published_or_final_version / Dentistry / Master / Master of Dental Surgery

Dissertation submitted to the Faculty of Science, University of the Witwatersrand, in fulfillment of the requirements for the degree of Master of Science. Johannesburg, 2013. / The evolutionary genesis and the current adaptive significance of the use of the discontinuous gas exchange cycle (DGC) for respiration by insects is the subject of intense debate. Most current research centers on three adaptive hypotheses and one non-adaptive hypothesis; these are the hygric hypothesis, the chthonic hypothesis, the oxidative-damage hypothesis and the emergent-property hypothesis respectively. Workers of the harvester termite, Hodotermes mossambicus were selected as a model to test three of these hypotheses. The respiratory patterns of workers, investigated using flow-through respirometry, were obtained at 0 % relative humidity (RH), 100 % RH, at 100 % O2 and under varying temperature to evaluate the assumptions of the various hypotheses. A change in ambient humidity had no impact on metabolic rate (VCO2), coefficient of variation (CV) or the pattern of gas exchange but only influenced the amount of water loss experienced by workers. Major workers exposed to hyperoxia (100 % O2) responded by increasing spiracular control and constriction through the use of cyclic gas exchange thereby protecting their interior against the toxic effects of O2. As VCO2 increased in response to increasing temperature, the gas exchange pattern displayed by workers transitioned from a modified DGC through cyclic to continuous gas exchange. A true DGC, defined as showing all three phases and a CV value close to 2, was not expressed under any of the experimental conditions. The results of this study support the oxidative-damage and emergent-property hypotheses but not the hygric hypothesis. The workers of H. mossambicus spend only brief periods above ground before returning to the refuge of their underground nests and as such there is probably little selective advantage to the DGC for limiting respiratory water loss. The conclusion drawn from the study of termite workers is that changes in respiratory patterns are most likely an emergent property of the insects’ nervous and respiratory systems and spiracular control also serves to limit oxidative damage.

Water moves in soil in response to potential gradients. The basic equation for this movement is the generalized flow equation: v = - KV0 in which v is volume of water passing through a unit area in unit time, K is the conductivity coefficient, V is the gradient operator (vector), and V 0 is the potential gradient.

<p>Accelerated life testing (ALT) is commonly used to obtain the reliability (lifetime) of a system in a short period by applying severe stress factors (i.e. temperature, humidity, chemical etc.,) to the system. Accelerated life testing was done on 2N3904 N-P-N bipolar junction transistor. After accelerated life testing, the acceleration factor is calculated through statistical analysis to predict the lifetime of a system at a certain operating condition. Multiple temperature and water immersed experiments were conducted on transistors at various temperatures. With the obtained results, analysis was done on the data to predict lifetime of transistors at different environmental conditions. Effects of the environmental conditions on the transistors were also discussed. Corrosion effects on transistors were studied. ALTA software is used to analyze the experimental data and to calculate the lifetime of the transistors. From the obtained failure data prediction of mean time to failure at various temperatures was done. </p>

Leishmania caracteriza-se por apresentar duas formas morfologicamente distintas em seu ciclo de vida: promastigotas e amastigotas. As formas promastigotas vivem tubo digestório do vetor flebotomíneo, sob as condições de pH 7,0 e temperatura ambiente, ao redor de 25ºC. As formas amastigotas são encontradas no interior dos fagolisossomos de macrófagos infectados onde encontram um ambiente de pH ácido e temperatura ao redor de 34ºC. Leishmania utiliza arginina para a síntese de poliaminas, que desempenham papel fundamental no crescimento, diferenciação celular e sucesso da infecção. A tomada de arginina em L. (L.) amazonensis é feita pela proteína transportadora de aminoácidos - amino acid transporter-like 3 (AAP3), codificada por duas cópias do gene (5.1 aap3 e 4.7 aap3) dispostas em tandem no genoma. Os transcritos de 5.1 aap3 e de 4.7 aap3 apresentam 98% de identidade entre as ORFs, mas diferem nas 5\' e 3\' UTR. O objetivo do presente trabalho foi avaliar se os sinais de temperatura, pH e privação de arginina disparam a regulação da expressão de aap3 em formas promastigotas e amastigotas. Para isso avaliamos o nível dos transcritos e realizamos ensaios de tomada de arginina em células submetidas à privação ou suplementadas com arginina, nas temperaturas de 25°C ou 34°C em pH 7,0 ou 5,0. Constatou-se em formas promastigotas que o transcrito 5.1 aap3 apresentou maior abundância em relação a 4.7 aap3, e que a privação promoveu o aumento da tomada do aminoácido quando os parasitos eram mantidos em pH 7,0 a 25°C, corroborando dados anteriores do nosso grupo. Demonstramos que a mudança de temperatura foi um fator importante para o aumento do número de cópias de 5.1 aap3 em promastigotas privadas, principalmente quando associadas com o pH 5,0. Além disso, o aumento da temperatura favoreceu a tomada de arginina, corroborando com a elevação do número de cópias observada para o transcrito 5.1 aap3. Em amastigotas-like, mantidas a 25°C e pH 7,0 a privação reverteu a expressão de 5.1 aap3 para o mesmo perfil observado para promastigotas. Contudo, não observamos um favorecimento na tomada de arginina. Ainda em amastigotas, o tratamento a 34°C e pH 7,0 favoreceu a tomada de arginina, porém não observamos um aumento correspondente na quantificação do transcrito. O transcrito 4.7 aap3 não apresentou alteração significativa em qualquer tratamento em promastigotas e amastigotas. Os nossos resultados indicam que a variação de temperatura e do pH, além da privação de arginina, podem ser sinais importantes para regulação da expressão diferencial de aap3, principalmente a cópia 5.1 aap3, de forma a assegurar a oferta de arginina em formas promastigotas previamente à entrada no hospedeiro mamífero e em formas amastigotas, na passagem para o vetor, assegurando o sucesso da infecção / Leishmania presents two morphologically distinct forms in its life cycle: promastigote and amastigote. The promastigotes live in the midgut of the sand fly vector under the conditions of pH 7.0 and room temperature, around 25°C. The amastigote forms are found inside the phagolysosomes of infected macrophages where they encounter an environment of acidic pH and temperature around 34°C. Leishmania uses the arginine to synthesize polyamines which play an important role in cell growth, differentiation and in the successful of infection. The arginine uptake in Leishmania (L.) amazonensis is made by an amino acid porter 3-like protein (AAP3), coded by a two copies gene (5.1 aap3 e 4.7 aap3) arranged in tandem in the genome. The transcripts, 5.1 aap3 and 4.7 aap3, present 98% of ORFs identity, but differ in the 5\' and 3\' UTR. The aim of this work was to evaluate if canges in temperature, pH and arginine deprivation represent signals to trigger the regulation expression of aap3 in promastigotes and amastigotes. For this, we evaluated the transcripts level and performed assays of arginine uptake in parasites subjected to arginine starvation or supplemented with arginine, at temperatures of 25°C or 34°C and pH 7.0 or 5.0. In promastigotes we verified that the transcript 5.1 aap3 showed higher abundance in relation to 4.7 aap3, and that the arginine starvation promoted an increase in the amino acid uptake when the parasites were maintained at pH 7.0 and 25°C, confirming previous data from our group. The change of temperature was an important factor to the increase of 5.1 aap3 transcripts - in starved promastigotes, particularly when associated with pH 5.0. In addition, the increase of the temperature led to an increase of the arginine uptake, correlated to the increase of 5.1 aap3 transcript. The amastigotes-like maintained at 25°C and pH 7.0 and submitted to the amino acid starvation reverted 5.1 aap3 expression profile to the same observed for promastigotes. However, those condictions did not favor an increase in arginine uptake. The treatment of amastigotes at 34°C and pH 7.0 facilitated the increased of arginine uptake, but did not correlated with the transcripts level. The 4.7 aap3 transcript did not change significantly in any promastigotes and amastigotes treatments. Our results indicate that variation in temperature and pH, in addition to arginine starvation may be important signals for regulating the aap3 expression, mainly the copy 5.1 aap3, in order to ensure the correct supply of arginine in the previous period in relation to the entry of the promastigotes into the mammalian host or to the amastigotes transition in the vector, ensuring the success of the infection.

Leishmania caracteriza-se por apresentar duas formas morfologicamente distintas em seu ciclo de vida: promastigotas e amastigotas. As formas promastigotas vivem tubo digestório do vetor flebotomíneo, sob as condições de pH 7,0 e temperatura ambiente, ao redor de 25ºC. As formas amastigotas são encontradas no interior dos fagolisossomos de macrófagos infectados onde encontram um ambiente de pH ácido e temperatura ao redor de 34ºC. Leishmania utiliza arginina para a síntese de poliaminas, que desempenham papel fundamental no crescimento, diferenciação celular e sucesso da infecção. A tomada de arginina em L. (L.) amazonensis é feita pela proteína transportadora de aminoácidos - amino acid transporter-like 3 (AAP3), codificada por duas cópias do gene (5.1 aap3 e 4.7 aap3) dispostas em tandem no genoma. Os transcritos de 5.1 aap3 e de 4.7 aap3 apresentam 98% de identidade entre as ORFs, mas diferem nas 5\' e 3\' UTR. O objetivo do presente trabalho foi avaliar se os sinais de temperatura, pH e privação de arginina disparam a regulação da expressão de aap3 em formas promastigotas e amastigotas. Para isso avaliamos o nível dos transcritos e realizamos ensaios de tomada de arginina em células submetidas à privação ou suplementadas com arginina, nas temperaturas de 25°C ou 34°C em pH 7,0 ou 5,0. Constatou-se em formas promastigotas que o transcrito 5.1 aap3 apresentou maior abundância em relação a 4.7 aap3, e que a privação promoveu o aumento da tomada do aminoácido quando os parasitos eram mantidos em pH 7,0 a 25°C, corroborando dados anteriores do nosso grupo. Demonstramos que a mudança de temperatura foi um fator importante para o aumento do número de cópias de 5.1 aap3 em promastigotas privadas, principalmente quando associadas com o pH 5,0. Além disso, o aumento da temperatura favoreceu a tomada de arginina, corroborando com a elevação do número de cópias observada para o transcrito 5.1 aap3. Em amastigotas-like, mantidas a 25°C e pH 7,0 a privação reverteu a expressão de 5.1 aap3 para o mesmo perfil observado para promastigotas. Contudo, não observamos um favorecimento na tomada de arginina. Ainda em amastigotas, o tratamento a 34°C e pH 7,0 favoreceu a tomada de arginina, porém não observamos um aumento correspondente na quantificação do transcrito. O transcrito 4.7 aap3 não apresentou alteração significativa em qualquer tratamento em promastigotas e amastigotas. Os nossos resultados indicam que a variação de temperatura e do pH, além da privação de arginina, podem ser sinais importantes para regulação da expressão diferencial de aap3, principalmente a cópia 5.1 aap3, de forma a assegurar a oferta de arginina em formas promastigotas previamente à entrada no hospedeiro mamífero e em formas amastigotas, na passagem para o vetor, assegurando o sucesso da infecção / Leishmania presents two morphologically distinct forms in its life cycle: promastigote and amastigote. The promastigotes live in the midgut of the sand fly vector under the conditions of pH 7.0 and room temperature, around 25°C. The amastigote forms are found inside the phagolysosomes of infected macrophages where they encounter an environment of acidic pH and temperature around 34°C. Leishmania uses the arginine to synthesize polyamines which play an important role in cell growth, differentiation and in the successful of infection. The arginine uptake in Leishmania (L.) amazonensis is made by an amino acid porter 3-like protein (AAP3), coded by a two copies gene (5.1 aap3 e 4.7 aap3) arranged in tandem in the genome. The transcripts, 5.1 aap3 and 4.7 aap3, present 98% of ORFs identity, but differ in the 5\' and 3\' UTR. The aim of this work was to evaluate if canges in temperature, pH and arginine deprivation represent signals to trigger the regulation expression of aap3 in promastigotes and amastigotes. For this, we evaluated the transcripts level and performed assays of arginine uptake in parasites subjected to arginine starvation or supplemented with arginine, at temperatures of 25°C or 34°C and pH 7.0 or 5.0. In promastigotes we verified that the transcript 5.1 aap3 showed higher abundance in relation to 4.7 aap3, and that the arginine starvation promoted an increase in the amino acid uptake when the parasites were maintained at pH 7.0 and 25°C, confirming previous data from our group. The change of temperature was an important factor to the increase of 5.1 aap3 transcripts - in starved promastigotes, particularly when associated with pH 5.0. In addition, the increase of the temperature led to an increase of the arginine uptake, correlated to the increase of 5.1 aap3 transcript. The amastigotes-like maintained at 25°C and pH 7.0 and submitted to the amino acid starvation reverted 5.1 aap3 expression profile to the same observed for promastigotes. However, those condictions did not favor an increase in arginine uptake. The treatment of amastigotes at 34°C and pH 7.0 facilitated the increased of arginine uptake, but did not correlated with the transcripts level. The 4.7 aap3 transcript did not change significantly in any promastigotes and amastigotes treatments. Our results indicate that variation in temperature and pH, in addition to arginine starvation may be important signals for regulating the aap3 expression, mainly the copy 5.1 aap3, in order to ensure the correct supply of arginine in the previous period in relation to the entry of the promastigotes into the mammalian host or to the amastigotes transition in the vector, ensuring the success of the infection.

Electrophysical agents (EPA) are a fundamental element of therapy practice and are vital for the treatment of a variety of conditions. Many of these agents employ some form of electromagnetic fields (EMF), in which radiofrequency (RF) is a major component. The therapeutic effects of RF are mainly linked to their effects on pain relief and potential effects on tissue repair. Although RF across various frequency ranges has been in use, reviews have shown that the frequency ranges currently used in therapy practice have narrowed to within 30 kHz-30,000 kHz (30 MHz). The most commonly used and hence the most commonly researched are shortwave therapies (SWT) that operate at 27.12 MHz, which is presently used predominantly in its pulsed form (PSWT). In addition to SWT, devices employing significantly lower RF ranges have also been used widely despite their lack of evidence. Capacitive Resistive Monopolar Radiofrequency (CRMRF) that operates at 448 kHz is one such RF. This programme of research was designed to investigate the physiological and clinical efficacy of CRMRF delivered using the 'Indiba Activ 902' device. The project also evaluated the scope and evidence for RF-based EPAs in therapy, through a comprehensive review of literature. A total of 120 relevant clinical studies on either acute (30 studies) or chronic (90 studies) conditions were reviewed. Notable evidence was identified for chronic OA knee and acute postoperative pain and wound healing. Some evidence also exists for chronic low back pain and healing of chronic wounds. Only eight studies reported devices that employed RF outside the shortwave frequency band. In a randomised crossover laboratory study on asymptomatic adults, the effects of contrasting doses of CRMRF on skin temperature (SKT), skin blood flow (SBF), nerve conduction velocity (NCV), deep blood flow and the extensibility of tissues were examined against a placebo dose and a control condition with no treatment. The study further compared CRMRF results with that of PSWT. The results showed that high (moderately thermal) and low (sub/minimally thermal) doses of CRMRF significantly enhanced and sustained SKT (p < 0.001), while only the high dose meaningfully increased SBF (p < 0.001). High dose PSWT increased SKT marginally (p < 0.001) but did not sustain it. Further, the high and low dose CRMRF significantly enhanced blood flow volume at depth (p=0.003), while PSWT failed to show any significant impact. None of the treatments significantly affected deep blood flow velocity, tissue extensibility or NCV. These results were reproduced on a cohort of patients affected by OA knee in a randomised controlled trial (RCT), and the effects appeared more pronounced in the patients than in the asymptomatic people. More importantly, the RCT showed that a four-week high dose CRMRF treatment (eight sessions) produced statistically and clinically significant gains in pain and function associated with OA knee in the short to medium term (p < 0.001), which was also significantly more pronounced than the gains produced by a placebo, or standard care (p=0.001for pain; p=0.031 for function). The findings of this study were considered promising. It is therefore suggested that CRMRF-based treatment can potentially be used as an adjunct to current therapeutic methods to enhance the clinical outcomes. However, further studies are needed to substantiate this, and the current results will provide credible baseline data for future research.