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Multidrug resistance (MDR) has become a major obstacle to chemotherapeutic cancer treatments. MDR is often a result of an up-regulation and overproduction of an ATP-binding cassette (ABC) transporter following chemotherapy. The phenomenon called collateral sensitivity (CS) is associated with MDR cancers and alleviates this condition in vitro. CS agents selectively kill MDR cancer cells over non-MDR cancer cells, often by up-regulating the overproduced ABC transporters. A common ABC transporter that is over-expressed in MDR cancer cells is P-glycoprotein, P-gp. The orphan drug tiopronin has been previously shown to mediate CS in a non-P-gp dependent manner. This means that the CS of tiopronin may be due to another ABC transporter or possibly not from an ABC transporter at all. I attempted to understand the mechanism of tiopronin’s CS capabilities and found that reactive oxygen species (ROS) playa major role in tiopronin-mediated CS. Specifically, superoxide (O2) as little effect on the CS of tiopronin while hydrogen peroxide (H2O2) affects tiopronin-mediated CS. However, H2O2 cannot induce CS by itself, indicating that H2O2 is likely an intermediate necessary for tiopronin’s CS capability but is not the direct cause of CS. Further research should be conducted to investigate the mechanism for tiopronin-mediated CS.

Stereotactic radiosurgery (SRS) is a form of radiation therapy that delivers a focused, highly conformal dose of radiation to a single intracranial volume, while minimizing damage to the adjacent nervous tissue. SRS ensures precise radiosurgical localization by immobilizing the patient’s skull in a specified fixed position and in turn precisely aiming a high dose of radiation at the neurological target. SRS can be delivered to the patient via 3 therapeutic devices: Gamma Knife (GK) radiosurgery, linear accelerator based treatment, and cyclotron-based proton beam. Published reports have not found statistically significant differences in terms of clinical outcomes when analyzing patients treated with either radiosurgical device. The GK is a cobal-60-based machine, with 201 separate 4 to 18 mm collimator openings that emits multiple gamma rays that converge on a target specified by computer planning. This thesis describes the clinical outcomes of patients treated with GK radiosurgery at the Gamma Knife of Spokane and Cancer Care Northwest for metastatic brain tumors, vestibular schwannomas, movement disorders, and trigeminal neuralgia. This thesis will not address specific treatment recommendations for other neurological disorders that may be treated using GK radiosurgery.

West Nile virus, an arbovirus classified in the genus Flaviviridae, presents a serious threat to humans and horses in Montana. The virus, first isolated in the West Nile district of Uganda in 1937, spread rapidly across the United States after its initial introduction to New York in 1999. In 2003 there were 222 human cases of West Nile in Montana. The overarching goal of the Carroll College West Nile virus study is to create a risk assessment map for the state of Montana. This will bring better awareness to the risk of West Nile infection based on geographic location and time. The specific goal of this project was to use microsatellite data to assess the genetic differentiation of the mosquito vector, Culex&tarsalis, between study sites across the state of Montana, and to evaluate if populations separated by greater geographic distance have higher FST values than those located closer together. Multiplex and individual locus PCR methods were used to amplify five microsatellite loci. HE (heterozygosity) values for each population were obtained, and FST values were obtained for betweenWpopulation comparisons. My hypothesis that FST values increase as geographic distance increases was not supported. Harlem and Medicine Lake had a lower FST than Medicine Lake and Bowdoin, which are separated by a smaller geographic distance. Three factors – small sample sizes, high frequencies of null alleles, and deviations from HardyW Weinberg equilibrium – are almost certainly confounding the data, making meaningful conclusions largely unfeasible to draw.

Although a number of genes and chromosomal abnormalities have been associated with the presence of chronic lymphocytic leukemia (CLL), no oncogenes or critical tumor suppressor genes (TSGs) have yet been implicated in its onset. In this study, we describe a chromosomal abnormality existing in a subset of white blood cells from an individual experiencing early stages of CLL: a balanced rearrangement involving the p-arms of chromosomes 1, 3, and 6. This mutation being both unique and balanced suggests that the genes disrupted by the translocation breakpoints may be critical to the initiation of CLL. Microdissection techniques were used to physically isolate chromosomal breakpoint regions, which were then analyzed via array comparative genomic hybridization and results confirmed by fluorescence in-situ hybridization. The translocation breakpoints were narrowed to contain WAS protein family member 2 (WASF2), AT hook DNA binding motif (AHDC1), and Gardner-Rasheed feline sarcoma viral oncogene homolog (FGR) genes on chromosome 1, a TSG cluster on chromosome 3, and the UHRF1 binding protein 1 (UHRF1BP1) gene on chromosome 6. Earlier research found that the FGR gene and 3p21.3 TSG cluster have properties that may promote neoplastic tendencies during abnormal expression. In conclusion, we suggest one or more of these genes is a candidate for initial and/or early development of CLL. Future work will be focused on further narrowing the breakpoints of this rearrangement and studying cellular implications involved in abnormal expression of these candidate genes.

Countries in the developing world continue to feel the effects of rapid population growth despite slowing growth in some regions. Due to limited funding and costs of fieldwork however, programs working to slow population growth have been hesitant to spend money to compare different methods. My study used computer modeling to create control models for Haiti and Niger, two countries with the highest fertility in their respective regions. Control models were compared to experimental models that took into account improvements in either education or family planning programs. The models revealed that in Haiti, helping women with no education achieve primary education would be the most effective method of reducing growth, given that helping women achieve secondary education led to population decline. In Niger, achievement of secondary education for all women would be most effective. These results may suggest that more emphasis should be placed on education as a strategy for slowing population growth in developing countries.

Glycosylation of the prion protein may be experimentally altered through expression in different insect cell lines. The presence or absence of several terminal sugars may or may not have an effect on the conversion efficiency of the prion protein into its infectious form. The main focus of this research was to characterize the prion protein as it is produced in two varying insect cell lines that are able to glycosylate proteins to different extents. Glycosidase treatment demonstrated that the expressed prion protein was glycosylated. Lectin blotting was used to assess the presence or absence of terminal sugar moieties existing on the prion protein. The differentially glycosylated prion proteins produced will allow insight into the effect of terminal glycosylation on prion protein conversion. This research may provide valuable insight into the still unknown prion interconversion process and disease transmission dynamics, and may have implications for human based prion disease.

This study had two major objectives: 1) to determine if IIL-73-74 larvae of the 5. arcticum complex at Wise River were a cytotype new to science and to determine the reproductive status of this taxon, and 2) to determine the reproductive status of .S'. arcticum types at the Clearwater River by subjecting the distributions of the autosomal polymorphism, IS-1, to equilibrium analysis. In both studies I used cytogenetic analysis to: compare the banding sequences of polytene chromosomes of salivary glands of larvae within the 5. arcticum compl ex, confidently identified larvae to sibling and cytotype, and scored autosomal polymorphisms such that reproductive status could be determined. For the study at Wise River, analysis of a possible new cytotype, IIL-73-74, was expanded. Although the lack of autosomal polymorphisms present within the samples prevented the analysis of reproductive status at Wise River, I concluded that the IIL-73-74 inversion characterized larvae that were a cytotype new to science. For the study of reproductive status and continuity of taxa, the Clearwater River in Missoula County, Montana was chosen. Previous analyses at the Clearwater River site indicated: 1) that at least two taxa of the S. arcticum, IIL-3 and IIL-22, were abundantly present, and 2) that heterozygosity of the wide-spread autosomal inversion polymorphism, IS-1, was nearly 50% so that reproductive status between the two taxa could be determined. This ongoing study also tests the Geographic Distribution/Taxon Age Continuum hypothesis (Shields, 2006) which suggests that taxa with broad, geographic distributions may be evolutionarily old and therefore are expected to be reproductively isolated in sympatry than presumably younger taxa which have more restricted distributions. The circumstance at the Clearwater River presents an “in between” situation in that a taxon with a broad geographic distribution, IIL-3, is sympatric with one having a very limited distribution, IIL-22. The data s

Formation and degradation of peroxisomes are not well understood, but their absence leads to serious consequences. In the study described in this thesis, 1 sought to (1) verify previous work indicating that switching from glucose-rich medium to lipid-rich medium induces peroxisome formation; (2) reveal that switching back from lipid-rich to glucose-rich medium induces peroxisome degradation; (3) test the hypothesis that Pex3p is a class II PMP that enters the peroxisome from the endoplasmic reticulum during de novo peroxisome formation; and (4) test the hypothesis that Pxalp is a class I PMP that enters the peroxisomal membrane from the cytosol. I followed the pattern of fluorescence in yeast cells expressing Pex3p-Green Fluorescent Protein (GFP) and Pxalp-GFP fusion proteins after cells were transferred either from a glucose-rich to a lipid- rich medium or vice versa. I succeeded in producing GFP transformants for two peroxisome genes. I also confirmed that switching to lipid-rich medium induced peroxisome formation and demonstrated that switching the peroxisomes back into the glucose medium triggered a rapid degradation. Evidence consistent with the hypotheses that Pex3p moves from the ER to the peroxisomal membrane and that Pxalp is transported from the cytosol to the membrane was obtained, but the overall support was weak.

This research applies the cytogenetic approach to investigate the reproductive status of a population of Simulium arcticum at the Clearwater River, Montana. Previous preliminary analysis at the site indicated: 1) the presence of two abundant taxa of the S. arcticum complex: IIL'3 and IIL'22, 2) a heterozygosity of at least 45% for the autosomal inversion IS'1, and 3) an abundance of larvae having polytene chromosomes of excellent quality. Since nothing was known about the reproductive status of the two prevalent taxa of S. arcticum at the Clearwater River prior to this study, I sought to gain insight into the -22 divergence process by testing the reproductive status of these taxa there. Since the IIL' cytotype has a very limited geographic distribution, I hypothesized that it would not be reproductively isolated from the more broadly distributed sibling, & arcticum sensu stricto (IIL'3). Of the 519 individuals analyzed, 17 different types existed although a majority (514 of the 519) was of the IIL'3 or IIL'22 types. About 80% were of the IIL'3 type and 20% were IIL’22. The distribution of genotypes for the IS'1 inversion indicated that these two types were in genetic equilibrium indicating that IIL' and IIL' are not reproductively isolated at the Clearwater River. This observation supports the Geographic Distribution/Taxon Age Hypothesis (Shields 2007). v

This thesis investigates the multiple drug resistant (MDR) characteristic within enteric Escherichia coli (E. coli) isolates and their ability to transfer this phenotype via resistant-plasmid (R-plasmid) conjugation. E. coli were isolated from bovine fecal samples in the western Montana region. These isolates were subjected to antibiotic resistance testing, then sorted and stored based on general resistance to two broad-spectrum antibiotics, ampicillin and chlorotetracycline. All isolates underwent molecular differentiation using RAPD PCR, then plasmid extraction using a Qiagen Maxi kit. These results were compared to determine if genetically similar isolates, as determined by RAPD PCR, were more likely to contain a plasmid. Isolates containing a plasmid were subjected to conjugation assays to determine transferability of plasmids. In the conjugation assay, each MDR donor isolate was mated with azide-resistant strain, J53AZr. The following hypotheses were addressed: 1) a strain of E. coli will demonstrate a MDR pattern with resistance to more antibiotics if a plasmid is present and 2) the ability to transfer the MDR characteristic to other strains lacking the plasmid is specific to the donor strain of E. coli and its MDR pattern designated by a plasmid. Plasmid presence in E. coli isolates increased with level of MDR. Results also demonstrated plasmid conjugation between E. coli isolates was more likely to occur as level of MDR increased; however the conjugation assay may require larger sample size to confirm the trend. The RAPD PCR method should also be altered to illustrate more accurate banding patterns and verify molecular differences between strains containing plasmids.

Marine sponges are known to produce or sequester compounds useful for defense against predation and to prevent fouling by parasites. Many of the compounds extracted from marine sponges have been shown to possess useful antitumoral properties. The present study investigates two species of freshwater sponges, Eunapius fragilis and Ephydatia muelleri, for possible cytotoxic compounds as determined by brine shrimp microassay. Results show that both species have cytotoxic compounds extractable in methanol and at least two cytotoxic compounds may be present in Eunapiusfragilis, both of which are extractable in hexane. The present study provides strong support for continued natural product research on freshwater sponges.

The genes OS-E and OS-F are expressed in the sensory nerve network of D. melanogaster's antennae and maxillary palps and are putative odorant-binding proteins. A transgenic construct used GAL4 to drive production of GFP in tissues where the gene of interest was being transcribed. Each gene drove production of GFP at a different time in pupal development. OS-E was found to fluoresce in sensory structures at 60 hours after pupation and OS-F fluoresced at 90 hours. Real-time quantitative PCR was used to study flies without gene insertions to determine the molecular nature of the expression of the two genes and consequently whether the developmental model shown by GFP was indicative of a natural process. PCR data were taken from a developmental time period when OS-E/GFP production was active and OS-F/GFP was not; these were compared to samples from adult flies. Concentrations of OS-E mRNA and OS-F mRNA were found to be surprisingly similar, indicating either that the GFP model is inaccurate, or that problems with the PCR reactions caused misrepresentative results.

The nucleoporin Nupl53 has a critical role in both import and export of cellular substances across the nuclear envelope. A unique RNA binding domain has been revealed in Nupl53 (Nupl53-RBD). In order to further understand the interaction between the RNA binding domain (RBD) of Nupl53 and RNA, a crystal structure of the domain would be invaluable. This can be accomplished only if a significant quantity of purified RBD is isolated. Several RBD constructs have been produced to aid in purification of this protein. In most cases, recombinant RBD preparations are fairly heterogeneous and thus unsuitable for structural studies. However, a GFP-RBD construct showed robust expression in E. coli and can be purified without degradation. Although crucial to production and purification, additional domains and tags are not desirable in the final protein sample used for X-ray crystallography. Thus, my goal was to improve the GFP-RBD construct by inserting a protease site that will enable cleavage of the protein once purification is complete, resulting in isolated, pure RBD. Using first QuikChange® and then a PCR based cloning strategy, a TEV protease site was inserted into the GFP- RBD construct. In the future, use of this construct could aide in elucidating how the Nupl53 RNA binding domain recognizes its targets. This could allow us to better understand, and ultimately control, nuclear pore function in the context of normal and disease states.

The numbers of amphibians are decreasing all over the globe, particularly in high elevation areas of the Andes. Glass frogs have been studied in tropical regions, but very little is known about the species in Ecuador. The objective of my study was to determine if habitat factors influence the selection of oviposition (egg laying) sites in Ecuadorian glass frogs in the Cordillera Oriental of the Andes Mountains, at the Yanayacu Biological Station. Two and a half km of one stream were selected as the study area because its bank vegetation and stream characteristics varied throughout its length. The stream was divided into four sections each about 300 meters long, and each night one section was searched for egg masses and adults. Egg masses of Cochranella wileyi were most abundant and were used for statistical analysis. Several habitat factors were examined that were thought to possibly have an effect on oviposition choice. Five factors were found to be positively associated with the location of egg masses: the number of ferns along the margin of the stream, the number of ferns overhanging the stream, canopy cover, and the amount of riffle area in the stream. The amount of grass along the stream margin was negatively associated with oviposition sites. Together, these results suggest that high gradient streams with canopy cover and a dense fem understory are necessary for breeding activity in this species.

Cytotoxic compounds, presumably functioning in defense against predators, parasites, and infectious organisms, are produced or sequestered by a variety of invertebrate animals. Marine sponges, in particular, have proven to be a rich source of these compounds, and many of the compounds or derivatives of them are valuable pharmaceutically. Preliminary work suggested that freshwater sponges may also harbor cytotoxic/ such compounds. In order to examine this possibility, I extracted specimens two species of freshwater sponges, Ephydatia muelleri and Spongilla lacustris, with a range of solvents and tested for cytotoxic activity using a brine shrimp survival assay. I confirmed that freshwater sponges contain cytotoxic activity and also began a characterization of the molecule (or molecules) responsible for this activity using partitioning, silica gel chromatography, and ultrafiltration. My results suggest that the active molecules are relatively polar and have molecular masses of 3000 - 30,000 Da.

The Office du Niger (ON) in Mali is one of the largest irrigation schemes in West Africa. In recent years a trend toward increased alkalinization and salinity due to poor irrigation, drainage, and fertilization practices has plagued the ON. To evaluate soil degradation in the ON, enzyme activities were measured among four agronomic treatments of a field trial established to measure the impact of fertility and irrigation practices on rice production. The properties used to evaluate soil quality include: pH, electrical conductivity, fluorescein diacetate activity and dehydrogenase activity. While no significant difference was observed among the four agronomic treatments, a significant difference in soil quality was found between the treated soils and the non- impacted soils. On average, the treated soils had a pH of 8.5 and an electrical conductivity of 427 mS/m. Increasing pH and electrical conductivity were both negatively correlated with microbial enzyme activities.

The goal of the present study was to both inhibit the a-CaM-Kinase II (cc-CaM-K II) enzyme, and to over-express it in neurons in order to observe if the enzyme has a role to play in memory as measured by behavioral and learning tests. An adeno-associated virus (AAV) containing either sense or anti-sense RNA to the a-CaM-K II protein was designed for the over-expression or suppression of the enzyme, respectively. Morriswater and open field mazes were used to test for the effects of the differences in protein concentration on memory and behavior. An increase in protein in the rats receiving sense RNA, as apparent in Western Blots, correlated with significantly greater time in the target quadrant when compared to the controls, as expected if a-CaM-K II protein was involved in the establishment of memory. Concentrations of a-CaM-K II protein in the hippocampi were determined 29 days after introduction of the virus using Western Blots. The concentrations of a-CaM-K II protein in the rats receiving sense RNA ( n = 6 ) were statistically larger (p

This study was designed to investigate a possible biogeographical origin and mechanism of dispersal ofthe Hawaiian fern Diplopterygium pinnatum. Molecular phylogenetic analyses were conducted on trnL-F and rbcL, cpDNA regions, for seven species of the genus Diplopterygium and three outgroups. Maximum parsimony and maximum likelihood analyses were used for phylogenetic reconstruction. Three hypotheses were tested: that the ancestor ofD. pinnatum originated from 1) an IndoPacific source and traveled to Hawaii via the jet stream, 2) an American source and traveled to Hawaii via the trade winds, or 3) an Austral source and traveled to Hawaii by a combination of an intertropical convergence zone (ITCZ) shift, Hadley cell air movement, and the trade winds. The two methods ofreconstruction, maximum parsimony and maximum likelihood, produced similar trees with similar support. The second hypothesis was rejected and the first and third hypotheses cannot be distinguished.

Selective estrogen receptor modulators (SERMs) either mimic or block the effects of estrogen (Goldstein, 1998). Tamoxifen, a SERM, blocks the effects of estrogen in breast tissue (Walling, 2005), but its effects in other tissues, specifically in the brain, are poorly understood. Tamoxifen may reproduce the effect of estradiol in the brain (Le Saux et al., 2005) or it may inhibit memory (Jenkins et al., 2004). This study used ovariectomized rats injected with pharmacological doses of estrogen or tamoxifen. Cognitive learning and retention were evaluated based on performance in the Morris water maze. The hypothesis tested was that tamoxifen would simulate the effects of estrogen in the brain, resulting in cognitive learning and retention similar to estrogen-injected rats but different from the non-stressed control group. While no statistically significant results were noted in the retention protocol, a statistically significant difference between the tamoxifen and the control groups on days four and five in the learning protocol was observed. However, when comparing average learning times, the results suggested that tamoxifen does not mimic the effects of estrogen in the brain.

The identification of sporadic point mutations in tumor-suppressor genes, which are responsible for regulating cell growth, has been linked to the early detection of cancer, as well as with identifying those individuals with a genetic predisposition to developing cancer. My research investigates the use of a single-step endonuclease V/ligase scanning assay in the detection of unknown point mutations in genomic DNA. Specifically, the research focuses on the K-Ras gene, which has been linked to several different types of cancer, including colorectal cancer. A universal PCR strategy using fluorescently labeled universal primers and unlabeled gene specific primers allowed for amplification ofK-Ras oncogene sequences. Amplification was followed by heteroduplex generation from the universal PCR products. This procedure resulted in products with one of two mismatches (A/C or G/T) that could be used for endonuclease treatment. Endonuclease V recognizes and cleaves base pair mismatches within the gene sequence, but it also cleaves some correctly paired bases. A highly specific ligase was used to reseal these miscleaves, thus increasing signal intensity. Capillary gel electrophoresis was employed to distinguish the single-stranded products of the EndoV/ligase assay based on fragment-size differences. The early detection and identification of mutations could lead to increased survival rates for individuals testing positive for those mutations. Further research should include the potential to transfer the assay to a microelectrophoretic device with possible clinical applications.

The secondary metabolites of marine sponges include many bioactive products (e.g., ara-C) that have found uses in the health field. Even though freshwater sponges share many characteristics with their marine cousins, little is known about their secondary metabolites. This study looks at the effects of the secondary metabolites extracted from three freshwater sponge species on the survival of bacteria, fungi and brine shrimp. It found that none of the extracts were toxic to the bacteria or fungi. Preliminary results did, however, show that all three species tested were active against the brine shrimp. This study suggests that the secondary metabolites of freshwater sponges should be tested more extensively to see if they contain useful bioactive compounds.

Chlorella sp. is a green alga that exists in endosymbiotic relationships with a number of freshwater invertebrates including the sponge, Ephydatia muelleri. Algae enter sponge cells through phagocytosis, but unlike most cells taken up by this process, the algal cells are not degraded. The mechanism by which Chlorella escapes degradation is currently unknown. One hypothesis is that the vacuoles containing algal cells do not progress along the degradative pathway but instead are maintained in an immature, pre-lysosomal state, displaying the early endosomal marker Rab5. In order to test this hypothesis, I localized Rab5 in sponge cells using immunocytochemistry. Two antibodies against human Rab5 were first shown by Western blotting to bind to a sponge protein having a molecular weight similar to that of mammalian Rab5. Both antibodies also recognized some higher molecular weight proteins. From these results, I concluded that there was sufficient cross reactivity to use the antibodies for immunocytochemistry. I was unable to localize Rab5 in cells using one of the antibodies; however, with the other, I detected antibody bound to algae-containing vacuoles. This latter result suggests that vacuoles containing symbiotic algae may express Rab5 and that maturation of the vacuoles may be hindered.

Microorganisms and their activities drive most ecosystems and therefore must be assessed in order to study ecosystem stability. Heavy metal contamination is known to have adverse effects on soil bacteria. In this study, bacterial populations from polluted (PV and PB) and non-polluted (NV) soils were compared in order to better understand soil resiliency, ecosystem stability, and the long-term effects of heavy metals have on bacteria. Biological parameters of soil quality including diversity, structure, activity, and heavy metal tolerance were compared and assessed. The numbers of Zn and Cu tolerant bacteria were determined by spread plating diluted soil samples onto agar plates containing 0.5,2, and 10 mM concentrations of the metals. Arginine and iodonitrotetrazolium assays were used to measure the extent of ammonification and dehydrogenase activity in the soil. And, 16S rDNA cloning and sequencing was used to identify 60 individual microorganisms from both the NV and PV soils. Neighbor-joining phylogenetic trees, constructed with the 16S rDNA clones, were then used to analyze differences in bacterial diversity and community structure. I hypothesized that the population in the NV soil would: 1) have higher activity levels, 2) be more diverse, 3) contain fewer metal tolerant microorganisms, and 4) show a different community structure than the bacterial populations isolated from the polluted soils. All hypotheses were found to be correct except for the hypothesis addressing the diversity of the two soils (NV and PV). It was found that both soils were very diverse in bacterial species and that 60 clones from each soil was not a big enough sample size to address the differences in diversity between the two bacterial populations isolated from the two soils.

The green alga, Chlorella, exists in a symbiotic relationship with the freshwater sponge, Ephydatia muelleri. Chlorella is taken up by phagocytosis and incorporated into phagosomes. However, the phagosomes containing the algae fail to fuse with lysosomes, resulting in a sustained symbiotic relationship. One possible explanation for the retention of algae in non-lysosomal vacuoles is that the phagosomes do not acquire the surface proteins needed for fusion with lysosomes. Among these proteins is Rab7, which has been suggested to regulate the transition from phagosome to lysosome. I hypothesized that the vacuole containing the symbiotic algae is early endosome-like and thus Rab7 is not present on the algal-containing vacuole membrane. Immunocytochemistry with antibody directed against human Rab7 was used in an attempt to localize Rab7 in the sponge cells. Western blot analysis was used to establish the specificity of the antibody. The results of both the Western blots and immunocytochemistry were inconsistent. Banding in the Western blots was faint, and the pattern of staining varied with the concentration of the primary antibody. Similarly, localization of Rab7 by immunocytochemistry was inconclusive. The inconsistency in both the Western blots and immunocytochemistry indicates that more research is needed to clarify the role Rab7 plays in establishing the symbiotic relationship between algae and sponge.

This research investigates the benefits of utilizing PCR/Ligase Detection Reaction multiplexing coupled with endonuclease V/AK16D ligase treatment in the detection of known and unknown point mutations within DNA. The research focused specifically on the K-ras gene, implicated in the development of certain cancers including colorectal cancer. Identification of point mutations within this gene could lead to early detection of cancer, as well as identify those with a predisposition to develop cancer. An LDR technique was utilized to identify specific, known mutations within the gene sequence, while the endonuclease/ligase treatment identified unknown mutations within the gene sequence. Capillary electrophoresis and slab gel electrophoresis were employed to distinguish the products of both EndoV/ligase and LDR, respectively, based on the fragments’ size differences. These methods have been found to be highly sensitive and have high throughput. Further research should explore the potential to transfer the gel electrophoresis separation to a microelectrophoretic device with the potential for clinical applications.

Controlled experiments in laboratory settings examining the effect of zinc pollution on bacterial communities in soil often neglect metal exposure over a period of time. Increasing levels of soil-zinc have been shown to increase the Zn tolerance of bacterial communities in soil and decrease diversity and change community structure of bacterial populations. Since soils are a critical component of terrestrial systems, the soil must be able to recover from a pollution event in order to maintain the quality of natural and agricultural systems. This study analyzed bacterial tolerance and diversity in soils containing 0,12.5,25, and 50 mM Zn after a year of incubation in a laboratory setting. Approximately 50 16S rDNA clones from each soil (0,25, and 50 mM Zn) were sequenced and analyzed by neighbor joining. There was an increase in tolerance with increasing Zn concentration in soil, and there was also an increase in tolerance from a year previous. Both the arginine and dehydrogenase assays showed a decrease in activity with increasing Zn concentration. The diversity of the communities changed little with 40 unique species in the control and 37 and 36 unique species in the 25 and 50 mM Zn amended soils, respectively. The structure of the communities also exhibited a small change as both the number of species in each taxon changed, and the taxa that were present changed with Zn concentration.

To date, the pathology of Alzheimer’s remains unknown, and many researchers have focused on the mitochondria as playing a significant role in its progression. In earlier studies, it was found that mRNA levels of mitochondrial proteins of Alzheimer’s Disease patients were altered in mouse and human models. Additional research is warranted to determine if the mRNA differences are translated to the protein level. It has been shown that mitochondrial genes are differentially expressed in Alzheimer’s patients when compared to age-matched controls. I propose the levels of the translated gene product will also be altered. To test this hypothesis a proteomic approach utilizing 2-D electrophoresis to separate mitochondrial proteins and compare expression between diseased and control samples was used. These results are preliminary and further trials will cement the results.

The retinoblastoma tumor suppressor protein, RB, is a negative regulator of the cell cycle that functions to prevent cells from entering S-phase when conditions are not appropriate for proliferation. It is thought that RB works to inhibit cellular proliferation in late Gi phase of the cell cycle. Several recent discoveries indicate that RB may actually inhibit proliferation in S-phase rather than Gi. One of the biggest of these discoveries is that Cyclin E, an initiator of DNA replication, is produced in the presence of active RB. Cyclin E is responsible for the binding of cyclin dependent kinases at the end of Gi and moving the cell into S-phase. At this point cyclin A, which is inhibited by active RB, takes over and continues the initiation of DNA replication. Because cyclin E is still produced in the presence of active RB, the cell cycle may not be inhibited until the initial stages of S-phase, where the lack of cyclin A prevents DNA replication. A second function of RB is to prevent cellular proliferation not only by cyclin A inhibition, but also through the depletion of dNTP pools. When nucleotide levels decrease below a certain threshold, proliferation can no longer proceed. In conjunction with this idea, it was recently discovered that the number of utilized origins changes with increases or decreases in nucleotide levels, but the cell doubling time and length of Sphase remain the same. Here I began preliminary testing on these two functions of active RB. First, RB actually inhibits cellular proliferation very early in the S-phase of the cell cycle, after primer formation but before processive elongation of replication forks. Second, alterations made in nucleotide pools in the DHFR locus will decrease or increase origin utilization.

In D. melanogaster, CycE and E2F1 are critical proteins in endocycle control. It is known that both of these proteins are required for endocycling and that cycE mRNA and protein oscillate in endoreduplicating tissues (ERT’s). The mechanism of endocycle regulation by E2F1 through CycE, however, has not been elucidated. Here, I implicate E2F1 protein oscillation in the fluctuation of two of its specific target genes, cycE and rnr2, which is then responsible for the G-S transition ofDrosophila ERT’s. The results also suggest that E2F1 control of S phase initiation within the endocycling cell is dependent on nutritional inputs.

Preliminary studies by Rhodes (2001) indicate that the IIL-3 and IIL-7 sibling species of the black fly, S. arcticum, occur at Little Prickly Pear Creek (LPPC), Lewis and Clark County, Montana. The occurrence and chromosomal diversity of S. arcticum in the various drainages east and west of the crest of the Rocky Mountains in Montana are currently being studied (Shields, unpublished data). As a part of this larger study, TenMile Creek (TMC), a tributary of LPPC, was chosen for this study. Correspondingly, I hypothesized that both the IIL-3 and IIL-7 siblings of S. arcticum would be present at TMC. The development and emergence of black flies was monitored from mid-May to the end of July of 2002. Modem cytogenetic methods were used to describe the banding sequences of the larval polytene chromosomes of the S. arcticum collected. Thus far, only the IIL-3 sibling of S. arcticum has been described at TMC. Further study both in early spring (March and April) and in late summer (August and September) is needed to completely describe the status of sibling species of S. arcticum at TMC.

Gadolinium, a Calcium-sensing Receptor (CaR) agonist, was applied in different concentrations to rat Superior Cervical Ganglia (SCG) to discover the dissociation constant and the maximum amount of CaR-mediated current that could be blocked by this agonist. It was hypothesized that these concentrations would be similar to those of previously determined concentrations for other neural tissues, suggesting a similar CaR complex shared by the tissues. The SCG were dissected from male rats, cultured, and neural activity was recorded through a modified patch-clamping technique. It was shown that gadolinium’s dissociation constant was in the hundreds of nanomolars (nM), and that the agonist could block all CaR-mediated current. Previous research has shown gadolinium blocking other important calcium channels, however, and ultimately a method of blocking only the current generated by the CaR is desired to gain control of excess neurological activity as seen in stroke, cerebral ischemia, and seizures.

Variation in the susceptibility to the lethal effects of Alzheimer's Amyloid Precursor Protein (APP) transgene exists among various mouse strains. Inbred FVB/N mice, expressing high levels of the transgene-encoded APP, die prior to 200 days, while inbred 129.Tg2576 mice carrying the transgene are far less susceptible. When the two strains are crossed, (FVB/Nxl29.Tg2576) FI mice survive, as does the 129.Tg2576 parent. Intercross and backcross offspring survived at rates of 60% and 35%, respectively, at 200 days signaling the presence of a polygenic trait. The goal of this study was to establish a linkage to genes affecting susceptibility to the APP transgene. The possible quantitative trait loci (QTL) were established using various genetic markers scattered throughout the genome. The presence of multiple QTLs is possible from the data obtained; however, an increased chance of type I errors (false positives) exists due to the large number of markers used for the genome scan.

This study compared 369 base pairs of the hypervariable control region of mitochondrial DNA in 137 brown bears (71 males, 66 females) of Glacier National Park to investigate dispersal patterns between the sexes. Since females establish home ranges close to their birth sites, we hypothesized a stronger phylogeographic pattern among females. Contrary to our expectations, males exhibited stronger phylogeographic patterning with one haplotype in males occurring exclusively in northern regions of the park, while a second occurred exclusively in the south. No phylogeographic patterning was observed among females. Male dispersal patterns could be influenced by gene flow from regions contiguous to the park. All brown bear haplotypes in Glacier National Park belong to clade IV of earlier studies and suggest a recent establishment of brown bears in this region.

A field experiment was performed to examine the effects of predation on the morphology of the spotted frog (Rana luteiventris). Seven ponds were randomly selected for survey in the Helena National Forest. Macroinvertebrate predators were counted and tadpoles were collected to measure morphological characteristics. Statistical analysis demonstrated that predation had a significant overall effect on tail morphology. Specifically, the higher number of predators in a pond, the longer the tail, the thicker the tail muscle, and the more narrow the mouth width of tadpoles. These induced changes presumably increase swimming speed, improving predator evasion. Interactions with other members of the aquatic community are likely to be influenced as well. This study provides further evidence for predator-induced plasticity in amphibian larvae.

The freshwater sponge, Ephydcitia muelleri, harbors symbiotic algae within its cells. These algae are taken up by phagoctyosis, but they fail to be broken down. One explanation for the lack of degradation is that their digestion is prevented at an unknown point along the phagocytic pathway. In an attempt to determine if the vacuoles containing algal endosymbionts had characteristics associated with early endosomes, I used immunocytochemistry to localize Rab 5 in sponge cells. Rab 5 is a protein that binds to membranes and functions in docking and fusion events early in the endocytic and phagocytic pathways. Immunoblots using antibody against human Rab 5 identified three proteins, with molecular weights of 26,000, 31,000, and 46,000, in homogenates of sponge tissue. The first has a molecular weight close to mammalian Rab 5. Immunocytochemical localization revealed deposits of reaction product on the membranes of the perialgal vacuoles and in the cytosol. These results indicate that Rab 5 could be bound to the surface of the vacuoles and that vacuoles housing endosymbiotic algae could be blocked at an early stage in the phagocytic pathway.

The butterflies of Lewis and Clark County were studied and then compared to Missoula County and the Province of Alberta. Missoula County is on the west side of the Continental Divide, while Alberta and Lewis and Clark County are east of the Divide. Believing that the Divide may limit east-west migration, I hypothesized that the fauna of Lewis and Clark County would more closely resemble the fauna of Alberta than that of Missoula County. For Lewis and Clark County, butterflies were captured, killed, mounted, and identified from eight sites. Collection took place from May 25 to July 27, 2001. My collection data of 69 species was combined with previous collection data from Dr. Steve Kohler (personal communication) and Dr. John Christenson (personal communication) for analyzing results. The combined data was used to create distribution maps and flight period tables for Lewis and Clark County. Lists were compiled for each ofthe three regions and then compared. While distribution maps reveal where certain species can be found, flight period tables represent the known times of emergence for different butterfly species. The comparison of compiled lists from Lewis and Clark County and Missoula County and Alberta showed Lewis and Clark County to more closely resemble Missoula County. These analyses allow for a more thorough understanding of the species found within Lewis and Clark County and for the comparison of Lewis and Clark County’s species to the other two regions.

There have been several studies done on the effects of restraint stress and other physical and psychological stressors on the immune system. In addition, female sex hormones (estrogen and progesterone) are known immunomodulators as well as affectors of the neuroendocrine system. There is little research, however, tying the three systems together. In this study, I used a simple leukocyte differential count to quantitatively analyze the immune response to restraint stress and to see in what way estrogen and progesterone alter that response in the prepubescent laboratory mouse (Mus musculus). My data show that estrogen and progesterone drastically alter the cellular immune response to restraint stress.

Amphibian populations are declining across the world. Implicated in this decline have been environmental pollutants and genotoxic agents such as pesticides, fertilizers, UV radiation, sewage contaminants, emissions of internal combustion engines, landfill run-offs, and industrial effluents. In Montana, abandoned mines have been shown to be a source of mine tailings in ponds and streams, resulting in low survival rates of certain frogs and toads. I examined survivorship, growth, and DNA fragmentation in Bufo boreas tadpoles after exposure to mining sediment at different pH levels. I used an alkaline single-cell gel (SCG) “comet” assay to quantify DNA fragmentation. This technique detects DNA fragments which, when electrophoresed, migrate from the nucleus of erythrocytes forming a “comet-with-tail.” When exposed to mining sediment, B. boreas showed significantly lower rates of survival and lower rates of growth compared to those exposed to control sediments (sand). In addition, I found a statistically significant increase in “comet” length-to-width ratios from 1.06 for those maintained in sand to 1.34 for those maintained in mining sediment. Moreover, in mining sediment treatments, the average length-to-width ratio was 1.29 for those at pH 5.0 compared to 1.39 for those at pH 7.0, which was also statistically significant. There was no difference in length-to-width ratio for different pHs in sand treatments. The results of the SCG assay indicate that this technique is sensitive to DNA damage resulting from mining sediment and that it may be used as an indicator of the genotoxicity of such environmental pollutants. The results also suggest that DNA damage could be a reason for declining amphibian populations in Montana ponds and streams that receive mine tailings.

The efficiency of therapeutic drugs is greatly reduced when they are administered systemically, while their potential for harm is distributed across the entire body. Synergistic relationships between high intensity focused ultrasound (HIFU) and agents of membrane disruption such as amphipathic polymers could selectively disrupt localized cell membranes and release bound chemical agents in the near vicinity to minimize side effects and increase the specificity of such agents. Human red blood cells were incubated with polypropylacrylic acid (PPAA) concentrations from 0.8 |J.g/ml to 1.6 p.g/ml, and then exposed to pulses o f high intensity focused ultrasound (HIFU) at intensities ranging from 572.68 W/cm2 to 1979.31 W/cm2. Release o f hemoglobin, a measure o f membrane disruption, was measured with UV spectrophotometry, and cavitation was quantified using LabView and MATLAB. Sham samples were run with no HIFU or no PPAA. Membrane disruption increased dramatically when both components were present. Polymer concentration did not appear to affect the extent of cavitation, while intensity of HIFU clearly was proportional to cell disruption. A synergistic effect was observed between these two agents, lending promise to the development of drug conjugation therapies.

The effect of sedimentation on fish populations, especially the effect on reproduction, has been one of the most significant problems surrounding sedimentation pollution. Sedimentation has deleterious effects on the hatching and survival of trout eggs by reducing the egg’s access to oxygen and nutrients. To test for the effects of sedimentation, five streams in the Townsend, Montana area were examined. The effects of total suspended solids (TSS) as well as the individual effects of different particle sizes (clay, silt, sand) on trout reproductive success were measured. The number of rainbow trout hatchlings was not significantly associated with sand (p=0.794), silt (p=0.815), or clay (p=0.140). However, there was a significant negative correlation between TSS and trout numbers (p=0.043). Collectively, all the particles contributed to the reduction in trout hatchling numbers. Despite testing of individual particles, the individual effects of different particle sizes could not be observed, possibly because they could not be sufficiently isolated from the other particles within the sediment. The TSS results show a strong correlation between the amount of fine sediment and reproductive success of rainbow trout, supporting previous studies. Sedimentation does indeed lower the survival rate of salmonid eggs.

The neutrophil is an integral part of the body’s line of defense against infectious microbial agents. Its microbicidal activity is carried out by NADPH oxidase, an enzyme that uses electron transfer to create superoxide (O2"), which is then transformed into various toxins that are responsible for the demise of the foreign microorganism. The oxidase system is composed of membrane and cytosolic components. One of the cytosolic components is a low molecular-weight member of the Rho family of GTPases, Rac2. A genetic link has recently been established between Rac2 and Chronic Granulomatomous Disease (CGD), a disease in which the body’s immune system is compromised as a result of a defect in the NADPH oxidase system. To further elucidate the role ofRac2, cytosolic subcellular fractions were immunoprecipitated and reconstituted in two different cell-free systems, SDS and McPhail, to determine the specific amount of Rac2 present, as well as how much of this protein can be depleted before immunity is compromised. Results indicated that immunoprecipitation (IP) of Rac2 was incomplete with concurrent IP of two other essential proteins, p47phox and p67phox, noted as well. The best results were obtained using 10% bovine serum albumin to block the sepharose beads, which were incubated with the cytosol separately from the antibody. It is possible that some Rac2 escaped IP or was present in the membrane prior to activation. IP of both Racl and Rac2 was completed to test a hypothesis that Racl was capable of substituting for Rac2 when its levels are diminished in the cell. Addition of purified Rac2 to IP cytosol demonstrated an optimal level of 1 pg, after which the increase in superoxide production began to level off. Finally, the GTP-dependency ofthis protein was studied using GTP-depleted cytosol in the McPhail cell-free assay. Control cytosol reached peak performance levels with the addition of GTP alone; GTP-depleted cytosol was shown to be dependent on the addition ofboth ATP

With greater emphasis being placed on molecular approaches to systematics, it is becoming increasingly important to be able to extract DNA from alcohol-preserved specimens, such as those maintained in museum collections. As a preliminary step to DNA sequence analyses o f alcohol-preserved freshwater sponge specimens, this study compared the purity and yield of genomic DNA isolated from alcohol-preserved specimens by three different DNA extraction methods. One method (standard method) made use of a proteinase K digestion in the presence of detergent and EDTA, followed by extraction with phenol-chloroform. A second method (CTAB method) involved proteinase K digestion in the presence of cetyltrimethylammonium bromide (CTAB), followed by extraction in chloroform. A third method employed a cell lysis solution containing guanidium thiocyanate, followed by extraction in phenol-chloroform. Analysis of isolates indicated (1) no difference in purity or yield between DNA extracted from alcohol-preserved and frozen samples and (2) no difference in purity among methods, although yield was highest using the CTAB method. Preliminary evidence also suggested that all DNA preparations contained appreciable amounts o f RNA.

In order to relate the three known populations of the freshwater sponge, Clypeatula cooperensis, to one another, sponge DNA was subjected to RAPDPCR. DNA was extracted using a Puregene kit from three specimens collected from each population. Following PCR optimization, DNA was amplified using random primers (OPA-10, 11, 12, 13, 18, and 20). Although reproducibility was a problem, preliminary results suggest that variability within populations is as high as variability among populations, raising the possibility that the three populations are genetically identical. This is an unexpected finding because one population is geographically isolated from the other two. If correct, one explanation is that the three populations have only recently become separated.

Over-expression of the ErbB receptor tyrosine kinase subfamily has been implicated in various types of human cancers. In the current study, ErbB-2, ErbB-3, and ErbB-4 tyrosine kinase domain cDNAs were isolated by RT-PCR and subcloned into a pFastBac-HT vector created through genetic engineering for the purpose of placing a his- tag on the ErbB protein receptors for purification. In isolating the ErbB-4 cDNA, an alternate receptor containing a 48 base-pair deletion in the tyrosine kinase region of the receptor was recovered. This result confirms an earlier report of an alternate splicing pattern in the ErbB-4 mRNA. Isolation of the ErbB TKD cDNAs allows for further research for the development of a diagnostic test of cancer tissues for over-expression of the ErbB receptors similar to the Herceptest, which tests for over-expression of ErbB-2.

Recent studies of female athletes have shown that females are more prone to knee injuries than their male counterparts. This phenomenon has been attributed to several factors including anatomical structure differences such as quadriceps-hamstring imbalances, type of activity (specifically cutting and planting motions), use of braces, and hormonal differences. It is known that hormones like relaxin have an obvious effect on ligament composition, but the effects of other hormones on ligaments has yet to be fully determined. One particular study reported that a female is most susceptible to knee injuries during the pre-ovulatory phase of her cycle, when estrogen levels are peaking. This project was an attempt to show the effects of estradiol on ligament strength and stretch. The role of progesterone as a counter-effector of estrogen receptors was also considered. The original hypothesis was that increased levels of estradiol would decrease the weight limit the medial collateral ligament could withstand before breaking. A second hypothesis was that the breaking point of the ligament would not be affected, but the elevated levels of estradiol would increase the amount of stretch in the ligament prior to breaking. Peak human estradiol levels were simulated in prepubescent rats and mechanical tests on the ligament were performed to measure breaking point and stretch. Combined levels of estradiol and progesterone were also given and the mechanical tests were repeated. It was concluded that increased levels of estradiol did not affect the breaking point but did increase the amount of stretch in the ligament prior to breakage. Progesterone did inhibit the effects of estradiol.

The biodiversity on Earth is in a state of decline. Because the full extent of the problem is not known, model systems are needed as environmental indicators. Amphibians are considered good indicators of wetland ecosystems. Many authors have agreed that a key factor driving population dynamics in a fragmented landscape is the ability of animals to move between habitat fragments. I studied the effects that a stream corridor has on the patch occupancy of breeding populations of western spotted frog (Rana luteiventris). I quantified landscape and habitat parameters in both Lump Gulch and Gravely Range landscapes. The Lump Gulch landscape consisted of wetlands connected by stream corridors, whereas the Gravely Range landscape consisted of glaciated kettle ponds, with no stream connectance. The area of wetland sites and the distance between sites did not have an influence on the patch occupancy of the different landscapes. By eliminating these factors my results suggest that stream corridors significantly influence the distribution of Rana luteiventris by providing a means to occupy and reoccupy wetland sites. In essence, the stream corridors between patches are precious resources and it is imperative that we preserve these habitats which are a means of dispersal across landscapes.

Unlike other freshwater sponges in the northern Rocky Mountains, Clypeatula cooperensis does not die back during the winter months but instead maintains its structure through the winter, although in an altered form. In addition, since this sponge does not produce gemmules, it apparently relies exclusively on sexual reproduction for propagation. In the present study, the morphology of the sponge was followed by light microscopy over the course of one year. Samples were collected at two-month intervals from August to May and at two-week intervals during June and July (when sexual reproduction occurs). During the winter, the sponge underwent a modified form of regression in which the choanocyte chambers disappeared, the mesohyl increased in density, and the volume of the canal system increased. Regression reached its peak in February. Afterwards the changes that gave rise to the regressed state were reversed: choanocyte chambers reformed and attached to the canal system; the mesohyl became less dense; and the volume of the canal system decreased. These changes were followed in June and July by gamete production, embryogenesis, and larval development. A previously undescribed type of cell, characterized by large eosinophilic granules, became concentrated in the dermal membrane during October. The role of these eosinophilic cells remains to be determined.

Saline seeps are a known environmental hazard in eastern Montana. Identifying the salinity threshold value for fish using a 96-hour acute toxicity test will allow fish salinity tolerance to be taken into account when biotic integrity is used to determine the health of a waterbody. Acute toxicity tests were run 10 fish per 8 gallons of test solution in glass aquaria. The test solution was made using Na2SC>4 dissolved in carbon filtered city water and was renewed after 48 hours. Mortalities were counted and removed every 6 hours for the first 24 hours and every 12 hours there after. Results obtained from the 96-hour acute toxicity test determined the LC50 range to be 10-20 g/L using the binomial method. The geometric mean being 14.1 g/L. Results from a follow up experiment showing 100% mortality at 13 g/L lead to the conclusion that the actual LC50 lies below 14.1 g/L. The calculated LC50 value of 14.1 g/L leads to the conclusion that northern redbelly dace are a salt tolerant species well adapted to the saline waters of eastern Montana. Affected streams in eastern Montana that have salinity levels regularly approaching 9 g/L are approaching toxic levels.

The herpes simplex virus (HSV-1) encodes a trans-activator protein VP16. VP16 activates the immediate early genes (IE) of the viral genome during lytic infection. VP16 has been heavily studied by mutational analysis as a model for transcriptional activation. The goal of this project was to examine two specific aspects of VP16. First, to compare the properties of a previously constructed insertion mutant, in1814, to other mutant strains constructed in our laboratory; this insertion was moved from strain 17 of HSV-1 into the KOS strain, the strain used in our lab. The second aspect of this examination is a mutational analysis of a possible cryptic N-terminal activation domain. To assess whether this region contributes to transcriptional activation of the IE genes, deletion mutants were constructed in the N terminus of VP16, with or without the deletion of the Cterminal activation domain. The constructed viruses will be verified for the proper mutations, before the properties of these viruses are characterized.