O estudo teve por finalidade avaliar a contaminação por aflatoxinas (AF) em três espécies de peixes no Estado de São Paulo, avaliando também a micobiota e a ocorrência das toxinas nas rações. Foram coletadas amostras de ração, em uso e em estoque, e amostras dos peixes lambari (Astyanax altiparanae), matrinxã (Brycon cephalus) e pacu (Piaractus mesopotamicus) em cinco pisciculturas localizadas no Estado de São Paulo. As amostras de ração foram avaliadas quanto à contaminação fúngica, classificando os Aspergillus quanto à espécie e ao potencial toxigênico. A contagem de bolores variou de 1,0 x 102 UFC/g até 4,0 x 104 UFC/g, sendo o maior valor encontrado em rações em uso. A atividade de água variou de 0,45 a 0,72. Na análise da micobiota da ração, o gênero Aspergillus foi encontrado em 100% das amostras avaliadas, além dos gêneros Penicillium, Fusarium e Cladosporium. Dentre os isolados de Aspergillus seção Flavi, 84,2% produziram aflatoxinas. Foram identificadas diferentes espécies de Aspergillus, sendo 42,1% classificados como Aspergillus flavus. A detecção e quantificação de aflatoxinas foram efetuadas por cromatografia líquida de alta eficiência (CLAE) em amostras de ração, tecido muscular e fígado dos peixes. AFB1 e AFG2 foram detectadas em 8,34% e 16,67% das amostras de ração, respectivamente. No tecido muscular, apenas uma amostra apresentou 5,4 µg AFM1/kg. Nas amostras de fígado, 50% apresentaram AFM1, variando de 2,3 a 17,1 µg/kg, e 16,67% apresentaram AFB1 em níveis de 8,9 a 12,7 µg/kg. Embora tenham sido encontrados níveis baixos de aflatoxinas na ração das propriedades investigadas, a detecção nos tecidos dos peixes sugere que os animais ingeriram a toxina em algum momento. Assim, pode-se concluir que o risco de contaminação por aflatoxinas em pisciculturas no estado de São Paulo existe e deve ser controlado. / The study aimed to evaluate the contamination by aflatoxins (AF) in three species of fish in the state of Sao Paulo, evaluating also the mycobiota and the occurrence of toxins in feed. It were collected feed samples, in use and stored, and fish samples: lambari (Astyanax altiparanae), matrinxã (Brycon cephalus) and pacu (Piaractus mesopotamicus) in five fish farming from state of Sao Paulo. Feed samples were evaluated for fungal contamination, classifying Aspergillus at species and their toxigenic potential. The mold count ranged from 1.0 x 102 CFU/g to 4.0 x 104 CFU/g, and the highest value was found in feed in use. The water activity ranged from 0.45 to 0.72. Regarding mycobiota analysis, the genus Aspergillus was found in 100% of the samples, as well Penicillium, Fusarium and Cladosporium genus were noted. Among the isolates of Aspergillus section Flavi, 84.2% produced aflatoxins. Different Aspergillus species were identified, with 42.1% classified as Aspergillus flavus. The detection and quantitation of aflatoxins in feed samples, fish muscle and fish liver were performed by high performance liquid chromatography (HPLC). AFB1 and AFG2 were detected in 8.34% and 16.67% of feed samples, respectively. In fish muscle, only one sample showed 5.4 µg AFM1/kg. In the liver samples, 50% presented AFM1, ranging from 2.3 to 17.1 µg/kg, and 16.67% had AFB1 at levels from 8.9 to 12.7 µg/kg. Despite the low levels of aflatoxin in the fish feed from the investigated properties, the detection in fish tissues suggests that animals could have ingested the toxin anytime. Thus, it can be concluded that the risk of aflatoxin contamination in fish farming in the state of Sao Paulo exists and it must be controlled.

O aumento da complexidade dos resíduos produzidos nos processos de mineração, que acompanha a crescente expansão deste setor no Brasil, tem revelado a necessidade de novas técnicas para o tratamento ecologicamente correto dos rejeitos de mineração. Desta forma, a biorremediação, uma técnica de baixo custo que utiliza microrganismos extremófilos na recuperação de metais tóxicos, se apresenta como um método economicamente viável no tratamento dos rejeitos contendo íons metálicos. Assim, mediante uma análise proteômica dos fungo isolados do ambiente de mineração, quando submetidos a estresse com ions de cobre, podemos compreender seu comportamento fisiológico no processo de biorremediação. Para isso, Aspergillus niger VC e Rhizopus microsporus VC, isolados do ambiente de mineração foram incubados junto a íons de cobre e mediante analise proteômica foram identificados biomarcadores de estresse oxidativo nos fungos. O proteoma realizado utilizando a técnica de NanoLC-ESI-Q-TOF identificou a expressão de proteínas que mudaram sob a influência do cobre em comparação a seus respectivos controles. Os resultados mostraram que as duas cepas obtidas do ambiente de mineração possuem diferentes mecanismos de resistência. Sendo assim, A.niger VC apresentou uma redução na expressão proteica, das quais 132 foram diferencialmente expressas na presença de íons Cu2+. Por sua vez, a cepa R.microsporus VC exibiu uma superexpressão proteica, com 389 proteínas expressas na presença do metal. Nestes cultivos foram identificadas proteínas relacionadas ao choque térmico e à adsorção de metais, conhecidas como proteínas de choque térmico (HSPs) e metaloproteínas, produzidas em resposta ao estresse imposto pela presença do agente indutor de estresse. No entanto, as enzimas envolvidas na defesa contra o estresse oxidativo, identificadas na ausência de metal, são um indicativo de adaptação metabólica em resposta ao ambiente de mineração. Assim concluímos que tanto A.niger VC como R.microsporus VC, são fungos que apresentam importantes características que permitem sua utilização como agentes de biorremediação dos rejeitos de mineração. / The increase in the complexity of the waste produced in the mining process, with the growing expansion of this sector in Brazil, has revealed the need for new techniques for the ecologically correct treatment of this toxic waste. Therefore, bioremediation, a low cost technique that uses endophilic microorganisms in the recovery of toxic metals, presented as an economically viable method in the treatment of metal ion-containing wastes. In this way, through proteomic analysis of the isolated fungus from the mining environment, when submitted to stress with copper ions, we can understand their physiological behavior in the bioremediation process. In this regard, Aspergillus niger VC and Rhizopus microsporus VC, isolated from the mining environment were incubated with copper ions and in result of proteomic analysis, biomarkers of oxidative stress were identified in the fungus. The proteome performed using the NanoLC-ESI-Q-TOF technique identified the expression of proteins that changed under the copper influence compared to their respective controls. The results showed that the two strains obtained from the mining environment have different resistance mechanisms. Thus, A.niger VC presented a reduction in protein expression, of which 132 were differentially expressed in the presence of Cu2+ ions. In turn, the strain R.microsporus VC exhibited a protein overexpression, with 389 proteins expressed in the metal presence. In these cultivations, proteins related to thermal shock and the adsorption of metals, known as HSPs and metalloproteins, produced in response to the stress imposed by the presence of the stress-inducing agent. However, the enzymes involved in defense against oxidative stress, identified in absence of metal, are indicative of metabolic adaptation in response to the mining environment. Therefore we conclude that both A.niger VC and R.microsporus VC are fungus that present important characteristics that allow their to be used as bioremediation agents for mining waste.

<p>Fungi display diverse growth forms. Some grow as unicellular yeasts, some grow as multicellular hyphae, while others switch between these two growth forms, i.e., the dimorphic fungi. Dimorphism is found in many pathogenic fungi, and it is thought to be a strategy to maximize their fitness during different stages of life cycles. The corn smut fungus <i>Ustilago maydis</i> serves as a renowned model organism for studying fungal dimorphism and its role in pathogenesis. However, knowledge only from the model species may not be expanded to other species unless multispecies studies have been demonstrated. In this dissertation, I performed comparative analyses to examine if knowledge from <i>U. maydis</i> is translational to other dimorphic fungi. First, a physiological study was conducted to find what can serve as a common signal for dimorphic transition of several Ustilaginomycotina species. I found that the lipid serves as a potential common cue for yeast-to-hyphal transition in most dimorphic species, while alternate types of energy-source carbohydrate do not affect fungal dimorphism. In addition, pectin and high temperature can also trigger filamentous growth in some Ustilaginomycotina species. Second, I performed comparative transcriptomics to determine if a mechanism for yeast-to-hyphal dimorphic transition is conserved across multiple dimorphic species. Three species of Ustilaginomycotina (<i>U. maydis</i>, <i>Tilletiopsis washingtonensis </i>and <i>Meira miltonrushii</i>) plus one species from Ascomycota (<i>Ophiostoma novo-ulmi</i>) were included in the analyses. I found that the similarity of transcriptomic alteration is not dependent on phylogenetic relatedness. Genes in amino acid transport and metabolism, energy production and conversion and cytoskeleton are commonly altered during the dimorphic transition of all studied species. Moreover, I discovered several core genes which can play a conserved role in transducing signals for the dimorphic transition. Finally, I performed comparative analyses of 190 fungal genomes to determine genomic properties that are associated with types of fungal growth form. I found that small genome size is a characteristic for yeast-like fungi. Few indicator genes, such as genes encoding proteins in the NADPH oxidase complex and cytoskeletons, which are predominantly lost in yeast-like fungi in both Ascomycota and Basidiomycota. However, many other genes are associated with types of growth form in a lineage-specific manner. Findings from this dissertation will serve as fundamentals for future research in fungal cell biology, especially in fungal dimorphism. Additionally, results from this study suggest cautions when extrapolating results from model species onto non-model species.</p>

During the development of the nervous system, neurons migrate to their final location and extend neurites that navigate long distances in the extracellular environment to reach their synaptic targets. The proper functioning of the nervous system depends on correct connectivity, and mistakes in the wiring of the nervous system lead to brain abnormalities and mental illness. Growth cones are motile structures located at the tip of extending neurites that sense and respond to guidance cues encountered along the path toward their targets. Binding of these cues to receptors located on growth cone filopodia and lamellipodia triggers intracellular signaling pathways that regulate growth cone cytoskeletal dynamics. Although studies on extracellular cues and their effects on neuronal guidance are well documented, less is known about the intracellular signaling mechanisms that regulate growth cone motility. This dissertation focuses on two signaling pathways and describes how they might be involved in determining growth cone morphology during neuronal development. The specific aims of this work address: (1) the role of phosphatidylinositol-3-kinase (PI-3K) and its downstream signaling pathway in regulating growth cone motility, and (2) the effect of nitric oxide (NO) release from a single cell on growth cone morphology of neighboring neurons. This study employs defined neurons from the pond snail, Helisoma trivolvis, to demonstrate that inhibition of PI-3K induces a concomitant increase in filopodial length and a decrease in the rate at which neurites advance. These effects are mediated through the lipid and protein kinase activities of PI-3K, and filopodial elongation is due to an increase in the rate at which filopodia elongate and the time that individual filopodia spend extending. Additionally, this study demonstrates that NO release from a single cell can affect growth cone dynamics on neighboring neurons via soluble guanylyl cyclase (sGC), and that NO has a physiological effect up to a distance of 100 ìm. Overall this study provides new information on cellular mechanisms regulating growth cone motility, and suggests a potential role of PI-3K and NO in neuronal pathfinding in vivo.

Sedan december 2003 har Kalmar läns slakteris (KLS) nya reningsverk varit i drift. Entreprenör för det nya reningsverket samt driftansvariga under det två första åren är Läckeby Water Group. Verket är av SBR-typ (Sekventiell Biologisk Rening) med biologisk kväverening och kemisk fällning av fosfor med hjälp av järnklorid. Från slakteriet leds avloppsvattnet genom en 2 km lång ledning ner till reningsanläggningen. Verket hade under 2004 problem med höga halter fosfor i utgående vatten, flertalet mekaniska haverier samt två perioder av slamsvällning. Slamsvällningen orsakades av filamentösa (trådformiga) bakterier, första gången av Thiothrix spp. och andra gången av Typ 021N. Syftet med examensarbetet var att finna orsaken till den senare slamsvällningen samt att söka förebyggande åtgärder mot Typ 021N. Examensarbetet utfördes genom litteraturstudier, laboratorieförsök, fullskaleförsök, genomgång av driftsdata samt mikroskopering av aktivt slam vid verket. Utifrån litteraturstudier konstaterades att filamentösa bakterier kan gynnas under perioder av låga syrehalter samt av låg näringstillförsel eftersom dessa bakterier har en högre tillväxthastighet vid låga substratkoncentrationer än flockbildande bakterier. Specifikt för Typ 021N är att dessa har möjlighet att utnyttja reducerat svavel som energikälla samt gynnas vid tillgång på korta lättnedbrytbara kolföreningar. Laboratorieförsök visade inte entydigt att låga fosfor eller syrehalter gynnade de filamentösa bakterierna. Inverkan av FeCl3, Ecofloc, PAXXL60, NaOCl och H2O2 studerades under korttids laboratorieförsök och effekten utvärderades i mikroskop. I några fall hämmades filamenten men aldrig utan att även påverka övriga mikroorganismer negativt. PAX-XL60 hämmade filamentförkomsten mest och påverkade andra organismer förhållandevis lite. Tillsats av PAX i filamenthämmande och flockbildande syfte utfördes därefter i fullskala. Effekten av tidigare tillsatser av NaOCl och H2O2 i filamenthämmande syfte studerades och visade sig ha givit varierande resultat. NaOCl visade sig effektivt bekämpa filamentösa bakterier i processen då inblandning skedde under rätt förutsättningar. Processdata för våren 2004 jämfördes med data från en period under hösten, vilken följdes av en slamsvällning. Perioderna visade stora skillnader m.a.p. syrehalt, temperatur, dosering av järnklorid och organisk belastning. En on-line mätning i inkommande vatten visade på mycket höga halter av svavelväte. Svavelväte bildas under anaeroba förhållanden t.ex. i stillastående avloppsvatten. Orsaker till slamsvällningen i september-oktober 2004 tros vara höga halter av svavelväte, perioder med låga syrehalter, höga vattentemperaturer samt tillgång på lättnedbrytbart organiskt material. Svavelvätet kan förslagsvis elimineras genom en tidsstyrd dosering av CaNO3 i inkommande ledning. Noggrann övervakning av syre samt tillgång på syre måste garanteras i processen. Det inkommande vattnets mikroflora kan förändras genom installation av en aerob selektor för att gynna de flockformande bakterierna. För att sänka fosforhalterna i utgående vatten samt att inte riskera fosforbrist i processen har en tillfällig efterfällning med extra tillsats av FeCl3 och polymer installerats. / In December 2003 the new plant treating slaughterhouse wastewater from KLS was taken into operation. Läckeby Water Group was entrepreneur and responsible for the maintenance during the following two years. The treatment plant is of SBR-type and has biological nitrate removal and chemical precipitation of phosphate with iron chloride. The wastewater from the slaughterhouse passes a 2 km long pipeline before entering the treatment plant. During 2004, the plant had problems with high levels of phosphorous in the effluent, several mechanical problems and two occasions of sludge bulking caused by filamentous bacteria. The first incident was caused by Thiothrix spp. and the second by Type 021N. The aim with the thesis was to find causes for the latest period of sludge bulking as well as investigate preparatory actions against Type 021N. The thesis included literature studies, laboratory and full-scale tests, evaluation of prior process data and continuous microscopic analysis of the activated sludge at the plant. The literature study showed that filamentous bacteria are favoured by low oxygen and low nutrient concentrations due to their possibly higher growth rate during low substrate concentrations. Type 021N, specifically, can use reduced sulphides as energy source and benefits from an excess of low molecular substrates. Laboratory experiments did not verify that the filamentous bacteria were favoured by low oxygen concentration or low phosphate levels. The effect of FeCl3, Ecofloc, PAX-XL60, NaOCl and H2O2 added to a bulking sludge was evaluated by microscopic analysis. No chemical was found to suppress the filamentous bacteria without also affecting the floc-forming bacteria negatively. PAX-XL60 showed the largest negative effects on filamentous bacteria and only a minor impact on other microorganisms. Full-scale tests with PAX were thereafter performed in order to suppress filamentous bacteria as well as flocculate particulate solids. The effect of earlier additions of NaOCl and H2O2 into the process gave varied results. NaOCl was efficient against filamentous bacteria when addition was made during correct circumstances. Process data from two separate periods during 2004 was compared. One period was followed by good effluent values and another period by a sludge bulking period. Large differences between the two periods were seen in oxygen conditions, temperature, FeCl3 dosage and organic load. Measurements on influent wastewater showed high levels of hydrogen sulphide, which can be produced during anaerobe conditions i.e. in stagnant sewage pipes. Likely causes for the sludge bulking in September-October 2004 were high levels of hydrogen sulphide in the influent, periods of insufficient oxygen concentrations, high water temperatures and access to easy degradable substrate. The hydrogen sulphide can be eliminated through time-controlled dosage of CaNO3 in influent pipeline. Sufficient oxygen levels must be guaranteed in the process. The microbiological fauna in influent can be changed by installation of an aerobe selector to benefit floc-forming bacteria. To lower the phosphorous levels in effluent water and not risk phosphorous deficiency in the process a post-precipitation have been installed. The post-precipitation include extra dosage of FeCl3 and polymer and a drum screen to minimize suspended solids.

Morphological transitions in Wangiella dermatitidis, a causative agent of human phaeohyphomycosis, influence virulence processes in this polymorphic fungus. My project first involved the cloning and characterizion of the β(1→3) glucan synthase gene WdFKS1, which encodes the enzyme's catalytic subunit, followed by cloning and characterizing the WdRHO1 gene, which encodes its regulatory subunit. To better understand the Rho-type GTPase-mediated regulation of cell polarity and its role in fungal morphological transitions, a homologue of WdRAC1 from a W. dermatitidis was subsequently identified by degenerate PCR and gene walking. Gene deletions of WdFKS1 and WdRHO1 in haploid W. dermatitidis were lethal, whereas the deletion of WdRAC1 was not. RNA interference on WdFKS1 mRNA expression resulted in incomplete septa and damaged cell wall integrity, as well as slow growth rate in W. dermatitidis. Overexpression studies, after site-specific integrations of WdRHO1 and WdRAC1 alleles under control of the glaA promoter into the nonessential WdPKS1 locus, showed the different alleles had different effects on the cell morphological development. For example, whereas overexpression of the wdrho1⁺ allele did not affect the growth rate of W. dermatitidis, the overexpression of wdrho1[superscript G14V], a constitutively active mutation, slowed growth and repressed true filamentous hyphal growth by promoting pseudohyphal growth. While the deletion of WdRAC1 did not affect growth, its loss retarded polarized hyphal growth in a hyphal-inducing minimal medium. Moreover, three new phenotypes of a previously derived WdCDC42 deletion mutant were discovered during this study: in the first, the wdcdc42[Delta] mutant displayed cell lysis when incubated in YPMaltose medium at 37°C; in the second, a dark budding neck abnormality was found after Calcoflour staining; and in the third, the wdcdc42[Delta] mutant displayed no branching during true hyphal growth. Interestingly, the overexpression of wdrac1[superscript G16V] complemented the second and the third phenotypes caused by the WdCDC42 deletion. In addition, the wdcdc42[Delta]/wdrac1[superscript G16V] double mutant unexpectedly displayed an interrupted carotenogenesis pathway. These results support that in W. dermatitidis, Rho-type GTPases play essential roles in growth rate determination and cellular morphogenesis, especially while producing polarized hyphal growth during its many morphological transitions. / text

Blooms of nitrogen fixing cyanobacteria (NFC) occur in many freshwater and marine systems, including the Baltic Sea. By fixing dissolved nitrogen, they circumvent general summer nitrogen limitation, while also generating a supply of novel bioavailable nitrogen for non-diazotrophic primary producers and ultimately supporting secondary production. Elucidating trophic links between primary consumers and NFC is essential for understanding role of these blooms for secondary production. However, until recently, there was no reliable method to quantify individual prey species for zooplankter feeding in situ. The development of PCR-based methods to detect prey-specific DNA in the diet of consumers, including microscopic animals, allows identification and quantification of trophic linkages in the field. Using molecular diet analysis in combination with egg production measurements, biochemical markers of growth and condition; and stable isotope approach, we explored a possibility to determine (1) whether cyanobacteria are grazed and assimilated by mesozooplankters (Papers I and II), (2) which species/groups are particularly efficient consumers of cyanobacteria (Papers II and III), and (3) how feeding on cyanobacteria affects zooplankton growth and development (Paper I and III). Taken together, these laboratory and field observations, provided evidence that NFC contribute to feeding and reproduction of zooplankton during summer and create a favorable growth environment for the copepod nauplii (Paper I). The favorable growth conditions for juvenile copepods observed during NFC blooms were hypothesized to be mediated by picoplankton that take up bioavailable nitrogen exuded from cyanobacterial cells. This hypothesis found support in Paper II that provided quantitative estimates for the direct picocyanobacteria → mesozooplankton pathway, with highest weight-specific consumption observed in nauplii. Further, using field observations on zooplankton and phytoplankton development during a growth season in the northern Baltic proper, we found that NFC nitrogen is assimilated and transferred to zooplankton via both direct grazing and indirectly through grazing on small-sized phyto- and bacterioplankton (Paper III). Finally, these and other findings emphasizing the importance of NFC for Baltic Sea secondary production during growth season were synthesized to show that diazotrophic nitrogen enters food webs already at bloom initiation (Paper III) and is transferred via multiple pathways to pelagic and benthic food webs and, ultimately, to fish (Paper IV). / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Accepted.</p>

The utilization of lignocellulosic residues to produce renewable energy is an interesting alternative to meet the increasing demand of fuels while at the same time reducing greenhouse gas emissions and climate change. Sweet sorghum bagasse is a lignocellulosic residue composed mainly of cellulose, hemicellulose, and lignin; and it is a promising substrate for ethanol production because its complex carbohydrates may be hydrolyzed and converted into simple sugars, and then fermented into ethanol. However, the utilization of lignocellulosic residues is difficult and inefficient. Lignocellulose is a very stable and compact complex structure, which is linked by β-1,4 and β-1,3 glycosidic bonds. Furthermore, the crystalline and amorphous features of cellulose fibers and the presence of hemicellulose and lignin make the conversion of lignocellulose into fermentable sugars currently impractical at commercial scale. The bioconversion of lignocellulose in nature is performed by microorganisms such as fungi and bacteria, which produce enzymes that are able to degrade lignocellulose. The present study evaluated the bioconversion of lignocellulosic residues of sweet sorghum into simple sugars using filamentous fungi directly in the hydrolysis of the substrate, without prior isolation of the enzymes. The fungus Neurospora crassa and some wild fungi (that grew naturally on sweet sorghum bagasse) were used in this investigation. The effect of the fungi on substrate degradation and the sugars released after hydrolysis were evaluated, and then compared with standard hydrolysis performed by commercial enzymes (isolated cellulases). In addition, different combinations of fungi and enzymes were used to determine the best approach. The main goal was to verify if the fungi were able to attack and break down the lignocellulose structure directly and at a reasonable rate, rather than by the current method utilizing isolated enzymes. The main finding of this study was that the fungi (N. crassa and wild fungi) were able to degrade sweet sorghum bagasse directly; however, in all of the cases, the hydrolysis process was not efficient because the hydrolysis rate was much lower than the enzymatic hydrolysis rate. Hydrolysis using a combination of fungus and commercial enzymes was a good approach, but still not efficient enough for practical use. The best results of combined hydrolysis were obtained when the substrate was under the fungus attack for three days and then, commercial enzymes with low enzymatic activity (7 FPU/g and 25 CBU/g) were added to the solution. These enzymes represent 10% of the current enzymatic activity recommended per gram of substrate. This process reached reasonable levels of sugars (close to 85% of sugars yield obtained by enzymatic hydrolysis); however, the conversion rate was still slower, making the process impractical and more expensive since it took twice the amount of time as commercial enzymes. Furthermore, the wild fungi able to degrade cellulose were isolated, screened, and identified. Two of them belong to the genus Aspergillus, one to the genus Acremonium, and one to the genus Rhizopus. Small concentration of spores-0.5mL- (see Table 4, CHAPTER III- for specific number of spores per mL) did not show any sugar released during hydrolysis of sweet sorghum bagasse. However, when the concentration of spores was increased (to 5mL and 10mL of solution), citric acid production was detected. This finding indicates that those wild fungi were able to degrade lignocellulose, even though no simple sugars were measured, citric acid production is an indicator of fungi growing and utilization of lignocellulose as nutrient. It is assumed that the fungi consume the sugars at the same time they are released, thus they are not detected. The maximum concentration of citric acid (~14.50 mg/mL) was achieved between days 8-11 of hydrolysis. On the other hand, before using lignocellulose, the substrate needed to be pretreated in order to facilitate its decomposition and subsequent hydrolysis. Sweet sorghum bagasse was washed three times to remove any soluble sugars remaining after the juice was extracted from the stalks. Then, another finding of this study was that the first wash solution could be used for ethanol production since the amount of sugars present in it was close to 13°Brix. The ethanol yield after 48 hours of fermentation was in average 6.82mg/mL, which is close to the theoretical ethanol yield. The other two washes were too dilute for commercial ethanol production. In terms of pretreatments, the best one to break down sweet sorghum bagasse was 2% (w/v) NaOH. This pretreatment shows the highest amounts of glucose and xylose released after hydrolysis. Unwashed and untreated bagasse (raw bagasse) did not show any sugar released. In terms of ethanol, 74.50% of the theoretical yield was reached by enzymatic hydrolysis, while 1.10% was reached by hydrolysis using the fungus N. crassa. Finally, it is important to remark that further investigation is needed to improve the direct conversion of lignocellulose into fermentable sugars by fungal enzymes. This approach is a promising technology that needs to be developed and improved to make it efficient and feasible at commercial scale.

Bright field cellular microscopy offers an image capturing method that is both non-invasive and simple to implement. However, the resulting micrographs pose challenges for image segmentation which are compounded when the subject cells are tightly clustered or overlapping. Filamentous cyanobacteria are a type of organism that grow as linearly arranged cells forming chain-like filaments. Existing methods for bright field cell segmentation perform poorly on micrographs of these bacteria, and are incapable of identifying the filaments. Existing filament tracking methods are rudimentary, and cannot reliably account for overlapping or parallel touching filaments. We propose a new approach for identifying filaments in bright field micrographs by combining information about both filaments and cells. This information is used by an evolutionary strategy to iteratively construct a continuous spline representation that tracks the medial line of the filaments. We demonstrate that overlapping and parallel touching filaments are handled appropriately in many difficult cases.

Ff filamentous bacteriophage infect male (F+) strains of Escherichia coli and are assembled at the cell membranes, by a secretion-like, non-lethal process. The pIII protein, located at one end of the virion-filament, is required at both the beginning and the end of the phage life cycle. During infection, the N-terminal domains of pIII, N2 and N1, bind to the primary and secondary host receptors, F pilus and TolA protein, respectively. At the end of the life cycle, the pIII C-domain mediates the termination and release of virions. Thus, both entry and release involve structural transitions of the virus coupled to membrane transactions of the virion proteins. "Unlocking” of the highly stable virion presumably results in membrane integration during entry, whereas a reverse event, “locking” of the virion, occurs upon detachment from the membrane at termination step of assembly/secretion. Recently, it was shown that the pIII C-domain plays an active role at the step of entry. This finding implicates the C-domain of pIII in “unlocking” of the virion, presumably resulting in the exposure of the membrane anchor at the very C-terminus of pIII (Bennett & Rakonjac, 2006). To further this work, this thesis has mapped the portion of the pIII C-domain required for infection, by constructing a set of nested deletions of the C-domain fused to the receptor binding domains N1 and N2, and then determined the infectivity of phage carrying the mutant proteins. This mapped the portion of the C-domain required for phage infection is different to that required for termination of assembly. The different requirement for entry and release suggests that the two processes are carried out by distinct mechanisms and/or depend on different sets of accessory proteins. In addition, a system was designed for the efficient production and purification of very short virions, the length of which is 1/20 that of the wild-type f1. These short virions, called microphage, are the first step towards the structural analyses of the phage termini cap structures, of which one contains pIII in the “locked” conformation.