Notre étude est destinée en premier lieu à l'évaluation paléo démographique des squelettes humains retrouvés sur le site de Pratu Pha afin d'extrapoler le potentiel des ossements humains dans le but de comprendre la nature bio-culturelle des adaptations de la population protohistorique de Pratu Pha. Les caractéristiques squelettiques observées ont été interprétées et comparées avec des données issues des populations squelettiques du Sud et du Sud-est asiatique, et en particulier avec la population protohistorique de Han Chiang (2100 av. J.-C-200 ap. J.-C.), dans le Nord-Est de la Thaïlande. Afin de saisir les possibles tendances micro-évolutionnaires dans la morphométrie squelettique en Thaïlande, certains résultats de recherche effectuée dans le sous-continent indien ont été mentionnés et utilisés dans cette étude avec pour objectif de projeter un possible scénario évolutionnaire en Thaïlande au cours des 5000 dernières années. Les échantillons de Pratu Pha comprennent à la fois des individus mâles et femelles de différents âges. Les deux (sexes) sont probablement présents dans la collection, même si nous ne pouvons pas l'affirmer avec certitude. La population squelettique peut, cependant, être prise comme une représentation transversale de la société disparue. Les observations paléo-pathologiques fournissent. les preuves de la vie quotidienne et des modes de vie des gens. Plusieurs sources de données indiquent que la vie quotidienne était physiquement intense. Même s'il existe des fractures apparues peu avant Je moment de la mort, il n')' a pas d'indications de guerre systématique ou d'autres violences interpersonnelles. Le sous-continent indien fournil un excellent tableau de squelettes humains appartenant à diverses phases culturelles. Ces populations comprennent un riche éventail d'adaptations culturelles, y compris la chasse et la cueillette au Mésolithique, l'urbanisation au Harappan, l'agro-pastoralisme au Néolithique-Chalcolithique, el l'économie de l'Age de bronze au Mégalithique. Les comparaisons transculturelles du cas Indien montrent des différences dans les caractéristiques crâniennes des populations préagricoles et celles des premières agro-pastorales, avec notamment deux changements significatifs dans la morphométrie crânienne. Cette hypothèse, testée avec succès dans les divers si les du sud-continent indien, mérite d'être essayée en Thaïlande. / The study primarily aims for a palacodemographic assessment of human skeletons recovered from the Pratu Pha site in order to project the research potential of human bones for understanding the nature of bio-cultural adaptations of the protohistork Pratu Pha population. The observed skeletal features are interpreted and compared with available skeletal data from South and South-cast Asian skeletal populations, esp. the protohistoric Ban Chiang population (2100 BCE-200 AD) of Northeastern Thailand. To understand possible microevolutionary trends in skeletal morphometry in Thailand, some results of research work undertaken for the lndian sub-continent are mentioned and used in this study for projecting possible evolutionary scenario in Thailand for the last 5000 years. The Pralu Pha sample comprises both male and female individuals of varying ages. Both are probably present in the collection, though this cannot be stated with certainty. The skeletal population can be therefore taken as cross-section representation of the bygone society. The palaeopathological observations provide evidence of the daily lives and ways of life of the people. Several lines of evidence indicate that daily life was physically strenuous. While there are occurrences of fractures in these remains, including one possible case of fracture near the lime of death, there are no indications of systematic warfare or other interpersonal violence. The lndian subcontinent provides an excellent array of human skeletal evidence belonging to various cultural phases. These populations include a rich spectrum of cultural adaptations, including hunting and gathering in the Mesolithic, urbanization in the Harappan, agro-pastoralism in the Neolithic ­Chalcolithic, and Iron-Age economy in the Megalithic. Cross-cultural comparisons in the Indian context show differences in cranial features of pre-agricultural and early agro-pastoral populations revealed by two significant changes in cranial morphometry. The hypothesis tested successfully for sites in the Indian sub-continent is worth trying in Thailand.

Os polihidroxialcanoatos (PHA) são poliésteres bacterianos. Na sua biossíntese, a PHA sintase incorpora monômeros 3HA à cadeia polimérica. O objetivo deste trabalho foi estudar o potencial da PHA sintase de 2 isolados do gênero Burkholderia sp. na produção de copolímeros. Construiram-se linhagens recombinantes que abrigavam os genes da PHA sintase classe I, em mutantes de Pseudomonas sp. e Burkholderia sacchari, que não acumulam PHA. Foram realizados ensaios de acúmulo de PHA usando glicose como fonte de carbono, apresentando a produção de unidades de 3HB, 3HO e 3HD nas linhagens recombinantes de Pseudomonas sp. As linhagens recombinantes de B. sacchari incorporaram como único constituinte P(3HB). Ensaios de acúmulo de PHA foram realizados nas linhagens recombinantes de B. sacchari, usando como co-substratos diferentes ácidos graxos, sendo detectada a incorporação de unidades de 3HV e 3HHx além do 3HB, quando foram fornecidos acido hexanóico e valérico. Estes resultados indicam que as PHA sintases classe I são capazes de incorporar diferentes unidades monoméricas. / The polyhydroxyalkanoates (PHA) are bacterial polyester. In their biosynthesis, the PHA synthase incorporates monomers 3HA to the polymer chain. The objective of this work was to study the potential of PHA synthase of 2 isolates of the genus Burkholderia sp. in the production of copolymers. Were constructed recombinant strains that housed the genes of PHA synthase class I mutants of Pseudomonas sp. and Burkholderia sacchari, which do not accumulate PHA. PHA accumulation assays were performed using glucose as carbon source, showing the production of units of 3HB, 3HO and 3HD in recombinant strains of Pseudomonas sp. The recombinant strains of B. sacchari incorporated as single constituent P(3HB). PHA accumulation assays were performed on the recombinant strains of B. sacchari, Using as co-substrates different fatty acids, being detected the incorporation of units of 3HV and 3HHx beyond the 3HB, when were supplied hexanoic acid and valerico. These results indicate that the PHA synthases class I are able to incorporate different monomer units.

PHAs are a class of biopolymers consisting of (R)-3-hydroxy-fatty acids and are produced by the majority of eubacteria and some archaeal bacteria as carbon storage material. In general, PHA is synthesised when a carbon source is available in excess while another essential nutrient is limited. The key enzyme of PHA biosynthesis, the PHA synthase, catalyses the polymerisation of the substrate (R)-3-hydroxyacyl-CoA to PHA accompanied by the release of coenzyme A. PHA is stored intracellularly as inclusions, the so-called PHA granules. When the external carbon source becomes exhausted, bacteria can metabolise these carbon inclusions by degradation of the polymer. PHA granules are water-insoluble, spherical inclusions of approximately 50-500 nm in diameter which consist of a hydrophobic polyester core surrounded by a phospholipid layer with embedded and attached proteins. One could consider isolated PHA granules as bio-beads due to their structure and size. In this study we tested if the PHA synthase can be used as an anchor molecule in order to display proteins of interest at the PHA granule surface. Furthermore, these modified PHA granules were analysed for their potential applicability as bio-beads in biotechnological procedures. The concept of using the PHA synthase as granule-anchoring molecule for display of proteins of interest was established by the functional display of the ß- galactosidase at PHA granules. This “proof of concept” was followed by the display of biotechnologically more interesting proteins. The IgG binding domain of protein A as well as streptavidin, which is known for its biotin binding ability, were fused to the PHA synthase, respectively, and therefore localised at the PHA granule surfaces during PHA granule assembly, resulting in functional bio-protein A -beads and bio-streptavidin-beads. Moreover, their applicability in biotechnological assays was demonstrated. Recently, we fused the green fluorescent protein (GFP) to the PHA synthase and demonstrated that the PHA granule assembly does not start randomly distributed in the cytoplasm but occurred localised at or near the cell poles. To further investigate if the localisation of the PHA granule formation process is due to polar positional information inherent to the PHA synthase, different mutated versions of the PHA synthase of Cupriavidus necator were created and analysed for a potential alteration in localisation. Furthermore, the phasin protein PhaP1 of C. necator was fused to HcRed, a far-red fluorescent protein, and localisation studies were accomplished when the fusion protein was expressed under different conditions in Escherichia coli.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Os caprinos sÃo considerados importantes fontes de carne, leite e pele, e, portanto, desempenham importante papel econÃmico especialmente na regiÃo nordeste do Brasil Diante disso, o desenvolvimento de sistemas de cultivo de folÃculos ovarianos pode otimizar potencial reprodutivo desta espÃcie. O objetivo deste trabalho foi avaliar o efeito da lectin PHA de Phaseolus vulgaris sobre o desenvolvimento de folÃculos ovarianos caprino cultivados in vitro. FolÃculos secundÃrios (~ 0,2mm) foram isolados de cÃrtex ovariano e individualmente cultivados por seis dias em α-MEM+ suplementado com 0, 1, 10, 50, 100 o 200Âg/mL da lectina PHA. Ao final do perÃodo de cultivo, os nÃveis de RNAm para FSH-R PCNA e NOS1 nestes folÃculos foram quantificados por qRT-PCR . Os resultados mostraram que, apÃs o cultivo, todos os tratamentos foram capazes de manter a sobrevivÃncia folicula AlÃm disso, nÃo foram observadas diferenÃas significativas entre os tratamentos e o control no aumento do diÃmetro folicular durante o cultivo. Entretanto, a PHA na concentraÃÃo de 10Âg/mL promove uma maior taxa de formaÃÃo de antro em relaÃÃo ao controle (α-MEM+ Nessa concentraÃÃo houve maior expressÃo dos nÃveis de RNAm para FSH-R e PCNA. Em conclusÃo, a PHA na concentraÃÃo de 10Âg/mL mostrou aÃÃo biolÃgica na formaÃÃo antro estimulou um aumento nos nÃveis de expressÃo de RNAm para FSH-R e PCNA em folÃculo secundÃrios caprinos cultivados in vitro por 6 dias. / Goats are considered important sources of meat, milk and skin, and therefore play an important economic role, especially in northeastern Brazil. Thus, the development of culture systems can optimize the reproductive potential of this species. This study aimed to evaluate the effect of lectin PHA of Phaseolus vulgaris on the development of caprine secondary follicles cultured in vitro. Secondary follicles (~0,2mm) were isolated from the cortex of caprine ovaries and individually cultured for six days in α-MEM+ supplemented with PHA at concentrations of 0, 1, 10, 50, 100 or 200Âg/mL. The levels of mRNA for R-FSH, PCNA and NOS1 in these follicles were also evaluated. The results showed that, after culture, all treatments were capable of maintaining the follicular survival. Moreover, there was no significant difference among all treatments regarding follicular growth. However, PHA at concentrations of 10Âg/mL increased the antrum formation rate when compared with control. In this concentration, there was a higher expression of mRNA for R-FSH and PCNA. In conclusion, PHA at concentration 10Âg/mL showed a biological action in antro formation and stimulated an increase of levels of expression of RNAm in caprine ovarian follicles cultured for six days.

Os polihidroxialcanoatos (PHA) são poliésteres bacterianos. Na sua biossíntese, a PHA sintase incorpora monômeros 3HA à cadeia polimérica. O objetivo deste trabalho foi estudar o potencial da PHA sintase de 2 isolados do gênero Burkholderia sp. na produção de copolímeros. Construiram-se linhagens recombinantes que abrigavam os genes da PHA sintase classe I, em mutantes de Pseudomonas sp. e Burkholderia sacchari, que não acumulam PHA. Foram realizados ensaios de acúmulo de PHA usando glicose como fonte de carbono, apresentando a produção de unidades de 3HB, 3HO e 3HD nas linhagens recombinantes de Pseudomonas sp. As linhagens recombinantes de B. sacchari incorporaram como único constituinte P(3HB). Ensaios de acúmulo de PHA foram realizados nas linhagens recombinantes de B. sacchari, usando como co-substratos diferentes ácidos graxos, sendo detectada a incorporação de unidades de 3HV e 3HHx além do 3HB, quando foram fornecidos acido hexanóico e valérico. Estes resultados indicam que as PHA sintases classe I são capazes de incorporar diferentes unidades monoméricas. / The polyhydroxyalkanoates (PHA) are bacterial polyester. In their biosynthesis, the PHA synthase incorporates monomers 3HA to the polymer chain. The objective of this work was to study the potential of PHA synthase of 2 isolates of the genus Burkholderia sp. in the production of copolymers. Were constructed recombinant strains that housed the genes of PHA synthase class I mutants of Pseudomonas sp. and Burkholderia sacchari, which do not accumulate PHA. PHA accumulation assays were performed using glucose as carbon source, showing the production of units of 3HB, 3HO and 3HD in recombinant strains of Pseudomonas sp. The recombinant strains of B. sacchari incorporated as single constituent P(3HB). PHA accumulation assays were performed on the recombinant strains of B. sacchari, Using as co-substrates different fatty acids, being detected the incorporation of units of 3HV and 3HHx beyond the 3HB, when were supplied hexanoic acid and valerico. These results indicate that the PHA synthases class I are able to incorporate different monomer units.

O lixo urbano tem sido apontado como um dos maiores poluentes ambientais. O lixo plástico chega a representar 20% do volume do lixo doméstico. Como alternativa aos plásticos petroquímicos, produtos plásticos menos agressivos ao meio ambiente e mais biodegradáveis têm sido estudados, entre eles os polihidroxialcanoatos (PHA). Os PHA são poliésteres biodegradáveis acumulados como material de reserva por inúmeras bactérias e que possuem aplicabilidade comercial bastante abrangente. Recentemente, os actinomicetos passaram a ser estudados para a produção destes polímeros. Em trabalho prévio, 53 novas linhagens de actinomicetos produtoras de biopolímeros foram isoladas de solo. Neste trabalho foi feita a seleção das bactérias quanto aos polímeros produzidos em diferentes de carbono. Das quatro linhagens selecionadas, duas foram analisadas quanto à produção de um novo polímero. Nas outras duas linhagens foram amplificados e estudados os genes sintetizadores dos polímeros. Em todas as linhagens foram feitas análises taxonômicas e cultivos em rejeitos industriais. / The urban waste has been described as one of the largest environmental pollutants. The plastic garbage can represent up to 20% of the volume of household waste. As an alternative to petrochemical plastics, plastic products less damaging to the environment and more biodegradable have been studied, among them polyhydroxyalkanoates (PHA). The PHA is a biodegradable polyester material accumulated as a reserve material by many bacteria and they have very broad commercial applicability. Recently, the actinomycetes have been studied for the production of polymers. In previous work, 53 new strains of actinomycetes producers of polymers were isolated from soil. In this work the bacterial the selection of bacteria was made concerning the polymers production on different carbon. Of the four strains selected, two were analyzed for the production of a new polymer. In the other two strains were amplified and studied the genes of polymers synthases. In all lineages were analyzed taxonomically and in cultivation on industrial waste.

The aim of this diploma thesis was study of the isolation of polyhydroxyalkanoates using a commercial surfactant and selected biosurfactants, which were sophorolipids, coconut soap, lecithin, lauryl glucoside, coco glucoside and cocamidopropyl betaine. PHA was isolated from Schlegelella thermodepolymerans DSM 15344, where the amount of NaCl in the production medium was first optimized. The molecular weight of the isolated polymer was analyzed by SEC-MALS. The largest structural changes were recorded for coconut soap. Possible impurities in the isolated polymer were analyzed by infrared spectrometry (ATR-FTIR). The least protein contamination of the polymer was recorded with coconut soap. From the above biosurfactants, coconut soap, lauryl glucoside and coco glucoside were selected, because the highest purity of PHA was obtained. However, coconut soap had the greatest potential. The isolation temperature and surfactant concentration were optimized for selected biosurfactants. The best results were obtained at 90 °C and a concentration of 5 g/L. Furthermore, the versatility of the isolation method was tested using selected biosurfactants on various thermophilic microorganisms, which were Chelatococcus composti DSM 101465, Schlegelella thermodepolymerans DSM 15264, Tepidimonas taiwanensis LMG 22826 and Aneurinibacillus thermoaerophilus H1.

This diploma thesis deals with identification of bacteria which are capable of producing polyhydroxyalkanoates (PHAs). Work included testing variety of genera including Pseudomonas, Lactobacillus, Bifidobacterium, thermophilic cultures and samples gathered from natural sources. Bacteria were investigated by molecular technique polymerase chain reaction – PCR. An amplification of the PHA synthase gene (phaC) was analyzed. In the first reaction phaC and 16S rRNA genes were tested at the same time. 16S rRNA gene is used as control for bacterial DNA and as an identification tool for natural source samples. This multiplex PCR used multiple primers in PCR mix. Second reaction search for amplicon specific for catalysing biosynthesis mcl-PHA (phaC1). The presence of the PHA synthase gene was verified in 11 samples which were Bifidobacterium breve CCM 7825T, Lactobacillus rhamnosus CCM 1825T, Lactobacillus zeae CCM 7069T, Lactobacillus delbrueckii subsp. bulgaricus CCM 7190T, Lactobacillus plantarum CCM 7039T, Pseudomonas gessardii, Pseudomonas fulva, Arthrobacter protophormiae, Curtobacterium flaccumfaciens, Mycobacterium neoaurum and Staphylococcus lentus.

I framtiden kommer avfall att behöva hanteras cirkulärt och resurser tas om hand för att möta hårdare framtida miljökrav. Ett miljöproblem som lyfts de senaste åren är nedskräpning där plast ofta nämns som ett exempel. Plast är ett viktigt material och används i stora delar av samhället. Det är hållbart och har ett högt nedbrytningsmotstånd vilket har bidragit till materialets popularitet. Dock leder dess egenskaper till problem om materialet hamnar i naturen eller havet. Ett alternativ till konventionellt tillverkad plast som senare år har fått uppmärksamhet är bioplast. Ett sätt att tillverka bioplast är från polymerer som kallas polyhydroxyalkanoater (PHA). PHA är biologiskt lättnedbrytbara, biobaserade och förnyelsebara polymerer och benämns som framtidens polymerer av forskare. Polymererna kan tillverkas från mixade bakteriekulturer som bland annat hittas i skogsindustriella avloppsvatten. Fördelar med att använda en mixad istället för en monobakteriekultur är reducerade kostnader i produktionen. De höga kostnaderna för produktionen är en nackdel och lyfts fram som ett hinder för att en storskalig industriell produktion kan ske lönsamt enligt tidigare studier. Kostnaderna för extraktionen av polymererna utgör ungefär 70 % av de totala produktionskostnaderna. I det här arbetet har sex olika teoretiskt uppbyggda system som producerar färdiga PHA polymerer från skogsindustriellt avloppsvatten undersökts med Gruvöns bruks biologiska reningsanläggning, Multibio, som utgångspunkt. Systemen som undersöktes var kombinationer av olika sätt att fermentera avloppsvattnet och därefter extrahera polymererna. Målet var finna det system som var mest ekonomiskt försvarbart med ett syfte att analysera olika system som kan bidra till en ekonomisk hållbar tillväxt. För att beräkna flöden av massa och energi upprättades en beräkningsmodell i Matlab Simulink, där dagsdata från 2019 matades in. Resultaten från mass- och energianalysen importerades därefter till Microsoft Excel och en kalkyl byggdes. Resultaten visade att systemet med fermentering vid inloppstemperaturen på flödet och en kombination av högtryckshomogenisering och enzymextraktion producerade upp till 650 ton PHA per år och även generade en vinst på cirka 20 miljoner kronor om året. Med hjälp av nuvärdesmetoden itererades en högsta tillåten grundinvestering fram för att systemet ska vara avbetalat efter 15 år som slutade på cirka 150 miljoner kronor. I studien förekommer osäkerheter som efter analyser och diskussion är rekommenderade att utredas vidare som bland annat efterhanteringen av de nya kemikalier som uppstår med det nya systemet och indata på priser till kalkylen. Av studiens resultat dras slutsatsen att potential finns för att produktion av PHA från skogsindustriellt avloppsvatten kan ske med en ekonomisk hållbarhet men att fortsatta studier är nödvändiga på grund av osäkerheterna i arbetet. / In the future, waste will need to be handled in a circular manner and resources be taken care of to meet the tougher environmental requirements of the future. An environmental problem that has been highlighted in recent years is littering where plastics are often cited as an example. Plastic is an important material and is used in large parts of society. It is durable and has a high degradation resistance which has contributed to the material's popularity. However, its properties often lead to complications if the material ends up in nature or the ocean. An alternative to conventionally manufactured plastics that has received attention in recent years is bioplastics. One way to make bioplastics is from polymers called polyhydroxyalkanoates (PHA). PHAs are biodegradable, biobased and renewable polymers and are referred to as the “polymers of the future” by researchers. The polymers can be made from mixed bacterial cultures which can be found in forest industrial wastewater. Benefits of using a mixed rather than a monobacterial culture are the reduced costs in production. The high cost of production is a disadvantage and is highlighted as an obstacle to a large-scale industrial production being profitable according to previous studies. The cost of extraction of the polymer represents about 70% of the total production cost. In this work, six different theoretically constructed systems that produce ready-made PHA polymers from forest industrial wastewater have been investigated using Gruvön mill's biological wastewater treatment plant, Multibio, as a starting point. The systems investigated were combinations of different ways of fermenting the wastewater and then extracting the polymers. The goal was to find the system that was most economically defensible with the aim of analysing different systems that could contribute to economically sustainable growth. To calculate mass and energy flows, a model was established in Matlab Simulink, where daily data from 2019 were used. The results of the mass and energy analysis were then imported into Microsoft Excel and a spreadsheet was built. The results showed that the system of fermentation at the inlet temperature of the flow and a combination of high-pressure homogenization and enzyme extraction produced up to 650 tonnes of PHA per year and generated a yearly profit for about 20 million SEK. Using the present value method, the maximum allowable basic investment was iterated until the system would be paid off after 15 years, which ended at about 150 million SEK. The study contains uncertainties, that after analysis and discussion are recommended to be further investigated, such as the handling of the new chemicals that arise with the new system and input on prices for the calculations. The study concludes that there is potential for production of PHA from forest industrial wastewater with economic sustainability, but that further studies are necessary due to the uncertainties in the thesis.

In vivo production of medium-chain-length poly(3-hydroxyalkanoates) (MCL-PHA) containing a side chain carboxyl group from azelaic acid (AzA), a nine-carbon α,ω-dicarboxylic acid, was investigated using Pseudomonas citronellolis DSM 50332 in a phosphate (P)-limited chemostat. Co-feeding with nonanoic acid (NA) and inhibition of β-oxidation with acrylic acid (AA) were strategies that were used to stimulate the incorporation of carboxylated monomers, but both were unsuccessful. P. citronellolis DSM50332 was capable of growing on AzA as a sole source of carbon and energy, indicating that enzymes in β-oxidation utilized AzA and its derivatives. However, the MCL-PHA produced from AzA comprised 3-hydroxyoctanoate (C8) and 3-hydroxydecanoate (C10) monomers, which was consistent with precursor supplied via the de novo fatty acid biosynthesis pathway. This evidence suggests that one or more of 3-ketoacyl-CoA reductase (FabG), enoyl-CoA hydratase (PhaJ) and PHA synthase (PhaC) of this organism do not have the low specificity required to utilize a carboxylated substrate. Future work involving mutations may broaden the substrate specificity of these key enzymes to overcome this obstacle. Two-stage high-cell density carbon (C)-limited chemostat cultivation of P. putida KT2440 was examined for MCL-PHA production from nonanoic acid (NA) at high intracellular polymer content and volumetric productivity. Growth conditions stimulating good PHA production were first established in single-stage chemostat, which yielded 63.1 wt% PHA containing 90 mol% C9 units and a productivity of 1.52 g L-1h-1 at a dilution rate of 0.30 h-1. This productivity was higher than any value reported in literature for continuous MCL-PHA production systems and comparable to the upper range of fed-batch results. Two-stage production yielded promising results, notably the increase in polymer content from the first to second stage. However, complications involving foaming and an unexplained decline in PHA content adversely affected system performance. The best PHA content and overall productivity were 58.5 wt% and 0.76 g L-1h-1, respectively. Nonetheless, the results demonstrate the potential to achieve high PHA content without the need for pure oxygen at high dilution rates, warranting further investigation focusing on the optimization of growth conditions. / Thesis (Master, Chemical Engineering) -- Queen's University, 2011-12-19 15:48:21.808