This diploma thesis is an experimental study of grease lubrication in a starved elastohydrodynamic lubricated (EHL) contact. Lubricating film thickness is measured with tribological experimental device ball-on-disc using optical interferometry and high speed camera. The experiment is aimed at a comparison of results acquired by ball-on-disc device and full scale axial bearing. In addition, this paper also investigates the influence of number of the rolling elements (overrolling time) on the resulting film thickness.

Mitochondria are organelles that contain their own genomes (mtDNA) however, the majority of the mitochondrial proteome is encoded by nuclear genes and imported into the mitochondria for assembly into various components. Mitochondria adapt metabolism and biomass to changes in cellular protein synthesis rates accompanying growth. The signaling mechanisms that precede or initiate a mitochondrial expansion program to coordinate mitochondria-to-nuclear communication during development is not well-understood. C. elegans undergo long bouts of starvation in their natural environment upon hatching and remain developmentally arrested as L1s (also known as “L1 diapause”) until they encounter food sources. Prolonged L1 diapause leads to manifestation of age-related phenotypes and mitochondrial remodeling. The mitochondrial unfolded protein response (UPRmt) is a transcriptional response mediated by the bZip protein ATFS-1. ATFS-1 scales mitochondrial expansion with protein synthesis during normal development by regulating genes involved in mitochondrial biogenesis. Here, we demonstrate that ATFS-1 is required for growth and establishment of mature germline upon exiting from starvation-induced L1 arrest. Starvation survival as well as mtDNA depletion during L1 arrest is independent of ATFS-1. Interestingly, we found that the mitochondrial-localized function of ATFS-1 is required for the recovery and expansion of mtDNA following feeding. Lastly, we demonstrate that ATFS-1 functions downstream of the insulin-IGF signaling pathway to regulate mtDNA quantity. The insulin receptor DAF-2 senses nutrient fluctuations and hypomorphic mutation in DAF-2 causes an increase in mtDNA level partly regulated by mitochondrial-localized ATFS-1. Together, our data indicate the physiological relevance and significance of UPRmt in recovering mitochondrial mass when growth and development resumes following starvation.

The Nit1C cluster is a conserved gene cluster of seven genes that confers bacterial growth on cyanide as the sole nitrogen source. Bacteria with this ability are referred to as cyanotrophs. To date, the linkage between Nit1C and cyanotrophy has only been demonstrated for environmental isolates but the cluster also exists in certain medically related bacteria. In this study, a nosocomial isolate, Acinetobacter haemolyticus ATCC 19194, carrying Nit1C also displayed the ability to grow on cyanide. Growth on cyanide was accompanied by the induction of the cluster as was the mere exposure of cells to cyanide. Expression of the cluster was determined by measuring the activity of the nitrilase (NitC) coded for by the cluster and by transcriptional analysis (qRT-PCR). However, a disconnect between nitC message and NitC protein was observed depending on the phase of the growth cycle, the disconnect being related to proteolytic digestion of the NitC protein. Ironically, the cluster was also discovered to be upregulated in the absence of cyanide under nitrogen starvation conditions paralleling biofilm formation. The basis of the genetic linkage to cyanotrophy is not understood but taken together with results showing that nitrogen starvation and biofilm formation are also physiologically associated with Nit1C expression, points to a critical role for the cluster in stress-induced adaptation.

Streptococcus mutans is a secondary colonizer of the dental plaque biofilm and is the primary causative agent of dental caries. Sugar metabolism is central to S. mutans growth and survival. S. mutans produces lactic acid as an end product of sugar metabolism, which results in dissolution of the tooth enamel, leading to dental cavities. Sucrose metabolism also results in the formation of extracellular dextrans that are a key component of the extracellular matrix that encases the bacteria in the biofilm. The availability of sugars is dependent on diet, on competition with other bacteria and on the location of the bacteria within the dental plaque. I hypothesize there are distinct subpopulations of S. mutans within biofilms that respond differently to environmental conditions. I have identified several genetic markers that are helping us identify and characterize some of these subpopulations, and how they react to starvation and to the restoration of nutrients in single species biofilms of S. mutans. Two of the loci that were identified as markers via microarray analysis are rpsT and pdh. rpsT encodes a small ribosomal protein which is strongly expressed during exponential growth, when the cells are producing high levels of ribosomes. The other marker, pdh, is a four-gene operon encoding the pyruvate dehydrogenase complex; pdh is upregulated in late stationary phase. Our laboratory has recently shown that expression of the pdh operon is important for long-term survival in stationary phase, where a subpopulation (~0.5%) is dividing, forms long chains and expresses pdh. In the current studies, rpsT and pdh promoters driving expression of gfp were used to identify the exponential phase subpopulation (rpsT) and a subpopulation capable of surviving in late stationary phase (pdh). In addition, I developed an unstable variant of GFP by fusing a proteolytic tag sequence to the C-terminus of GFP (encoded by ugfp). When the rpsT promoter was inserted upstream, uGFP was produced and subsequently degraded within about 1.5 hours of translation. This behavior allowed us to distinguish exponentially growing cells, as the signal diminishes once the cells entered stationary phase. In biofilms that had been starved for 10 days, there was no expression of PrpsTugfp. I observed that when sucrose was added to these biofilms, some bacteria within the biofilm microcolonies underwent fast exponential-like growth indicated by expression of PrpsT-ugfp. Within 24 hours of the sucrose addition, most growth had ceased and fluorescence had decreased. Using a Ppdh-gfp construct in bacteria in 10-day starved biofilms, fluorescence was observed in long chains of cells within the biofilms indicating slow growth. I hypothesized that the pdh-expressing cells were capable of responding to sucrose restoration and would be one of the principal subpopulations to do so. However, when sucrose was added, these fluorescing chains did not exhibit any growth, while other non-fluorescing bacteria within the biofilm clearly responded to the sucrose by growing. This was unexpected since inactivating the pdh operon leads to drastically reduced survival. It is concluded that pdh plays a role in long term survival, but pdhexpressers do not appear to respond to sugar restoration. This led me to hypothesize that the pdh-expressing population is interacting with other populations of cells in some capacity, enabling them to survive. To determine if this was the case, we performed a mixed culture experiment with wild-type S. mutans and the pdh null mutant. I observed that when these two strains were grown in co-culture, the pdh null mutant survived at low levels, for over 30 days, while this mutant by itself typically did not survive past ten days. This result indicates that the wild-type strain was able to interact with the mutant, leading to increased survival. In biofilms, it seems possible that the pdh-expressing cells secrete a substance or directly interact with other cells, somehow promoting their survival in the starved biofilm. The fluorescent constructs appear to mark distinct populations of cells that respond in different ways to sugar availability, suggesting that S. mutans forms a mixed population of cells able to grow in the presence of sugar or survive prolonged sugar starvation. These studies demonstrate that indeed subpopulations of cells do exist within biofilms, and their interactions may be more complex than previously thought. / Microbiology and Immunology

This study quantifies the rate of bed bug nymphal development, mortality, fecundity and survivorship during starvation for wild caught resistant populations. I then compare some of these characteristics with two susceptible strains. I found that resistant populations develop faster and exhibit less mortality per life stage than susceptible populations. However, there were no significant differences in the total number of eggs produced by the resistant females from the field strains during the 13 feedings/oviposistion cycles (P = 0.106). On average, resistant females from the field strains produced 0.74 eggs per day. Susceptible strains survived a significantly longer time without feeding (89.2 d and 81.4 d) than the resistant strains (RR, ER). The mean duration of adult life (from the day the female becomes an adult until the day she dies) for (RR) strains was 118.7 d ° 11.8 SE. The intrinsic rate of increase r or average daily output of daughter eggs by female was 0.42. The net reproductive rate Rₒ, indicated that one live female egg would, on the average, be replaced by approximately 35 females. Resistant and susceptible populations were found to be different in terms of development, survivorship, and fecundity. The differences between susceptible and resistant strains could be explained by a trade-off between the alleles that confer resistance and the fitness in the population. When compare the stable age distribution of a pyrethroid susceptible strain (HS) and a resistant strain (RR) there were not significant differences (?°= 9.0066, df = 6, P = 0.1732) in the stable age distribution, basically both strains were dominated by the egg stage. No significant difference was found in the expected reproductive contribution of the various life stages to future population size between the two strains (?°= 1.5458, df = 6, P = 0.9564). Despite this, the reproductive contributions of life stages other than eggs were generally higher for the HS strain than for the RR strain. For both strains changes in P? for the adult stage are expected to have the greatest impact on?? compared with changes in P? for the other life stages. The key to the reduction of the populations of bed bugs lies with the reduction of survival of the adults. / Master of Science in Life Sciences

No description available.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Introduction: Breast cancer is a major contributor to mortality in women worldwide. Although, anthracyclines, such as doxorubicin, are among the most valuable treatments for breast cancer, their clinical use is limited due to detrimental side-effects such as cardiotoxicity. Additionally, evidence suggests that cancer cells are becoming increasingly resistant to chemotherapeutic agents. The consequence of poor vascularisation within tumours subsequently leads to a nutrient deprived microenvironment which cancer cells are known to adapt to via metabolic remodelling and increasing autophagy. Autophagy is an intracellular degradation system, which is induced as a survival mechanism in response to starvation and other environmental stressors. Recent studies have shown that starvation protects non-tumourigenic cells against chemotherapy-induced cell death. Furthermore, patients who starved prior to chemotherapy reported reduced side-effects. However, these studies investigated the effects of long-term starvation, which maybe clinically challenging. Therefore, this concept, under shorter and more tolerable periods of starvation still needs to be investigated. We hypothesis, that short-term starvation will sensitize breast cancer cells to doxorubicin-induced cell death. In order to test this hypothesis this study was approached by the following aims: (i) to establish a time point at which MCF12A breast epithelial cells are protected against starvation; (ii) to determine the effect of short-term starvation on doxorubicin induced cell death; (iii) to assess autophagy and; (iv) to assess these above mentioned aims using an in vivo model. Methods: MDAMB231 cells and MCF12A cells were starved for 0, 3, 6, 12, 24 and 48 hours using Hanks Balanced Salt Solution. Cell viability was assessed using the trypan blue, MTT and Caspase-Glo assays. MDAMB231 cells and MCF12A cells were subjected to the following conditions: (1) control; (2) 5 μM doxorubicin; (3) starvation of 3 hours and (4) a combination of starvation and doxorubicin. Following treatment an MTT assay to assess cell viability was performed. MDAMB231 cells were further examined using Live-Cell Imaging and western blot analysis. C57BL6 tumour bearing mice were treated with doxorubicin (5 mg/kg) or in combination with starvation of 24 hours. Upon termination of the protocol, tumour tissue was assessed using western blot analysis. In both in vitro and in vivo analyses cleaved-caspase 3 and cleaved-PARP were used as markers for apoptosis, LC3 and p62 as autophagic markers and p-AMPK and p-mTOR as markers of oxygen and energy sensing, respectively. Results and discussion: Three hours of starvation was chosen for in vitro experiments since no significant reduction in cell viability or increases in apoptosis occurred at this time-point in the normal MCF12A breast epithelial cells. As expected, doxorubicin induced a significant decrease in cell viability in the cancerous MDAMB231 cells. Short-term starvation in combination with doxorubicin treatment caused a further significant decrease in cell viability in MDAMB231 cells compared to the doxorubicin group alone. Interestingly, starved MCF12A cells were protected against doxorubicin-induced cytotoxicity as cell viability significantly increased. A significant decrease in autophagy was further observed with the combined treatment of doxorubicin and starvation which corresponded with a significant increase in cell death. In contrast, although the in vivo study also demonstrated a significant elevation in cell death and autophagy in response to doxorubicin treatment, the combined treatment (starvation and doxorubicin) did not have an additive effect when compared to the doxorubicin group alone. Conclusion: Our in vitro results clearly demonstrate that short-term starvation sensitizes breast cancer cells to doxorubicin-induced cell death. Additionally, decreased levels of autophagy appear to contribute to this phenomenon of sensitization. Although doxorubicin treatment resulted in increased apoptosis in vivo, 24 hours starvation in combination with doxorubicin did not sensitize the tumours to doxorubicin treatment. Thus, for future in vivo studies more time points should be considered in order to translate the beneficial effects of short-term starvation observed in our in vitro study to an animal model. / AFRIKAANSE OPSOMMING: Inleiding: Borskanker is ‘n belangrike faktor wat bydrae tot sterftes in vrouens wêreldwyd. Alhoewel antrasikliene soos doxorubicin, waardevol is vir die behandeling van borskanker, word die kliniese gebruik daarvan beperk deur newe-effekte soos kardiotoksisiteit. Verder, word daar al hoe meer bewys dat kankerselle toenemend weeerstandbiedend word teen chemoterapeutiese middels. Swak vaskularisasie van tumore lei tot ‘n mikro-omgewing met beperkte voedingstowwe waaby kankerselle kan aanpas deur middel van metaboliese hermodelering en ‘n toename in autofagie. Autofagie is ‘n intrasellulêre degraderingsisteem wat as ‘n oorlewingsmeganisme aangewend word tydens verhongering en ander omgewingstressors. Onlangse studies het getoon dat verhongering nie-tumourigeniese (normale) selle teen chemoterapie-geïnduseerde seldood beskerm. Verder is daar ook geraporteer dat pasiënte wat gevas het voor chemoterapie, verminderde newe-effekte getoon het. Hierdie studies het egter gefokus op ‘n relatief lang-termyn vas, wat klinies nogal uitdagend kan wees. Daarom moet hierdie konsep nog op korter, meer hanteerbare tye getoets word. Ons hipotese is dus dat kort-termyn vas borskankerselle kan sensitiseer tot doxorubicin-geïnduseerde seldood. Om hierdie hipotese te toets, is die volgende doelwitte gestel: (i) om ‘n tydspunt te bepaal waar MCF12A borsepiteelselle beskerm is teen verhongering; (ii) om die effek van kort-termyn verhongering op doxorubicin-geïnduseerde seldood te toets; (iii) om autofagie te karakteriseer in ons model en; (iv) om hierdie doelwitte ook in ‘n in vivo model te toets. Metodes: MDAMB231 en MCF12A selle is verhonger vir 0, 3, 6, 12, 24 and 48 ure deur van Hanks se gebalanseerde soutoplossing gebruik te maak. Sellewensvatbaarheid is bepaal deur middel van trypan blou, MTT en die Caspase-Glo tegnieke. MDAMB231 en MCF12A selle is onderwerp aan die volgende omstandighede: (1) kontrole; (2) 5 μM doxorubicin; (3) verhongering van 3 ure en (4) ‘n kombinasie van verhongering en doxorubicin. Na behandeling is die sellewensvatbaarheid deur middel van die MTT tegniek bepaal. MDAMB231 selle is verder ondersoek deur middel van “Live-Cell Imaging” en die westelike klad tegniek. C57BL6 tumor-draende muise is behandel met doxorubicin (5 mg/kg) of met ‘n kombinasie van verhongering van 24 ure en doxorubicin. Aan die einde van die protokol, is die kankerweefsel geanaliseer deur die westelike klad tegniek. In beide in vitro en in vivo analises, is gekliefde- caspase 3 en -PARP as merkers vir apoptose, LC3 and p62 as merkers vir autofagie en p-AMPK en p-mTOR as suurstof- en energie sensors respektiewelik gemeet. Resultate en bespreking: Vir die in vitro eksperimente, is ‘n tydspunt van 3 ure gekies as gevolg van die feit dat geen afname in sellewensvatbaarheid en ‘n toename in apoptose in hierdie tydsgleuf tydens verhongering in die normale MCF12A borsepiteelselle plaasgevind het nie. Soos verwag, het doxorubicin behandeling ‘n insiggewende afname in sellewensvatbaarheid in die kankeragtige MDAMB231 selle veroorsaak. Die kombinasie-terapie van verhongering en doxorubicin het ‘n verdere verhoging in seldood teweeg gebring in die MDAMB231 selle, maar het die normale MCF12A borsepiteelselle teen doxorubicin-geïnduseerde toksisiteit beskerm. Die kombinasie-behandeling is ook geassosieer met ‘n afname in autofagie. Alhoewel, die in vivo studie ook getoon het dat doxorubicin alleen insiggewende hoeveelheid seldood teweeggebring het, het die kombinasie-behandeling nie die additiewe effek, soos in die in vitro studie, teweeg gebring nie. Gevolgtrekking: Die in vitro resultate het duidelik getoon dat kort-termyn verhongering borskankerselle kan sensitiseer vir doxorubicin terapie. Verder het dit geblyk dat ‘n afname in autofagie tot die fenomeen van sensitisering bygedrae het. Alhoewel doxorubicin behandeling in vivo tot ‘n toename in apoptose in die tumor gelei het, het die kombinasie behandeling nie die kankerweefsel ten op sigte van doxorubicin gesensitiseer nie. Daar sal dus vir toekomstige in vivo studies meer tydsgleuwe van behandeling ondersoek moet word om die optimum verhongeringsperiode te vind sodat die in vitro resultate ook in vivo van toepassing kan wees. / NRF and CANSA for financial support

Drought-induced tree mortality is an emerging global phenomenon that appears related to climate change and rising temperatures in particular, and may be an early indication of vegetation change. However, vegetation response to climate change is uncertain, particularly for future novel climates. Notably, no current models of vegetation change attempt to mechanistically predict plant mortality, and in particular, mortality of trees, which exerts strong influences on ecological function. Resolving uncertainties surrounding the physiological mechanism and temperatures sensitivity of tree mortality is a current challenge in global change ecology. The objectives of this dissertation were to 1) consider tree mortality consequences for earth system processes related to carbon, water, and energy exchange that include climate regulation; 2) explore tree mortality effects on the water cycle by developing hypotheses and research needs; 3) quantify the temperature sensitivity of drought-induced tree mortality and gain insight into the physiological mechanism of mortality; 4) quantify the relationships among temperature, stored carbohydrate resources, and gas exchange to further elucidate physiological tree mortality mechanisms; and 5) quantify the sensitivity of two species of pine seedlings to progressively elevated temperatures and relate mortality to the effect of temperature on carbon metabolism. Major findings of this dissertation relate to the temperature sensitivity, physiological mechanism, and implications of tree mortality. Assessment of the potential consequences of tree mortality for earth system processes documented the contrasting influences of tree mortality on the terrestrial C cycle and land-surface energy exchange, the balance of which will determine the net effects on climate regulation (Appendix A). Following a survey of the ecohydrology literature, thresholds for tree mortality to cause watershed changes were hypothesized at ~20% loss of canopy cover, ~500 mm of annual precipitation, and whether flows are snowmelt dominated (Appendix B). Elevated temperature (~+4°C) accelerated tree mortality by 28% during experimental drought, a difference related to cumulative respiration dynamics in piñon pine (Appendix C). Stored carbohydrate resources were declined during lethal drought but were not entirely depleted prior to mortality (Appendix D). Seedlings exhibited progressive declines in time-to mortality with increased temperatures, a response related to C metabolism (Appendix E).

The distribution of resources is vital to any system or society. This is particularly true of social insect colonies where independent access to resources is not available to all members. Only a fraction of individuals are responsible for obtaining food for the colony from outside the nest. Surprisingly little is known about how this food is subsequently distributed to members inside the nest. The work in this thesis is focused around a set of food distribution experiments conducted using four colonies of the ant Temnothorax albipennis. The study applies a well-used technique in a new way to investigate the distribution of food under two different scenarios: feeding under normal conditions and famine relief feeding after a period of starvation. All ants in each colony are marked and then individually tracked recording every feeding interaction to obtain a complete network of food transmission. This work has shown that all four colonies efficiently relieved the famine within 30 minutes of introducing new food. This process was facilitated by workers abandoning their spatial structure and expanding their space use; feeding multiple recipients from a single donor; and simultaneously spreading stored food and new food. Recruitment of foragers did not play a major role in relieving the famine but foragers were responsible for most of the first round of feeding. The study revealed that not all members received the same amount of food and most ants received food in multiple feeding interactions. The transmission pathways used to distribute the food present an opportunity for harmful substances to spread. The pathways are explored in this context to see whether the colonies might aim to minimize the spread by partitioning the pathways or maximise spread by mixing to promote social immunity. The study reveals behavioural differences between the four colonies which are likely to result from the inherent variation in demographic and geometric properties. These differences highlight the flexibility of ant colonies during problem solving under different conditions.

No description available.