The evolution of immune escape during HIV infection is well documented, yet some CD8+ T-cell epitopes remain conserved even in the presence of strong cognate responses. This report investigates the frequency of sequence variability within the HLA-A2 restricted immunodominant epitope SLYNTVATL (SL9). Sequencing results, from 15 HIV+ HLA-A2+ individuals, support a very high degree of conservation of this epitope sequence with only focused, conservative changes evident in proviral quasispecies. These observations suggest that changes to the SL9 sequence may have deleterious effects on viral replication and are therefore limited in vivo. To investigate the sequence constraints imposed on the SL9 epitope by HIV replicative fitness, HIV plasmid clones incorporating synonymous and non-synonymous mutations throughout the SL9 epitope region were constructed. Growth of the resultant congenic viruses in mono-infection assays indicated a wide diversity of SL9 variant viruses that grew efficiently. Furthermore, dual-infection viral competition assays demonstrated that SL9 variants known to arise in vivo were equally fit to consensus SL9 virus and several novel epitope variants were also able to compete effectively. Collectively, the data reported in this thesis reveal that the viral pressures acting to conserve the SL9 epitope sequence are less than previously assumed and highlight the lack of understanding of protective CD8+ T-cell action during HIV infection.

The aims of this thesis were to characterize the molecular ecology of the hyporheic zone – between dissolved organic matter (DOM) and microbes – and to test whether seasonal and spatial patterns existed in correlation with seasonal ecosystem processes. The hyporheic zone is an area of vertical integration between groundwater and surface water, and lateral integration between terrestrial and stream ecosystems. Colonization corers were used to collect in situ DOM and bacterial communities from the hyporheic sediments of two streams that varied in hydroperiod (i.e., permanent vs. intermittent). DOM was collected using passive samplers and analyzed using 1H NMR and fluorescence spectroscopy; bacteria were characterized using terminal-restriction fragment length polymorphism. At the permanent site, bacteria correlated significantly with seasonal environmental factors including: fall communities with DOM concentration; spring and winter communities with nitrate concentrations; and summer communities with temperature. Bacterial communities at the intermittent site were significantly correlated with flooding as a function of hydrologic connectivity. Sediment communities were discriminated between hyporheic sediments and interstitial porewaters, and shared several operational taxonomic units (OTUs). Sediment communities were more distinct when hydrologic connectivity was low, and porewater communities changed dramatically upon flooding. Fifteen out of 259 OTUs were shared across aquatic sediments, interstitial porewater and watershed soil samples. DOM was spatially and seasonally dynamic in both sites. Five key DOM groups described using 1H NMR spectroscopy revealed spatial differences between the permanent and intermittent sites. EEM-PARAFAC models confirmed that despite significantly different molecular components, the relative sources of DOM at both sites were similar, including humic-like terrestrial sources and tyrosine (microbial) sources. This study provides new knowledge on both organic matter dynamics and bacterial communities in a dynamic aquatic ecotone, and also confirmed the hypothesis that bacterial communities correlated significantly with ecosystem processes within a watershed.

The evolution of immune escape during HIV infection is well documented, yet some CD8+ T-cell epitopes remain conserved even in the presence of strong cognate responses. This report investigates the frequency of sequence variability within the HLA-A2 restricted immunodominant epitope SLYNTVATL (SL9). Sequencing results, from 15 HIV+ HLA-A2+ individuals, support a very high degree of conservation of this epitope sequence with only focused, conservative changes evident in proviral quasispecies. These observations suggest that changes to the SL9 sequence may have deleterious effects on viral replication and are therefore limited in vivo. To investigate the sequence constraints imposed on the SL9 epitope by HIV replicative fitness, HIV plasmid clones incorporating synonymous and non-synonymous mutations throughout the SL9 epitope region were constructed. Growth of the resultant congenic viruses in mono-infection assays indicated a wide diversity of SL9 variant viruses that grew efficiently. Furthermore, dual-infection viral competition assays demonstrated that SL9 variants known to arise in vivo were equally fit to consensus SL9 virus and several novel epitope variants were also able to compete effectively. Collectively, the data reported in this thesis reveal that the viral pressures acting to conserve the SL9 epitope sequence are less than previously assumed and highlight the lack of understanding of protective CD8+ T-cell action during HIV infection.

The aims of this thesis were to characterize the molecular ecology of the hyporheic zone – between dissolved organic matter (DOM) and microbes – and to test whether seasonal and spatial patterns existed in correlation with seasonal ecosystem processes. The hyporheic zone is an area of vertical integration between groundwater and surface water, and lateral integration between terrestrial and stream ecosystems. Colonization corers were used to collect in situ DOM and bacterial communities from the hyporheic sediments of two streams that varied in hydroperiod (i.e., permanent vs. intermittent). DOM was collected using passive samplers and analyzed using 1H NMR and fluorescence spectroscopy; bacteria were characterized using terminal-restriction fragment length polymorphism. At the permanent site, bacteria correlated significantly with seasonal environmental factors including: fall communities with DOM concentration; spring and winter communities with nitrate concentrations; and summer communities with temperature. Bacterial communities at the intermittent site were significantly correlated with flooding as a function of hydrologic connectivity. Sediment communities were discriminated between hyporheic sediments and interstitial porewaters, and shared several operational taxonomic units (OTUs). Sediment communities were more distinct when hydrologic connectivity was low, and porewater communities changed dramatically upon flooding. Fifteen out of 259 OTUs were shared across aquatic sediments, interstitial porewater and watershed soil samples. DOM was spatially and seasonally dynamic in both sites. Five key DOM groups described using 1H NMR spectroscopy revealed spatial differences between the permanent and intermittent sites. EEM-PARAFAC models confirmed that despite significantly different molecular components, the relative sources of DOM at both sites were similar, including humic-like terrestrial sources and tyrosine (microbial) sources. This study provides new knowledge on both organic matter dynamics and bacterial communities in a dynamic aquatic ecotone, and also confirmed the hypothesis that bacterial communities correlated significantly with ecosystem processes within a watershed.

Lake Zapotlán is a small (1100 ha) endorheic lake in western Mexico that is internationally recognized by RAMSAR. It receives point source pollution from partially treated sewage from two surrounding cities, as well as non-point sources, including urban runoff, agricultural runoff, erosion and consequent deposition of sediment as a result of deforestation surrounding the Lake. The purpose of this study was to determine the severity of pollution in the Lake through measurement of nutrient and bacteria levels and assess for potential human health and ecological risks in Lake Zapotlán. Results found that nutrient levels have increased since 1994 and that they are high enough to cause eutrophication problems. Partially treated wastewater contributes over 30 tonnes of phosphorus to the Lake each year. E. coli levels were extremely high and could pose a health risk to those participating in recreational activities on the Lake.

Lake Zapotlán is a small (1100 ha) endorheic lake in western Mexico that is internationally recognized by RAMSAR. It receives point source pollution from partially treated sewage from two surrounding cities, as well as non-point sources, including urban runoff, agricultural runoff, erosion and consequent deposition of sediment as a result of deforestation surrounding the Lake. The purpose of this study was to determine the severity of pollution in the Lake through measurement of nutrient and bacteria levels and assess for potential human health and ecological risks in Lake Zapotlán. Results found that nutrient levels have increased since 1994 and that they are high enough to cause eutrophication problems. Partially treated wastewater contributes over 30 tonnes of phosphorus to the Lake each year. E. coli levels were extremely high and could pose a health risk to those participating in recreational activities on the Lake.

Doctor of Philosophy / Department of Biology / Walter K. Dodds / We used a series of whole stream experiments to quantify the impacts of inorganic-nitrogen concentration on stream nitrogen cycling and transport in prairie streams. We conducted 15NO3- stable isotope tracer experiments to measure the nitrogen cycling dynamics in 9 streams with a wide range (over 5 orders of magnitude) of nitrate concentrations. The major nitrogen-transforming processes, including uptake, nitrification, and denitrification, increased approximately 2 to 3 orders of magnitude and did not show signs of Michaelis-Menten type saturation across streams. Denitrification only accounted for a small proportion of total nitrate uptake. The observed functional relationships of biological nitrogen transformations and chronic nitrate concentration were best described by a Log-Log relationship. A series of inorganic-nitrogen addition experiments were conducted to quantify the impacts of acute nitrogen inputs on nitrogen cycling. These experiments showed that uptake saturated in response to short-term pulses of nitrogen. Ambient concentrations of ammonium and nitrate were less than their respective half-saturation coefficients, and uptake rates were less than 5% of Vmax, suggesting severe limitation of nitrogen. The saturation of uptake due to acute nitrogen inputs contrasts with uptake associated with chronic inputs of nitrate, which was not found to saturate. Chamber experiments and whole-stream ammonium addition experiments demonstrated that uptake and mineralization of ammonium varies spatially within the stream channel, occurring predominantly in riffles as opposed to pool habitats. The total transport distance of nitrogen and carbon within prairie streams was estimated based on field measurements and nutrient spiraling theory. Transport of organic nitrogen was dominant in prairie streams, as compared to inorganic nitrogen transport, both in terms of total concentration and transport distance. These results indicate that although carbon and inorganic-nitrogen were highly conserved in these headwater streams, organic-nitrogen was much more readily transported.

Master of Science / Department of Biology / Walter K. Dodds / North America has lost more than 95% of the original tallgrass prairie because of heavy land conversion, making prairie streams some of the most endangered habitats in North America. In order to effectively manage aquatic systems and improve biotic integrity of prairie streams research is needed that assesses the ecosystem characteristics of natural systems and evaluates the influence of anthropogenic alteration. We described the ecosystem characteristics of six ephemeral headwater streams draining tallgrass prairie within the Osage Plains of southwest Missouri. NO-3-N among all sites ranged from 1.56-91.36 μg L-1, NH+4-N ranged from 5.27-228.23 μg L-1, soluble reactive phosphorus ranged from below detection (1.0 μg L-1) to 41.22 μg L-1, TN ranged from 113.82-882.89 μg L-1, and TP ranged from 8.18-158.5 μg L-1during baseflow conditions. TN:TP molar ratios ranged from 22:1 to 53:1 indicating possible P was limiting relative to N in some streams. TSS during baseflow conditions ranged from 0.27-31.80 mg L-1. Autotrophic and heterotrophic comparisons of our study sites and reference sites classified our study streams as oligo-, meso-, and eu-autotrophic (N= 1, 4, and 1, respectively) and oligo-, meso-, and eu-heterotrophic (N= 4, 1, and 1, respectively). This study suggests that good water quality and moderate heterotrophic condition, with greater GPP resulting from an open canopy, are common conditions of tallgrass prairie streams. We also investigated interactions between land use/land cover, discharge rate, hydrologic alteration, and in-stream total suspended solids concentration in 23 Kansas- Missouri streams. Most streams had break points in the TSS loading rates at discharge rates exceeded <25% of days. Our estimates showed that 88% of the total annual TSS load occurred during the 11% of days with the greatest discharge rates. Buffered streams with greater percentages of grass and/or forest riparian areas had lower breakpoint values (indicating greater discharge rates were required to transport solid particles) and lower regression intercepts, which correlated to lesser TSS concentrations relative to unbuffered streams during high discharge days. In addition, grass buffered streams had smaller flood peaks and slower rise rates and forest buffered streams had less frequent floods, which lead to less total TSS transport.

We deployed depth-specific drifters in the western and eastern parts of the South Arm basin of Lake Opeongo and collected zooplankton samples at west and east fixed stations and at additional up- and downwind locations at three depths of the epilimnion under a range of wind conditions. Water currents had highest association with the immediate wind direction and the direction they travelled was dependent on wind strength. Along the main west-east fetch large zooplankton resided high in the epilimnion and were transported eastwards by strong surface currents where they accumulated. Small zooplankton were more uniformly distributed with depth and their accumulation patterns and transport mechanisms are less clear. Along shorter fetches oriented off-angle with the main one accumulations of zooplankton occurred at all downwind locations under heavy winds although the patterns are more variable and complex. These downwind accumulations likely create high quality habitat for warm water fish.

Nous avons étudié l’application de plusieurs mesures d’isotopes stables afin de caractériser les processus du cycle de l’azote et les sources d’azote dans les lacs tempérés à diverses échelles spatiales et temporelles. Les résultats d’une étude à travers 65 lacs sur un gradient trophique ont démontré que le ratio d’isotopes stables d’azote (δ15N) des sédiments de surface est un indicateur de l’importance relative des sources d’azote anthropique, mais que ce ratio peut être altéré par la diagenèse. La mesure du δ15N des sédiments demeure néanmoins un outil permettant de déterminer à long terme le changement des charges en azote anthropique aux écosystèmes lacustres et les causes de l’eutrophisation de ces systèmes. Nos résultats d’une étude sur la variation saisonnière de plusieurs isotopes stables dans trois lacs peu profonds situés sur un gradient trophique et ayant différents régimes de stratification ont démontré que cette approche est prometteuse dans les lacs mésotrophes et stratifiés. Dans ces systèmes, le δ15N de la matière organique particulaire (MOP) aurait le potentiel de déterminer les sources d’azote assimilées par le phytoplancton. Cependant les mesures d’isotopes stables du carbone (δ13C) et du ratio C:N indiquent que les apports de matières organiques du bassin versant peuvent altérer les relations observées. Nous avons également constaté une déviation de la relation 1:1 entre les isotopes stables d’azote et d’oxygène (δ18O) du nitrate (NO3-) indiquant son assimilation et sa nitrification simultanée. Cette application est particulièrement prometteuse puisque la nitrification est méconnue dans les lacs et peut exacerber les effets de l’eutrophisation. / We studied the application of multiple stable isotopes measures with the overall objective of improving our understanding of nitrogen cycling and sources in temperate lakes over different spatial and temporal scales. Results from our study across 65 lakes on a trophic gradient demonstrated that surface sediment nitrogen stable isotope ratio (δ15N) is an indicator of the relative importance of anthropogenic N loads, but that diagenesis can alter this ratio. Nevertheless, sediment core δ15N is a powerful proxy for the determination of longterm changing anthropogenic N loads to lake ecosystems and the causes of lake eutrophication. Results from our second study on multiple stable isotopes seasonal variation in three shallow lakes along a trophic gradient and with different stratification regimes have demonstrated that such an approach is particularly promising in mesotrophic and stratified lakes. In these systems, our results showed that the suspended particulate organic matter (SPOM) δ15N could be used to assess the nitrogen sources assimilated by phytoplankton. However, measurement of carbon stable isotopes (δ13C) and C:N ratios from the SPOM showed that the observed relationships can be altered by watershed derived organic matter. We have also found a deviation from the 1:1 relationship between nitrogen and oxygen stable isotopes (δ18O) of nitrate (NO3-) indicating a simultaneous NO3- assimilation and nitrification in our stratified system. The application of dual nitrate isotopes is promising in the study of nitrification since this process is not well understood in lakes and can increase the severity of eutrophication symptoms.