategies. Based on the analysis of stool samples by HPLC MS / MS, the study confirmed the effect of selected probiotics on the modification of metabolic pathways and the proper functioning of the microbiota per se. The greatest effects were observed on tyrosine metabolism, tryptophan metabolism, arachidonic acid metabolism and bile acid biosynthesis metabolism. A positive effect of selected probiotic bacteria was found in the memory component of cognitive functions. Significant improvements were observed in verbal learning and verbal memory. In subjects with mild cognitive impairment, the progression of cognitive deficit has been slowed. Significant improvement was also observed for executive functions. These findings were not followed by a subjective...

This thesis is focused on the possibility of analysis of volatile organic compounds produced by monocytes during sepsis. Method of comprehensive two-dimensional gas chromatography with mass spectrometric detection was chosen for this purpose. Content of the first part was the optimization of the method of two-dimensional gas chromatography for the determination of volatile organic compounds. In this part were gradually adjusted parameters of the gas chromatography method to achieve the maximum efficiency. Further were adjusted conditions of samples preparation. Content of the second part was the usage of already optimized method for the analysis of the samples set of monocytes. Samples were subjected to the action of different inhibitors of the immune system and stimulators simulating bacterial or yeast infection. Based on this analysis were identified some compounds, which are produced by monocytes under condition simulating the infection.

Tato diplomová práce se zabývá rekonstrukcí dynamického modelu produkce polyhydroxyalkanoátů (PHA) termofilní bakterií Schlegelella thermodepolymerans. První kapitola poskytuje čtenářům krátký úvod do systémové biologie a matematické teorie grafů. Na ni navazuje druhá kapitola zabývající se různými přístupy v dynamickém modelování, včetně běžně používaných nástrojů pro dynamickou analýzu komplexních systémů. Třetí kapitola pak sleduje další pojmy a možnosti týkající se analýzy modelu. Následující kapitola se zaměřuje na metabolomiku a často používané laboratorní techniky a pátá kapitola je pak věnována polyhydroxyalkanoátům, zejména jejich chemické struktuře a vlastnostem. V kapitole šesté je navržen obecný booleovský model pro produkci PHA termofilními bakteriemi. Kapitola sedmá se poté zaměřuje na zdokonalení modelu se zaměřením na S. thermodepolymerans. Výsledný dynamický model je podroben analýze a výsledky jsou diskutovány.