Humans directly or indirectly cause changes in the environment, and urbanisation is currently one of the most important threats to biodiversity. Urbanisation exposes organisms to novel pressures that are drastically different from those in their native habitats, as human modification rapidly and dramatically changes natural environments, alters habitats as well as shifts resources and predator communities. Organisms can cope with the novel challenges by modifying their behaviour, physiology, morphology and cognition. To understand the impacts of urbanisation on phenotypic traits, the goal of my research was to study the social and survival strategies of the Indian rock agama, Psammophilus dorsalis. Using a combination of field and laboratory-based experiments, I examined differences in signal-receiver dynamics in communication, anti-predator strategies, stress physiology, and spatial learning. Social interactions in this species involve rapid physiological colour changes and behavioural displays. My work shows that colour patterns are diametrically different between courtship and aggressive interactions. Males change their dorsal body region to red, and their lateral body region to black when courting females, whereas these regions turn yellow and orange respectively when fighting with competitive males. Regardless of social context, suburban males express lower colour contrast and are also slower to change colours than rural males. Using robotic lizard stimuli, I found that receiver responses match the population-specific intensity of male signals. For the first time in any lizard species, I find that perception and responsiveness to motion and colour are lateralized in different ways. Psammophilus dorsalis is left visual field dominant when responding to social display colours, but motion stimuli elicit similar responses from both visual field. Along with shifts in colour signalling strategies, stress physiology and social behavioural display was also affected by urbanisation. Suburban males had significantly higher circulating corticosterone levels during both control conditions and immediately following social interactions compared to rural males. Proportion and rate of courtship displays was also significantly lower in suburban males compared to rural males. In the field, escape strategies of males, but not females differed between suburban and rural populations, such that suburban males were more tolerant of simulated predator attacks than rural males. As expected from their cryptic body patterns, females, regardless of habitat, relied more heavily on crypticity rather than flight to minimize predation risk. Suburban males also had stronger cognitive skills, as spatial learning and reversal learning in suburban males was faster than in rural males. In sum, differences in these behavioural, physiological, and cognitive responses of suburban and rural populations of lizards demonstrated in my thesis, indicate human-induced changes in selective pressures that support shifted survival and reproductive strategies. Psammophilus dorsalis promises to be an excellent system to further examine the specific selective pressures that shift in urban landscapes. The study of multiple integrated phenotypic traits in response to urbanisation gives a broader perspective as to how a species can flexibly adapt to rapid environmental disturbances, which is currently one of the greatest threats to biodiversity worldwide.

As enzimas CYP450 são as principais enzimas metabolizadoras de fármacos. Elas são codificadas por genes que apresentam polimorfismos gênicos que lhes confere características fenotípicas diversas. Estes fenótipos são: Metabolizadores Lentos ou PM, Metabolizadores Normais ou EM, Metabolizadores Intermediários ou IM e Metabolizadores Ultra-rápidos ou UM. A Farmacogenética é a ciência que estuda estas variações gênicas e sua relação com a resposta terapêutica no organismo. Entre as enzimas CYP450 se encontram as enzimas CYP2D6, que são responsáveis pelo metabolismo de 25% dos fármacos clinicamente prescritos. O objetivo principal deste estudo foi a identificação dos polimorfismos mais importantes deste gene: CYP2D6*1, CYP2D6*2, CYP2D6*3, CYP2D6*4, CYP2D6*5, CYP2D6*6, CYP2D6*10, CYP2D6*17 e CYP2D6*41 pelos métodos de Genotipagem (PCR Tetra Primer e Seqüenciamento) e Fenotipagem (analisado pelo Índice Metabólico) em 75 voluntários sadios da região de Campinas. Para a caracterização da Fenotipagem foi usada a substância teste Dextrometorfano (DM). Esta foi monitorada por espectrometria de massa mediante a determinação da concentração do seu principal metabólito o Dextrorfano (DX), que foi extraída das amostras de urina. Os resultados foram comparados entre estas duas metodologias e apresentaram alta correlação. Os resultados obtidos são a identificação das freqüências dos alelos *1, *3 e *4 pelo método PCR Tetra Primer (30.66%, 1.3% e 14%, respectivamente). O método de seqüenciamento detectou também outros alelos que não foram detectados pela PCR Tetra Primer. A avaliação do número de cópias do gene CYP2D6 também foi avaliada, detectando em um voluntário 3 cópias do gene CYP2D6, característica de metabolizadores Ultra-rápidos. Podemos afirmar que os métodos usados forneceram perfis dos polimorfismos de maneira rápida e prática. / The CYP450 enzymes are the major drug metabolizing enzymes. They are encoded by genes that show genetic polymorphisms which gives them several phenotypic characteristics. These phenotypes are Poor Metabolizers or PM, or Extensive Metabolizers or EM, Intermediate Metabolizers or IM, and finally, Ultra-rapid Metabolizers or UM. Pharmacogenetics is the science that studies these genetic variations and its relationship to therapeutic response in the body. One of CYP450 enzymes is CYP2D6 enzyme, which are responsible for the metabolism of 25% of clinically prescribed drugs. The main objective of this study was to identify the most important polymorphisms of this gene: CYP2D6 * 1, CYP2D6 * 2, CYP2D6 * 3, CYP2D6 * 4, CYP2D6 * 5, CYP2D6 * 6, CYP2D6 * 10, CYP2D6 * 17 and CYP2D6 * 41 by genotyping methods (PCR Tetra Primer and Sequencing) and phenotyping (by metabolic rate monitoring) in 75 healthy volunteers in Campinas region.To characterize the phenotyping was used to test substance Dextromethorphan (DM). This was monitored by mass spectrometry by determining the concentration of its major metabolite the Dextrorphan (DX), which was extracted from urine samples. The results were compared between these two methods and showed high correlation. We can obtain the identification of allelic frequencies of alleles * 1, * 3 and * 4 by Tetra Primer PCR (30.66%, 1.3% and 14% respectively). The sequencing method has also detected other alleles that were not detected by PCR Tetra Primer. The assessment of the number of copies of the CYP2D6 gene was also assessed. This method detected a volunteer which carrying three copies of CYP2D6 gene, characteristic of Ultra-rapid metabolizers. We can say that the methods used in this study provide polymorphism profiles quickly and conveniently.

Pseudomonas aeruginosa is one of the most studied biofilm-forming organisms and has emerged as a model organism in the study of surface- and biofilm-induced gene expression. The transition from a planktonic to a biofilm mode of growth results in diverse changes in gene expression, which causes the attaching cells to become phenotypically and metabolically distinct from their planktonic counterparts. In this study, a proteomic approach was used to study differences in protein profiles obtained from 18-h old P. aeruginosa PAO1 (DSM 1707) planktonic, surface influenced planktonic (SIP) and biofilm populations grown in batch in the absence or presence of a glass wool substratum. Glass wool as an attachment substratum not only supported growth of biofilms, but it also allowed for the separation of the biofilm biomass from the surrounding surface influenced planktonic (SIP) cells for further characterisation. Comparative analysis of the respective proteomes indicated striking differences in the protein patterns of planktonic, biofilm and SIP cells and several uniquely expressed proteins were seen on the 2-DE protein maps of the respective populations. Whereas a general down-regulation of protein expression was seen in the biofilm cells, in SIP cells, expression of the proteins was generally up-regulated. The results confirmed that the biofilm population differs from the planktonic population and indicated that the SIP population is not merely a mixture of planktonic and biofilm cells but rather a unique phenotype. Several differentially expressed protein spots were selected and identified using a combination of N-terminal protein sequencing and peptide mass fingerprinting. The proteins comprised mostly of outer membrane or membrane-associated proteins. Based on these analyses, a mutant P. aeruginosa strain, deficient in outer membrane protein OprG, was generated and its ability to form biofilms on a glass wool substratum was compared with that of the wild-type P. aeruginosa strain. The mutant strain was attachment-proficient but biofilm-deficient, suggesting that OprG plays a role in P. aeruginosa biofilm development under the culturing conditions used in this study. / Thesis (PhD (Microbiology))--University of Pretoria, 2007. / Microbiology and Plant Pathology / unrestricted

The amount of available protein sequences is rapidly increasing, mainly as a consequence of the development and application of high throughput sequencing technologies in the life sciences. It is a key question in the life sciences to identify the functions of proteins, and furthermore to identify the phenotypes that may be associated with a loss (or gain) of function in these proteins. Protein functions are generally determined experimentally, and it is clear that experimental determination of protein functions will not scale to the current { and rapidly increasing { amount of available protein sequences (over 300 million). Furthermore, identifying phenotypes resulting from loss of function is even more challenging as the phenotype is modi ed by whole organism interactions and environmental variables. It is clear that accurate computational prediction of protein functions and loss of function phenotypes would be of signi cant value both to academic research and to the biotechnology industry. We developed and expanded novel methods for representation learning, predicting protein functions and their loss of function phenotypes. We use deep neural network algorithm and combine them with symbolic inference into neural-symbolic algorithms. Our work signi cantly improves previously developed methods for predicting protein functions through methodological advances in machine learning, incorporation of broader data types that may be predictive of functions, and improved systems for neural-symbolic integration. The methods we developed are generic and can be applied to other domains in which similar types of structured and unstructured information exist. In future, our methods can be applied to prediction of protein function for metagenomic samples in order to evaluate the potential for discovery of novel proteins of industrial value. Also our methods can be applied to the prediction of loss of function phenotypes in human genetics and incorporate the results in a variant prioritization tool that can be applied to diagnose patients with Mendelian disorders.

Indiana University-Purdue University Indianapolis (IUPUI) / During gestation, the maternal liver undergoes various adaptive changes to cope with the in-creasing physiological and metabolic demands from both maternal and fetal compartments. Among these changes are robust growth and changes in transcriptome profile. However, how these events happen, and other aspects of this physiological phenomenon remains unexplored. Therefore, we aimed at further understanding how maternal liver responds to pregnancy. We used BrdU labeling combined with a virus-based tracing approach to quantify the percentage of maternal hepatocytes undergoing DNA synthesis and division over the course of gestation in mice. We found that ~50% maternal hepatocytes entered S-phase but, unexpectedly, did not undergo cytokinesis. This strongly suggests that maternal hepatocytes in fact undergo endoreplication instead of hyperplasia, as believed previously. Pericentral Axin2+ hepatocytes were reported to behave as liver stem cells responsible for liver homeostasis and turnover. We generated an in vivo fate-tracing mouse model to monitor the behavior of these cells in the maternal liver. Our results showed that they did not proliferate during pregnancy, homeostasis, and following par-tial hepatectomy. Curiously, we uncovered that, hepatocytes exhibit developmental phenotypes at mRNA level pre-pregnancy and at both mRNA and protein level during pregnancy. In the non-pregnant state, hepatocytes reserved mRNA expression of liver progenitor marker genes Cd133 and Afp, which are localized in the nuclei, without protein translation. During gestation, maternal hepatocytes displayed cytoplasmic translocation of Cd133 and Afp transcripts, con-comitant with corresponding protein expression. Overall, all maternal hepatocytes became CD133+, and a subset of them express AFP. Addi-tionally, in non-pregnant livers, mRNA of Epcam, another liver progenitor marker, was ex-pressed within majority of hepatocytes, whereas its protein was solely translated in the pericen-tral region. In contrast, by end-gestation, EPCAM protein expression switched to the periportal region. These observations indicate that maternal hepatocytes exhibit heterogeneous develop-mental phenotypes, partially resembling fetal hepatocytes. It is intriguing why mature hepato-cytes dedifferentiate into a progenitor state in response to pregnancy. AFP is considered to be produced primarily from fetal liver and thus is used to evaluate fetal development health. A potential clinical relevance of our data is that we identified maternal liver as a new source of AFP. The hippo signaling pathway has been shown to potently control liver growth and hepato-cyte heterogenicity. Surprisingly, we found that pregnancy neither altered the expression nor activities of the components of this pathway and its effector YAP1/TAZ. This finding indicates that pregnancy-induced maternal liver growth is not driven by hippo-YAP1 pathway. However, we demonstrate that the presence of YAP1 is essential for CD133 protein expression in mater-nal hepatocytes. Collectively, we revealed that, as pregnancy advances, maternal hepatocytes likely undergo endoreplication and display developmental phenotypes. Mechanistically, YAP1 dictates the expression of CD133, contributing to the pregnancy-dependent phenotypic changes of maternal hepatocytes.

Purpose of review: This article critically reviews the utility of “phenotypes” as behavioral descriptors in aging/HIV research that inform biological underpinnings and treatment development. We adopt a phenotypic redefinition of aging conceptualized within a broader context of HIV infection and of aging. Phenotypes are defined as dimensions of behavior, closely related to fundamental mechanisms, and, thus, may be more informative than chronological age. Primary emphasis in this review is given to comorbid aging and cognitive aging, though other phenotypes (i.e., disability, frailty, accelerated aging, successful aging) are also discussed in relation to comorbid aging and cognitive aging. Recent findings: The main findings that emerged from this review are as follows: (1) the phenotypes, comorbid aging and cognitive aging, are distinct from each other, yet overlapping; (2) associative relationships are the rule in HIV for comorbid and cognitive aging phenotypes; and (3) HIV behavioral interventions for both comorbid aging and cognitive aging have been limited. Summary: Three paths for research progress are identified for phenotype-defined aging/HIV research (i.e., clinical and behavioral specification, biological mechanisms, intervention targets), and some important research questions are suggested within each of these research paths.

Three greenhouse experiments were conducted with three phenotypes of alfalfa (Medicago sativa L.) obtained from a potassium (K)-deficient field and with their diallel crosses grown on low K soil. The first experiment was conduc ted to study the partitioning and broad-sense heritability of K a nd sodium (Na) between leaves and stems of the three phenotypes which were deficient in K and exhibited normal (N), marginal chlorotic (M), and white spot chlorotic (W) leaflets. The second experiment was conducted to study the partitioning of K and Na in leaves, stems, and roots as influenced by 32 alfalfa crosses obtained from diallel crossing of the mother plants of the three phenotypes. The objectives of the third experiment were to study the effects and interactions of nine alfalfa crosses and three soil K and Na levels on transpiration, biomass, and elemental composition of alfalfa components. The three phenotypes showed no variations in their leaf and stem K concentrations but varied in their ability to partition Na between the leaves and stems. Phenotype M accumulated more Na compared to N and W phenotypes. The Na trait was highly heritable in the broad sense. The K and Na concentrations varied among the diallel crosses. Crosses with M as the maternal parent had high Na concentrations in leaves while stems and roots accumulated lesser amounts. In contrast, the remaining crosses had higher Na concentrations in roots and lower and least amounts in stems and leaves, respectively. Significant genetic variation among alfalfa crosses from a single cultivar was observed for transpiration, biomass production, plant water-use efficiency, elemental concentrations, and K utilization efficiency. Leaf and stem biomass and K concentrations in alfalfa components increased in response to increasing soil K levels. The Na concentrations in stems and roots fell in response to increasing soil K levels and increased in response to Na application. The K utilization efficiency of alfalfa increased with increase in soil Na levels, indicating partial Na substitution for K. The differences among alfalfa phenotypes and crosses from a single cultivar in their Na accumulation and translocation were thought to be governed by plant genetics rather than the direct effect of K availability.

Thioredoxin is a small ubiquitous oxido-reductase found in all species. The highly conserved active site, which facilitates thioredoxins redox activity, contains two redox active cysteine residues. Thioredoxin has numerous protein substrates to which it donates H+ ions and it can also function as a free radical scavenger. Through these activities thioredoxin is able to influence the redox state of not only its protein targets, but also the entire cellular environment. Thioredoxin has been implicated in many biological functions, and one mechanism by which it influences these functions is through interactions with a number of transcription factors including NF-kappa-B and p53. Thioredoxin also has numerous extracellular biological roles. It has been shown that thioredoxin is actively secreted from a number of normal and transformed cell lines including fibroblasts and activated B and T cells. This study investigates the role of thioredoxin in embryonic implantation and cancer cell metastasis, two physiological functions which rely on the same basic processes. Thioredoxin expression has previously been shown to be increased in many cancers. However it has not yet been established whether this increase is a causative or a side effect of the cancerous phenotype. Similarly thioredoxin expression has previously been shown to be increased during different phases of the oestrus cycle and pregnancy. This thesis describes the role of thioredoxin in embryonic implantation using a marmoset model. A thioredoxin cDNA was isolated and subsequently sequenced. Preliminary antibody experiments indicated that the anti human thioredoxin monoclonal antibodies available in our laboratory would recognise marmoset thioredoxin. Subsequently immunocytochemistry using anti human thioredoxin antibodies was carried out on sectioned marmoset uterus and embryonic tissue. The results indicated that thioredoxin is expressed by cells at the embryonic-maternal interface of early implantation sites. Further studies demonstrated that thioredoxin is also expressed and secreted by cultured blastocysts in vitro. This thesis also describes the role of thioredoxin in cancer cell metastasis. Results of this study indicate that thioredoxin is actively involved in facilitating the invasive phenotype of breast cancer cells. The two cell lines utilised were MCF-7, a well differentiated, relatively non-invasive breast cancer cell line; and MDA-MB-231, a poorly differentiated, highly invasive breast cancer cell line. The cell lines were transfected with thioredoxin sense, antisense and 1SS (encodes thioredoxin with both active cysteine residues mutated to serine residues and is thus redox inactive) constructs. The results demonstrate that when endogenous thioredoxin levels are increased, i.e. transfected with a sense thioredoxin construct, the invasive breast cancer cell line MDA-MB-231 becomes more invasive, conversely when endogenous levels are decreased, i.e. transfected with antisense or 1SS constructs, the invasive capacity of these cells decreases. However, when the endogenous level of thioredoxin was manipulated in the relatively non-invasive cell line MCF-7 very little effect was observed. Results also indicate that thioredoxin has the ability to act as a chemoattractant for actively invading breast cancer cells. Both of these functions appear to be dependent on thioredoxin's redox activity. Additional studies described in this thesis have shown that thioredoxin is involved in the regulation of Sp1 in vitro. Sp1 is a transcription factor known to regulate the transcription of a number of genes whose products are intimately involved in the invasive phenotype. The results in this study suggest that Sp1 DNA binding is regulated by thioredoxin such that when reduced by the enzyme its binding to DNA is facilitated. Results also indicate that Sp1 may regulate the transcription of thioredoxin by binding to Sp1 sites within the thioredoxin promoter.

Linhagens de camundongos AIRmax e AIRmin diferem quanto a reatividade inflamatória aguda às partículas de Biogel. A AIR divergente nessas linhagens está bem estabelecida, no entanto, diferenças na resposta inflamatória crônica ao Biogel ainda não foram descritas. Assim, nós decidimos verificar se o processo de seleção que modificou a resposta inflamatória aguda nessas linhagens, também afetou o desenvolvimento da resposta inflamatória crônica. A infiltração de células no exsudado da linhagem AIRmax foi maior que na linhagem AIRmin em ambos os períodos avaliados (48h e 30d), e no período de 48 horas, os animais AIRmax apresentaram alta produção de citocinas no exsudato inflamatório. Essa linhagem mostrou ainda maior número de genes ativados envolvidos na transdução de sinal, resposta imune e inflamatória. Alguns genes envolvidos com a resposta inflamatória aguda, também apresentaram diferenças após 30 dias. Esses resultados indicam que o processo de seleção para a inflamação aguda pode ter afetado também o desenvolvimento da resposta inflamatória crônica ao Biogel. / AIRmax and AIRmin mouse lines differ in terms of acute inflammatory response after Biogel injection. The distinct AIR in these lines is well established, however, differences in late or chronic inflammatory response to Biogel were not described yet. Thus, we decided to check if the genetic selection that modified the acute inflammatory response in these lines, also affected the development of a chronic inflammatory response. We found that AIRmax mice had higher cellular influx in the inflammatory exudate than AIRmin mice in both analyzed periods (48h and 30d) and that after 48 hours of Biogel injection, AIRmax mice showed higher cytokine levels in inflammatory exudates. This line also showed higher number of up regulated genes in AIRmax than in AIRmin mice involved with inflammatory response, immune response and signal transduction. Some acute inflammatory response genes also showed differences on day 30. Our results indicate that the genetic selection for acute inflammatory response may also have affected the chronic inflammatory response to Biogel.

O presente trabalho tem os seguintes objetivos: propor uma sistemática para o estudo e a interpretação da estabilidade e adaptabilidade fenotípica, através de duas técnicas de análise multiway (PARAFAC e Tucker3); propor a construção de um gráfico, denominado de Triplot, que possibilita avaliar as relç]oesoes entre os 3 modos (genótipos, locais e anos); implementar uma rotina computacional para a análise de dados, segundo os modelos multiway; implementar uma rotina computacional para a construção do Triplot. Os dados a serem uti- lizados são relativos a experimentos com 13 genótipos de feijão que foram conduzidos em 9 ex- perimentos distintos constituídos pelos anos agrícolas de 2000/2001, 2001/2002 e 2005/2006, pelos municípios de Dourados e Aquidauana, sendo que os experimentos foram instalados na época das águas (Dourados)e também na época da seca (Dourados e Aquidauana). Cada local é constituído de município e uma época de instalação. Os resultados indicaram que o gráfico triplot e joint plot, facilitam o entendimento da interação tripla e traz ao pesquisador informações mais reais sobre a interação tripla, do que a modelagem AMMI de duas entradas; o gráfico triplot, ajuda a identificar genótipos, locais e anos estáveis, dentro de um grande grupo de genótipos, locais e anos; de uma maneira geral recomenda-se, utilizar o triplot e o joint plot juntos, para obter melhores interpretações dos resultados; dentre os genótipos estudados, o genótipo 6 é o que menos contribui para a interação e o os genótipos 12, 9 e 5 são os que mais contribuem para a interação. / The present work has the following objectives: to propose a systematics for the study and the interpretation of the phenotypic stability and adaptability, through several multiway models (PARAFAC and Tucker3); to propose a graphic, called of Triplot, that it makes possible to evaluate the relations between the 3 ways (genotypes, locations and years); to implement a computational routine for the data analysis, according multiway models; to implement a computational routine for the construction of Triplot. The used data are relative the experiments with 13 genotypes of beans that had been lead in 9 experimental distinct ones constituted by agricultural years of 2000/2001, 2001/2002 and 2005/2006, by Dourados and Aquidauana cities, where the experiments had been installed at the time of waters (Dourados) and also at the time of dries (Dourados and Aquidauana). Each location is constituted of city and time of installation. The results indicated that the graphic triplot and joint plot, facilitate the agreement of triple interaction and bring to the researcher more real information about triple interaction, of what AMMI model of two way; the graphic triplot, helps to identify stabels genotypes, locations and years, inside of a great group of genotypes, location and years; in a general recommend to use triplot and joint plot together, to get better interpretations of the results; the genotype 6 is what less contributes for the triple interaction and genotypes 12, 9 and 5 are the that more contribute for the interaction.