Recent studies have discovered that LPS-treated Sprague-Dawley rats induced a reduction (phase I), followed by an augmentation (phase II), and decrease again (phase III) in the power density of the vasomotor component (0-0.8 Hz) in systemic arterial pressure (SAP). It was reported that the vasomotor components were related to the brain stem, even closely related to the rostral ventrolateral medulla (RVLM). But the molecular mechanism involved in the death progression of rat brain stem is mostly unknown. We used suppression subtractive hybridization (SSH) and library construction to find differentially expressed genes between phase I and phase II of LPS-treated RVLM. At present, we have found some genes that are differentially expressed between phase I and phase II of LPS-treated RVLM. Some genes are up-regulation expression and others are down-regulation expression. Thus, these genes may be involved in the molecular mechanism of the death progression in the rat brain stem.

As its objective this study develops a modus operandi for the interior designer who must understand and work with both additively and subtractively mixed colors in constructing interior color schemes.

Dermatofitoses são comumente causadas por fungos que parasitam pele e unha de humanos, cuja propagação depende do contato entre os hospedeiros infectados e não infectados. Muitos fatores contribuem para a patogenicidade dos dermatófitos, dentre eles, a capacidade de se instalar no ambiente ácido da pele se reveste de importância. Sendo assim, para ser bem sucedido, o dermatófito precisa ter capacidade aderente, germinação e penetração rápida das hifas e, portanto, dispor de uma maquinaria metabólica que atue de forma eficiente em pH ácido. A fim de identificar genes supostamente expressos nos passos iniciais da infecção, submetemos a linhagem H6 do dermatófito T. rubrum ao pH ácido por 30 minutos e 1 hora e isolamos dessas condições experimentais os transcritos com elevada expressão, empregando a metodologia de Biblioteca Subtrativa Supressiva (SSH). Obtivemos um total de 234 unigenes cujos transcritos revelaram ampla diversidade funcional. Esses transcritos estão envolvidos em 13 processos celulares diferentes, tais como, metabolismo, defesa e virulência, síntese de proteínas e transporte celular. Desses, confirmamos por Northern blotting, os genes que expressam as proteínas carboxipeptidase S1, acetoamidase, aconitase, dessaturase, a proteína TINA, transportador de aminoácidos, fator de alongamento alfa 1, proteína ribossomal L10, e uma proteína hipotética. Nesses experimentos também foi utilizada a linhagem de T. rubrum pacC-1, que tem o seu gene pacC rompido, com o objetivo de verificar se estes genes isolados seriam regulados pela proteína PacC. O gene pacC codifica uma proteína homóloga ao regulador transcricional PacC/Rim101p da conservada via de sinalização do pH. Verificamos que o gene pacC se expressa preferencialmente em pH 8.0 e que embora o padrão de processamento da proteína PacC seja dependente do pH a forma íntegra da proteína PacC foi identificada tanto em pH ácido como alcalino. Por outro lado, o mutante pacC-1 apresentou diminuida capacidade infectiva em fragmentos de unha humana quando comparado com a linhagem selvagem. Além disto, a atividade queratínolitica do mutante também se mostrou diminuída quando comparada ao controle, confirmando o papel da proteína PacC na capacidade infectiva do T. rubrum. / Dermatophytosis is commonly caused by fungi that parasite human skin and nail, whose propagation depends on the contact between infected and noninfected hosts. Many factors contribute to the pathogenicity of the dermatophytes. Among them, the capacity to install in the skin´s acid ambient bears great importance. Thus, in order to be successful, the dermatophyte needs to have adhering capacity, fast germination and penetration of hyphae and, therefore, needs to afford a metabolic machinery which acts efficiently in acid pH. In order to identify genes supposedly expressed in the initial steps of infection, we submitted the strain H6 of the dermatophyte T. rubrum to the acid pH for 30 minutes and 1 hour and isolated, from this experimental conditions, the transcripts with high expression, employing the suppression subtractive hybridization (SSH). We obtained a total of 234 unigenes whose transcripts revealed a wide functional diversity. These transcripts are involved in 13 different cell processes, such as metabolism, defense and virulence, protein synthesis and cell transport. Among these, we confirmed through Northern blotting the genes which express the proteins carboxipeptidase S1, acetamidase, aconitase, fatty acid desaturase, NIMA interactive protein (TINA), amino acid permease, elongation factor 1-alpha, 60S ribosomal protein L10 and a hypothetical protein. In these experiments, we also used the T. rubrum pacC- 1 strain, which has its pacC gene disrupted, aiming at verifying whether these isolated genes would be regulated by the PacC protein. The pacC gene encodes a protein homologous to the PacC/Rim101p transcriptional regulator of the conserved route of pH signaling. We verified that the pacC gene is expressed preferentially in both pH, and that although the processing pattern of the PacC protein is dependent on the pH, the full form of the PacC was identified as alkaline. On the other hand, the pacC-1 mutant presented diminished infecting capacity in human nail fragments when compared to the wild strain. Moreover, the keratinolytic activity of the mutant also seemed diminished when compared to the control, confirming the role of the PacC protein in the infecting capacity of T. rubrum.

Malignant mesothelioma (MM) is an aggressive tumour, which is highly associated with previous asbestos exposure and is resistant to most conventional anticancer therapies. Previous studies have used a mouse model of to 01 p effective approaches to induction of anti-tumour immunity using modification of tumour cells by the introduction of genetic constructs expressing genes such as that for B7-1 so that tumour growth can be inhibited in vivo. Transfectant clones, AC29 B7-7 and AC29 B7-6, which showed equal levels of expression of B7-1 but were markedly different in tumorigenicity were assessed using suppression subtractive hybridization (SSH) in order to isolate transcripts which may have been differentially expressed in the two clones. SSH allowed isolation of a number of cDNAs which were apparently differentially expressed in the cell lines. These required characterisation in order to determine their possible relevance to tumorigenicity. Two cDNAs designated as 7-7-76 and 7-7-43 had been isolated previously and the aim of this project was to characterise these cDNAs by sequencing, searching for their homology relationships and investigating gene expression profiles. Preliminary searches revealed that clone 7-7-43 had homology to cyclin-dependent kinase regulatory subunit 1 which plays a role in the cell cycle. On the other hand, clone 77-76 showed only homology to an EST of hypertension related protein and therefore, further investigation was required to obtain the identity of clone 7-7-76. The first part of this project was to in investigate and evaluate gene expression on clone 7-7-43, using both relative RT-PCR and Northern blotting.' In the second part of this project, a more intense study of clone 7-7-76 was conducted. Clone 7-7-76 was investigated for its homology relationships and its gene expression profile. / Results obtained from relative RT-PCR suggested no difference in the expression of the either eDNA clone (7-7-43 and 7-7-76) between the MM clones AC29 B7-6 and AC29 B7-7, the cells used to derive these clones by SSH. Therefore, it was concluded that neither clone 7-7-43 nor 7-7-76 was differentially expressed in MM cells of differing immuno enicit RACE was employed in order to derive a longer sequence of clone 7-7-76 and the newly derived sequence of 7-7-76 was again used to search for homologies using a wider range of sequences for human and other species. These investigations on clone 7-7-76 showed it to correspond to the sequence of human mitofusin 2 which is involved in determining mitochondrial morphology The results determined in this project suggest that clones 7-7-43 and 7-7-76 are not differentially expressed in the range of MM cell lines tested. The data have however highlighted the potential of the SSH technique to easily derive cDNA clones worthy of investigation, but underline the possibility of false positive clones being isolated. The need for an efficient, accurate screening procedure such as real-time PCR is acknowledged.

In most mammals, sexual fate is determining genetically by the presence of the SRY gene which encoded the testis-determining factors on the Y chromosome. Likewise, avian sex is determined genetically. At day 3.5 (stage 22; HH) in chicken embryogenesis, the gonadal primordium begins forming. Thus, to identify the novel sex-determinating genes in early chicken embryos, subtractive cDNA libraries from male-minus-female (M-F) and female-minus-male (F-M) of 3 Dpc. embryos were established. Both collected male and female chicken total mRNAs were purified using Dynabeads. After a blund-end restriction endonuclease Rsa I digestion of cDNA, adaptor ligation for tester cDNA was performed. When first and second cDNA hybridization was finished, those nonredundant cDNA between tester and driver will be amplified by two rounds of PCR. Subsequently, TA-cloning was performed and the cDNA fragments were PCR-amplified using M13 primers. PCR products of Clones were first screened by differential screening hybridization to decrease false positive inserts. Then, gene annotation was carried out by data-mining in public databases, GeneBank (NCBI, USA). Finally, 40 known and 71 novel transcripts of M-F cDNA library, 88 known and 128 novel transcripts of F-M cDNA library were identified. In M-F subtracted library, 4 identified known genes were located on Z sex chromosome such as WD repeat domain 36 (WDR36), PC4 and SFRS1 interacting protein 1 (PSIP1), serum response factor binding protein 1 (SRFBP1) and glycine dehydrogenase (decarboxylating) (GLDC). Another two identified known genes, laminin alpha 1 (LAMA1) and leukocyte cell derived chemotaxin 1 (LECT1) were reported be relate to cell differentiation and development. In F-M subtracted library, only Wpkic-8 was located on W sex chromosome. Other identified genes like slowmo homolog 2 (Drosophila) (SLMO2), collagen, type IV, alpha 1 (COL4A1), anterior gradient 2 homolog (Xenopus laevis), transcript variant 2 (AGR2), solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 6 (SLC25A6) and prolyl endopeptidase (PREP) were also found expressed higer in human ovary then testis. PREP was proposed that it may play a role in mediating sperm death by regulating the levels of thyrotropin-releasing hormone analogs and in mediating sperm death associated with necrozoospermia. These transcripts located on W or Z sex chromosome identified from subtracted libraries may play an important role in sex determination mechanism.

Anxiety disorders are one of the most prevalent mental disorders in the world. While normal anxiety serves as an important protective mechanism, pathological anxiety characteristic of an anxiety disorder is both maladaptive and disruptive. The majority of studies have focused on the neurotransmitter systems associated with the actions of known anxiety drugs. This focus may likely limit the exploration of mechanisms underlying anxiety disorders. This project aims to examine changes in gene expression that may underlie higher or lower levels of inherent anxiety. Using a well-established behavior test for anxiety, the elevated plus maze, we identified male Wistar rats exhibiting inherently high- or low-anxiety levels. Brain regions known to mediate anxiety, the amygdala, hippocampus and nucleus accumbens, were dissected and total mRNA isolated. The mRNA was converted to cDNA via reverse transcription-polymerase chain reaction (RT-PCR). Then, the cDNA was used in suppression subtractive hybridization, a technique used to compare two complete populations of cDNAs and identify cDNAs that are upregulated in one population in relation to the other. In this project suppression subtractive hybridization was used to compare high- and low-anxiety cDNA populations. The upregulated cDNAs were amplified in a PCR reaction that enables rare transcripts to be identified. The PCR products from the suppression subtractive hybridization were cloned and used to create two cDNA libraries for high- and low-anxiety related genes. These clones were sequenced to show over 1000 genes upregulated in high- and low-anxiety. The gene list was then subjected to bioinformatic analysis to identify one candidate to be studied in further detail. <p>The prion protein was identified as a potential candidate. Examination of the literature sparked an interest in studying other prion-like proteins, more specifically the cytoplasmic polyadenylation element binding protein (CPEB). The CPEB protein is a potent regulator of mRNA translation in both mature oocytes and the adult brain. While unphosphorylated the CPEB protein keeps specific mRNAs dormant in the cytoplasm. In its phosphorylated form CPEB catalyzes polyadenylation of the mRNA, leading to protein synthesis. p*PCR was used to show the presence of CPEB mRNA transcripts in the rat hippocampus. CPEB protein expression was examined in the brain samples isolated from control, high- and low-anxiety rats. It was found that CPEB was significantly upregulated in high- and low-anxiety rats compared to control. The protein expression of an upstream kinase, Aurora A kinase, and a downstream target, Calcium/Calmodulin Dependent Kinase II (CaMKII), was also investigated. The results from Aurora A kinase were inconclusive. CaMKII, on the other hand, was significantly upregulated in high-anxiety over both control and low-anxiety. These results suggest that CPEB may catalyze increased translation of mRNAs in high-anxiety while acting as a repressor of those same mRNAs in low-anxiety. <p>Recent studies have suggested that CPEB protein plays an important role in synaptic plasticity. The regulation of synaptic plasticity, and its impact on learning and memory, is believed to be a key mechanism behind the maintenance of anxiety disorders. Therefore the results of this study suggest a new molecular mechanism in the development of anxiety disorders.

Anxiety disorders are one of the most prevalent mental disorders in the world. While normal anxiety serves as an important protective mechanism, pathological anxiety characteristic of an anxiety disorder is both maladaptive and disruptive. The majority of studies have focused on the neurotransmitter systems associated with the actions of known anxiety drugs. This focus may likely limit the exploration of mechanisms underlying anxiety disorders. This project aims to examine changes in gene expression that may underlie higher or lower levels of inherent anxiety. Using a well-established behavior test for anxiety, the elevated plus maze, we identified male Wistar rats exhibiting inherently high- or low-anxiety levels. Brain regions known to mediate anxiety, the amygdala, hippocampus and nucleus accumbens, were dissected and total mRNA isolated. The mRNA was converted to cDNA via reverse transcription-polymerase chain reaction (RT-PCR). Then, the cDNA was used in suppression subtractive hybridization, a technique used to compare two complete populations of cDNAs and identify cDNAs that are upregulated in one population in relation to the other. In this project suppression subtractive hybridization was used to compare high- and low-anxiety cDNA populations. The upregulated cDNAs were amplified in a PCR reaction that enables rare transcripts to be identified. The PCR products from the suppression subtractive hybridization were cloned and used to create two cDNA libraries for high- and low-anxiety related genes. These clones were sequenced to show over 1000 genes upregulated in high- and low-anxiety. The gene list was then subjected to bioinformatic analysis to identify one candidate to be studied in further detail. <p>The prion protein was identified as a potential candidate. Examination of the literature sparked an interest in studying other prion-like proteins, more specifically the cytoplasmic polyadenylation element binding protein (CPEB). The CPEB protein is a potent regulator of mRNA translation in both mature oocytes and the adult brain. While unphosphorylated the CPEB protein keeps specific mRNAs dormant in the cytoplasm. In its phosphorylated form CPEB catalyzes polyadenylation of the mRNA, leading to protein synthesis. p*PCR was used to show the presence of CPEB mRNA transcripts in the rat hippocampus. CPEB protein expression was examined in the brain samples isolated from control, high- and low-anxiety rats. It was found that CPEB was significantly upregulated in high- and low-anxiety rats compared to control. The protein expression of an upstream kinase, Aurora A kinase, and a downstream target, Calcium/Calmodulin Dependent Kinase II (CaMKII), was also investigated. The results from Aurora A kinase were inconclusive. CaMKII, on the other hand, was significantly upregulated in high-anxiety over both control and low-anxiety. These results suggest that CPEB may catalyze increased translation of mRNAs in high-anxiety while acting as a repressor of those same mRNAs in low-anxiety. <p>Recent studies have suggested that CPEB protein plays an important role in synaptic plasticity. The regulation of synaptic plasticity, and its impact on learning and memory, is believed to be a key mechanism behind the maintenance of anxiety disorders. Therefore the results of this study suggest a new molecular mechanism in the development of anxiety disorders.

OBJECTIVE: To explore a novel virtual inspection approach with a 3D metrology software to provide a non-destructive in situ analysis in digital workflow. Also, to evaluate the fit discrepancies of lithium disilicate crowns by using such a novel virtual measuring technique. MATERIALS AND METHODS: Maxillary arch typodont was used to design abutment for tooth #8 and #14 (hand prepared) and #4 and #10 (titanium custom abutment). All four abutments were placed into a duplicated maxillary arch solid stone model for scanning with laboratory scanner. Four crown patterns were designed and exported as STL files. The internal control group consists of the four original digital STL files and the external control group which was the 32-milled lithium disilicate crowns (IPS e.max® CAD, Ivoclar Vivadent, Inc.), eight patterns for each tooth. Thirty-two pressable wax patterns (8 of each) was fabricated for each of the three different technique systems. Two printed wax systems, Varseo Wax CAD/Cast (BEGO) and Press-E-Cast (EnvisionTec). Two milled wax systems Harvest Wax (Ivoclar Vivadent, Inc.) and Polycon Cast (Straumann), and a set of conventional cutbacks of 1.5mm with applied marginal wax. All patterns were pressed into lithium disilicate crowns, then fine polished and scanned. Each file was imported into a quality control metrology software (Geomagic Control X, 3D Systems) for marginal fit and internal fit evaluation with respective digital abutment. RESULTS: Mean of marginal gap for all groups were all lower than the preset gap space of 40 microns. Statistically significant differences in the fit accuracy were found among tooth number, technique system and measurement locations, but the differences are in clinically acceptable range. New scope of analyzing a restoration in a 3D fashion can help solve clinical complications. The study has shown that lower marginal gap does not necessary indicates a better fit restoration, as every level of the crown should be evaluated for. CONCLUSION: This novel inspection method can be used as a replacement of fit checker and help clinician to work in a full digital workflow. Lithium disilicate restorations fabricated through printed wax pattern, milled wax pattern and conventional hand wax are all clinically acceptable techniques. / 2019-09-26T00:00:00Z

Dermatofitoses são comumente causadas por fungos que parasitam pele e unha de humanos, cuja propagação depende do contato entre os hospedeiros infectados e não infectados. Muitos fatores contribuem para a patogenicidade dos dermatófitos, dentre eles, a capacidade de se instalar no ambiente ácido da pele se reveste de importância. Sendo assim, para ser bem sucedido, o dermatófito precisa ter capacidade aderente, germinação e penetração rápida das hifas e, portanto, dispor de uma maquinaria metabólica que atue de forma eficiente em pH ácido. A fim de identificar genes supostamente expressos nos passos iniciais da infecção, submetemos a linhagem H6 do dermatófito T. rubrum ao pH ácido por 30 minutos e 1 hora e isolamos dessas condições experimentais os transcritos com elevada expressão, empregando a metodologia de Biblioteca Subtrativa Supressiva (SSH). Obtivemos um total de 234 unigenes cujos transcritos revelaram ampla diversidade funcional. Esses transcritos estão envolvidos em 13 processos celulares diferentes, tais como, metabolismo, defesa e virulência, síntese de proteínas e transporte celular. Desses, confirmamos por Northern blotting, os genes que expressam as proteínas carboxipeptidase S1, acetoamidase, aconitase, dessaturase, a proteína TINA, transportador de aminoácidos, fator de alongamento alfa 1, proteína ribossomal L10, e uma proteína hipotética. Nesses experimentos também foi utilizada a linhagem de T. rubrum pacC-1, que tem o seu gene pacC rompido, com o objetivo de verificar se estes genes isolados seriam regulados pela proteína PacC. O gene pacC codifica uma proteína homóloga ao regulador transcricional PacC/Rim101p da conservada via de sinalização do pH. Verificamos que o gene pacC se expressa preferencialmente em pH 8.0 e que embora o padrão de processamento da proteína PacC seja dependente do pH a forma íntegra da proteína PacC foi identificada tanto em pH ácido como alcalino. Por outro lado, o mutante pacC-1 apresentou diminuida capacidade infectiva em fragmentos de unha humana quando comparado com a linhagem selvagem. Além disto, a atividade queratínolitica do mutante também se mostrou diminuída quando comparada ao controle, confirmando o papel da proteína PacC na capacidade infectiva do T. rubrum. / Dermatophytosis is commonly caused by fungi that parasite human skin and nail, whose propagation depends on the contact between infected and noninfected hosts. Many factors contribute to the pathogenicity of the dermatophytes. Among them, the capacity to install in the skin´s acid ambient bears great importance. Thus, in order to be successful, the dermatophyte needs to have adhering capacity, fast germination and penetration of hyphae and, therefore, needs to afford a metabolic machinery which acts efficiently in acid pH. In order to identify genes supposedly expressed in the initial steps of infection, we submitted the strain H6 of the dermatophyte T. rubrum to the acid pH for 30 minutes and 1 hour and isolated, from this experimental conditions, the transcripts with high expression, employing the suppression subtractive hybridization (SSH). We obtained a total of 234 unigenes whose transcripts revealed a wide functional diversity. These transcripts are involved in 13 different cell processes, such as metabolism, defense and virulence, protein synthesis and cell transport. Among these, we confirmed through Northern blotting the genes which express the proteins carboxipeptidase S1, acetamidase, aconitase, fatty acid desaturase, NIMA interactive protein (TINA), amino acid permease, elongation factor 1-alpha, 60S ribosomal protein L10 and a hypothetical protein. In these experiments, we also used the T. rubrum pacC- 1 strain, which has its pacC gene disrupted, aiming at verifying whether these isolated genes would be regulated by the PacC protein. The pacC gene encodes a protein homologous to the PacC/Rim101p transcriptional regulator of the conserved route of pH signaling. We verified that the pacC gene is expressed preferentially in both pH, and that although the processing pattern of the PacC protein is dependent on the pH, the full form of the PacC was identified as alkaline. On the other hand, the pacC-1 mutant presented diminished infecting capacity in human nail fragments when compared to the wild strain. Moreover, the keratinolytic activity of the mutant also seemed diminished when compared to the control, confirming the role of the PacC protein in the infecting capacity of T. rubrum.

Polyamines play an important role in DNA, RNA and protein synthesis as well as a variety of other biological processes (cell division, differentiation and death) as outlined in Chapter 1. Assaraf and co-workers (1984) demonstrated that treatment with DFMO resulted in the inhibition of polyamine biosynthesis as well as schizogony arrest in P. falciparum. However, they did not elaborate on any other consequences that polyamine depletion could exert on the parasite. This dissertation aims to elucidate the significance of the inhibition of polyamine biosynthesis within P. falciparum by using differential transcriptome profiling. Suppression subtractive hybridisation generated transcripts which were potentially up-and down-regulated due to endogenous polyamine depletion within the human malaria parasite P. falciparum. The resulting transcripts were subjected to a restriction enzyme analysis and those with unique digestion profiles were selected and sequenced. The sequences were analysed using PlasmoDB to identify the genomic sequences to which they were best matched. To confirm that the selected transcripts were indeed differentially expressed a reverse virtual Northern dot blot was performed. Transcripts for proteins involved in protein processing, methionine and polyamine metabolism, various transporters, proteins involved in cellular differentiation and signal transduction were found to be upregulated in the absences of polyamines. This could be suggestive of a metabolic response induced by the parasite in order to overcome this deficiency. Polyamines seem to influence protein synthesis and haemoglobin degradation as well since depletion of endogenous polyamines within the parasite seems to result in increased food vacuole acidification, haemoglobin degradation, transport of proteins to the cytoplasm and protein synthesis and stabilisation. The majority of downregulated transcripts were found to be involved in cell-cell adhesion and erythrocyte invasion, protein processing and transport indicating that these processes are dependent on polyamines. Further validation of these findings by microarray as well as proteomic analysis will need to be undertaken. These results validate that polyamines do play an essential role in the cellular biology of the parasite. They also confirm that the inhibition of polyamine biosynthesis is a viable route to undertake in the search for new and improved antimalarial targets. This would be especially useful if it was combined with other antimalarials and their synergistic effects were investigated by transcriptomic, proteomic and bioinformatic analysis / Dissertation (MSc (Biochemistry))--University of Pretoria, 2007. / Biochemistry / MSc / unrestricted