Dissertations / Theses on the topic '069999 Biological Sciences not elsewhere classified'

The use of lipid profiles from the immobilised alga Selenastrum capricornuizim was investigated as a potential indicator of heavy metal pollution in freshwater environments. The toxicity of Cu", Zn 2 and Cd2t on algal growth was determined and the effective concentration inhibiting specific growth rate by 50 % (EC5 0) for each metal was found to be 124 pM Cu, 20 pM Zn and 5.7 pM Cd respectively. The Cu 24 EC50 value for immobilised cells was also shown to be 124 j.tM, suggesting that Cu exhibits similar toxic effects on growth in both free and immobilised cells. Studies of the effects of temperature and heavy metal exposure (Cu21, Zn 2 and Cd2 +) on S. caprzcornutum demonstrated that these factors altered the fatty acid and free sterol composition of free algal cells in batch culture. A shift in temperature from 25°C to 10°C led to an increase in the relative proportion of oleate and decrease in linoleate and parinate (18:4), together with a significant increase in the composition of ergostenol. Exposure to heavy metal ions led to an increase in oleate (with all three metals) and altered relative proportions of linoleate and parinate (changes being metal specific). Metal ion treatment also increased a22 desaturation of chondrillasterol. This characteristic lipid signature when S. capricornutum was exposed to heavy metal ions was significantly different from changes associated with other environmental factors. These changes in lipid composition upon heavy metal treatment were also observed during exposure of S. capricornuiwn to lower metal concentrations typically found in polluted environments. Studies of cells immobilised within alginate beads showed that gel confinement significantly affected the biochemistry and physiology of algal cells, with a reduction in growth rate and final cells numbers. Scanning electron microscopy demonstrated that growth was mainly limited to the bead periphery. Immobilisation altered the lipid of composition of cells as a consequence of alterations in membrane fluidity and membrane disruption. The Cu uptake from solution was greater in immobilised cells than free cells, thus gel confinement did not confer any protection to cells. The characteristic and significant changes within the lipid composition of free cells with Cu treatment were similarly observed in immobilised cells but were only apparent at 124 pM Cu. Thus lipid profiles, especially sterol composition of immobilised algal cells, may potentially be utilised as sensitive and novel indicators of heavy metal pollution in freshwater environments.

Despite improved knowledge and advanced treatments of high-grade gliomas, the overall survival rate of glioma patients remains low due to the recurrences and locations of the tumour. Evidence shows that the existence of a subpopulation of cells - cancer stem cells (CSCs) may be the major obstacle in treating gliomas. CD133 and nestin have been suggested as the markers of CSCs and natural stem cells. The primary focus of this study was to identify CD133+/nestin+ stem-like cells and discover their association with multidrug resistance (MDR) related genes, i.e. multiple drug resistance I (mdrl) gene and anti-apoptotic gene (bcl-2) in human glioma compared to normal brain tissues and cell lines. Glioma and normal astrocyte cell lines have been employed for CD133 isolating purposes to characterise the association with MDR related genotype and phenotype. The chemosensitivity of the isolated CD 133 population was investigated using chemosensitivity assay. Meanwhile, a serum deprivation method was established in this study to enrich and select CD 133+ CSCs in a glioma (GOS-3) cell line. As a secondary focus of this project, the possibility of immortalisation enzyme hTERT being a discriminative masker between normal and cancer brain stem cells and the transcriptional correlation between cd133 and bmi-lIc-myc oncogenes were investigated. For the first time, findings of the current study demonstrated that 1) there was an evident increase of CD133 gene expression in glioma compared to normal brain tissues where the latter expressed low levels of CD133, P-gp and Bcl-2 than glioma tissues, with an exception of nestin expression, 2) serum deprivation enriched CD133 expression and demonstrated a direct coexpression between CD133 and drug resistance in GOS-3 cells, 3) hTERT may not be a discriminative marker for normal and cancer brain stem cells, 4) although there was a strong transcriptional association between bmil and cmyc, there was an inverse transcriptional association between these genes and cd133 in serum deprived glioma cells, suggesting that bmil may not be essential for the maintenance of glioma stem cells, and 5) CD133+ glioma and normal brain cells showed a significantly high resistance towards chemotherapeutic drugs compared to the autologous CD133- cells. In conclusion, an improved understanding of molecules contributing to the maintenance of CSCs may lead to a combined treatment, targeting both CSCs and their protective MDR phenotypes leading eventually to attractive strategies for the treatment of gliomas.

In the mammal, anaesthetics are known to act via two distinct mechanisms, either increasing inhibition via GABAA receptors (eg. Na-thiopentone) or decreasing excitation via NIvIDA receptors (eg. ketamine). The aim of this thesis is to investigate the effects of both increased inhibition and decreased excitation at the synaptic level within an invertebrate model system, something which cannot readily be done in vertebrate systems. This was achieved by carrying out experiments using both the above mentioned anaesthetics on the whole animal, isolated brain and cultured neurons. In invertebrates it has been shown that GABA and Glutamate can be both excitatory and inhibitory, and injection of GABA into Lymnaea has been shown to result in behavioural changes in feeding, locomotion, escape reactions, male mating and respiration. Injection of Na-thiopentone into the whole animal was carried out in this investigation, in order to establish the anaesthetic response of the animal model to this barbiturate. The presence of gamma-aminobutyric acid (GABA) receptors has been demonstrated in a respiratory interneuron (RPeD1) both electrophysiologically and via molecular techniques, however inmiunostaining has proved negative in RPeD1 and follower cells VD2/3 (unidirectional excitatory synapse) and VD4 (mutual inhibitory synapse). This suggests that these neurons are not themselves GABAergic, although this investigation shows the responses of these neurons to bath and direct application of GABA. Na-thiopentone did not reliably anaesthetise Lymnaea upon injection into the sole of the foot, suggesting that Na-thiopentone binds to proteins within the snail, andlor has a low affininty for the GABAA receptor in Lymnaea. Other anaesthetic studies using propofol and ketaniine have also demonstrated a lack of anaesthetic response. RPeD1 hyperpolarised and became quiescent in response to the application of high concentrations of GABA (10 3-104M), however at lower doses (1O 8-1O 5M), no effect was observed (p < 0.05). Under these conditions simultaneous recordings from VD4 showed hyperpolarisation in response to the application of GABA, whereas VD2 and VD3 exhibited excitatory responses. Presynaptic picoinjection of GABA also resulted in hyperpolarisation and quiescence in RJeD1, but the simultaneous response in VD3 was not observed. Postsynaptic application of GABA directly to 'VD2, and VD4 however, resulted in responses similar to those seen in the whole brain. VD2 and 3 also receive input 2, which hyperpolarises RPeD1 and elicits an excitatory EPSP in VD2 and 3 as this is similar to the response observed in this experiment it is possible that the effects of input 2 on RPeD1 and VD2 & 3 are mediated by GABA. As RPeDI does not stain positively for GABA and hyperpolarises in response to the drug, it seems unlikely that the postsynaptie effects are due to presynaptic release of GABA. RPeD1 has been shown to form reciprocal synapses with VD4 both in vivo and in vitro. When perfused with GABA (lmJ'i4), both cells hyperpolarised reversibly. The postsynaptic response could be due to the action of GABA presynaptically inhibiting RPeDI, or directly on postsynaptic GABA receptors. However VD4 forms connections with other cells in the brain such as input 3 which may also have resulted in this inhibitory response. RPeD1 would however have received a simultaneous excitatory input from this interneuron. Attempts were made to establish the nature of the RPeD1JVD4 synapse in these experiments, but no synapses were evident. These experiments therefore confirm the presence of GABA receptors in RPeD1 and suggest theft presence in VD2, and VD4. This investigation confirms the findings of previous studies, that injection and bath perfusion of barbiturates does not lead to responses in Lymnaea comparable to that of the mammal. In addition to it's main target site, ketamine (a frequently used intravenous anaesthetic) has also been shown to act at cholinergic receptors. The effects of ketamine on learning and memory and apoptosis in the mammalian CNS are well recognised. Within the Lymnaea CNS, VD4 and LPeD1 form a unidirectional excitatory cholinergic synapse, and this was chosen to investigate the effects of ketamine on excitatory synaptic transmission, short term potentiation and synapse formation in the invertebrate animal model. Ketamine decreased synaptic transmission between VD4 and LPeD1 in a concentration dependent manner, but did not significantly affect short term synaptic plasticity (pc0.05). While neurite outgrowth remained extensive, actual sprouting was diminished by all doses of ketamine. Cells exhibited extensive veiling, which was not present in control cells. Percentage chemical synapse formation was reduced by all doses of ketamine, and in some cases inappropriate inhibitory chemical synapses were formed. Furthermore acute, clinically relevant levels of ketamine reduce excitatory cholinergic transmission between VD4 and LPeD1, but short term plasticity is unaffected. Nerve regeneration was seriously compromised, and formation of appropriate chemical synapses greatly reduced. This data has serious implications for the clinical - use of ketamine, particularly in pregnant women, children or critical care patients where nerve regeneration and synapse formation are of great importance and long term exposure common practice. In conclusion, this work supports that of other studies which have showed that invertebrates appear to be relatively insensitive to barbiturates, whereas ketamine appears to effect excitation in a manner similar to that in the mammal.

Antiulcer agents, notably inhibitors of gastric acid secretion, have been the most successful category of drugs to be discovered in recent years; and moreover, there are currently four such agents in the world list of top 25 best selling drugs. Histamine H2 antagonists have been the number one selling pharmaceutical product for more than a decade and inhibitors of the parietal cell HIC-ATPase, so called "proton pump inhibitors" (PPI), look set to continue this success. The proposed study was designed to establish the relative efficacy and mechanisms of action of three novel agents using both in vitro and in vivo models. The three compounds namely AG-1749 (Lansoprazole), PD-136450 and transforming growth factor alpha (TGF(x) were studied to evaluate their antisecretory and antiulccr activities. Lansoprazole, the second PPI to be developed for clinical use, is a non-competitive inhibitor of the H1C-ATPase and has recently been launched in a number of countries. PD-136450 is a competitive antagonist of central and peripheral cholecystokinin-B (CCK-B) receptors (gastrin receptor) and it under clinical development as an anxiolytic but which has actions on the stomach and pancreas. Anxiolytic drug is otherwise known as anti-anxiety drugs, which are used to treat anxiety disorders, like depression, panic disorders, phobias and many personality disorders. TOFu is a polypeptide growth factor, which acts at the EGF receptor and displays potent mitogenic and antisecretory activity. The initial study deals with the comparison of the three compounds with omeprazole and ranitidine in terms of their ability to inhibit acid secretion and their activity in a range of experimental ulcer models. Potency, duration of action and activity against a range of stimulants of acid secretion (histamine, pentagastrin and basal) was determined in anaesthetized rat models by establishing dose-response relationships. The compounds represent a spectrum of activities in as much as lansoprazole is a potent, long acting inhibitor, PD-136450 is an orally active but selective inhibitor, while TOFu has a very short duration and is only active after parenteral administration. In a view to find out the mechanism of action of these drugs on gastric acid secretion, isolated gastric glands from rabbits were employed as an in vitro technique using radiolabeled 14C-aminopyrine as a marker. The results show that lansoprazole was the most potent antisecretory agent compared to other two drugs. The second phase of the study deals with the activity of the three compounds against gastric ulcers induced by acid hypersecretion, indomethacin and stress. This study enabled us to assess the extent to which antisecretory activity per se compared with other actions such as wound healing (TGFa) or anxiolytic activity (PD-136450) contribute to ulcer healing. As other workers already established that prostaglandins and nitric oxide are involved in the cytoprotective activity, the present study investigated the influence of prostaglandin and nitric oxide by using indomethacin and L-NAME pretreatment on the cytoprotective activity of lansoprazole, PD-136450 and TGFcz. Moreover, the three drugs were tested for their activities on the mucus and bicarbonate production in the stomach. It was found that lansoprazole and TGFc increased the gastric mucus secretion while PD-136450 did not show any change. Moreover it was evidenced from this study that the protective activity of PD-136450 is associated with the influence of bicarbonate secretion from the pancreas. In conclusion, the results of this study have indicated that lansoprazole, PD- 136450 and TGFct are potent antisecretory and antiulcer agents which have great therapeutic importance.

Mature skeletal muscle fibres can be classified as type I, type IIa, type IIx or type IIb fibres according to the myosin heavy chain (MHC) isoform that they express. More broadly, type I fibres are classified as slow fibres and type IIa, IIx and IIb fibres as fast fibres. However, the phenotype of an adult skeletal muscle fibre is not fixed: it displays plasticity being capable of adapting to changing activity and loading levels by either transition towards a slower phenotype or transition towards a faster phenotype. Overall, the aims of these studies were to further investigate and define the signal transduction pathways implicated in the control of skeletal muscle fibre phenotype. The ability of a fast muscle to undergo a transition towards a slower phenotype in response to chronic low-frequency stimulation (CLFS) was assessed, via metabolic enzyme activity assays and NADH-TR staining, following blockade of the calcineurin signalling pathway. Metabolic enzyme assays and northern blots were employed to assess the changes in enzyme activities and MEC isoform expression levels following blockade of the calcineurin and ERK1/2 signalling pathways in primary cultures of rat myotubes. Differences in the levels of various signal transduction proteins/transcription factors between slow and fast muscle were investigated using western blotting. The nuclear translocation kinetics of NFAT and NF-κB in response to treatment with the calcium ionophore A23187 were investigated in L6 myotubes using immunocytochernistry. Calcineurin blockade using cyclosporin A failed to prevent a decrease in lactate dehydrogenase activity and an increase in NADH-TR staining intensity, both characteristics of a transition towards a slower phenotype, following CLFS of the fast rat tibialis anterior muscle. Blockade of the ERK1/2 pathway in primary cultures of rat myotubes using U0126 significantly decreased MHC Iβ mRNA levels and significantly increased MIHC IIx, MEC IIb and perinatal MHC mRNA levels. Calcineurin blockade significantly decreased MHC Iβ and embryonal mRNA levels and significantly increased MHC IIx mRNA levels. Calcineurin blockade also significantly increased the activities of lactate dehydrogenase and creatine kinase while ERK1/2 blockade significantly increased the activities of lactate dehydrogenase, creatine kinase, hexokinase, malate dehydrogenase and β-hydroxyacyl-CoA deydrogenase. ERK1/2 and NF-κB levels were found to be higher in slow muscle compared to fast muscle while calcineurin and p38α,β levels were higher in fast muscle compared to slow muscle. No nuclear translocation of NF-κB and only limited evidence for NFAT nuclear translocation was seen in L6 myotubes following treatment with A23187. Overall these studies further characterize the roles of the ERK1/2 and calcineurin pathways in the regulation of muscle phenotype suggesting that each pathway controls some, but not all, of the genes that are differentially expressed between slow and fast muscle fibres. Western blotting suggests further signal transduction protein/transcription factor targets, the functions of which may be explored in the future.

Based on previous studies of the novel Lactobacillus reuteri DPC16 strain, an in vitro investigation on the supernatant antimicrobial activity and the culture safety against normal gastrointestinal microflora and gastric mucus was done in this thesis. DPC16 cell-free supernatants (fresh and freeze-dried, designated as MRSc and FZMRSc) from anaerobic incubations in pre-reduced MRS broth, have shown significant inhibitory effects against selected pathogens, including Salmonella Typhimurium, E. coli O157:H7, Staphylococcus aureus, and Listeria monocytogenes. These effects were mainly due to the acid production during incubation as evidenced by the negation of such activity from their pH-neutral counterparts, and this acidic effect was shown to reduce the pathogen growth rate and decrease the total number of pathogen cells. By incubation of concentrated (11 g/L) resting cells in glycerol-supplemented MRS broth, another DPC16 cell-free supernatant (designated as MRSg) has shown very strong antimicrobial effect against all target pathogens. As indicated by a kinetic profile, this activity developed in a sigmoidal fashion as incubation proceeded, reaching to maximum activity after 6-8h and maintained at the same level thereafter. Further study has shown that the antimicrobial activity of this supernatant was pH-independent, effective across a pH range of 4.6 to 6.5, and acted on both Gram-negative and Gram-positive pathogens. Using the minimum effective dose, a time course investigation has provided evidence that this supernatant affected the growth of the target pathogens by elongating the lag phase and lowering the total cell number at the end of the incubation. Lastly, it was found that the strong antimicrobial effect of MRSg was bactericidal at high concentrations and bacteriostatic at low concentrations. However, it also found that the viability of DPC16 cells also decreased as incubation prolonged, which suggests that this glycerol-derived supernatant had a lethal effect to its own cells. Nevertheless, this lethal effect was exerted to a much lesser extent compared with that to the pathogens. The last DPC16 cell-free supernatant was designated as SGF, which was produced from secondary fermentation of the same resting cells in glycerol-water. SGF did not show a significant antimicrobial activity, which suggests that this specific strain is not capable of utilising glycerol in the absence of fermentable carbohydrates. The antimicrobial activity found in MRSg matched with previously published characteristics of reuterin, which is a unique antimicrobial substance synthesised by L. reuteri when incubated with glycerol. Therefore, a study on the production kinetics of reuterin by DPC16 was carried out. Supernatants of both MRSg and SGF were studied. Results showed that glycerol utilisation occurred in both MRSg and SGF, whereas the bioconversion of glycerol into reuterin was different. In MRSg, glycerol was constantly utilised by DPC16 resting cells, and by the end of an 18h incubation 85.8 mM of glycerol was utilised, where 72.8% was transformed into reuterin. The formation of reuterin initiated with an inclining production and reached the maximum rate of 10.9 mM/h after 6h of incubation, with the total production of 64 mM of reuterin at the end of the 18h incubation. This reuterin production in MRSg followed a similar pattern to that of its antimicrobial activity, which suggests a certain correlation between reuterin formation and the increase of antimicrobial activity in MRSg. Therefore, the major antimicrobial component in MRSg that accounted for its potent antimicrobial activity was very much likely to be reuterin. In SGF, however, detectable reuterin was negligible even though some glycerol may have been absorbed into the highly concentrated DPC16 resting cells. This has responded to the antimicrobial activity assay in that due to the lack of essential carbohydrate nutrient for normal cell metabolism, there was no glycerol utilisation and hence no reuterin synthesis. Having studied the antimicrobial activity of L. reuteri DPC16 and the production of antimicrobial-competent reuterin, two safety issues (the impact on growth on other normal commensal probiotics and mucin degradation activity) of this strain were assessed to further evaluate its probiotic potential. By using similar in vitro assays as in the antimicrobial test, the same set of DPC16 supernatants have demonstrated no adverse effect on the growth of either Lactobacillus acidophilus, Lactobacillus plantarum, Pediococcus acidilactici, or Bifidobacterium lactis DR10. No stimulatory effect was found either. By incorporating purified porcine gastric mucin into classic mucin-degradation assays in both liquid and agar media, DPC16 has not exhibited the same mucinolytic activity as that of the faecal flora cultures. Thus, it can be concluded that L. reuteri DPC16 is as safe to the host as normal commensal microflora.

The ecological importance of introduced mammalian predators is well acknowledged in New Zealand, however, little research has focused on the ecology of native avian predators and their role in communities. This study investigated the ecology of moreporks (Ninox novaeseelandiae) on Ponui Island, Hauraki Gulf, New Zealand between August 2007 and April 2008. The primary aim was to investigate the functional response of moreporks to availability of their prey. The contents of regurgitated morepork pellets were compared with relative abundance of prey taxa (invertebrates, small birds and rodents) over the study period. The diet consisted primarily of a range of invertebrate prey, particularly weta (Anostostomatidae and Raphidophoridae) and beetles (Coleoptera). Small numbers of vertebrate prey were recorded including rodents and birds. A positive relationship between the percentage contribution to pellet samples of certain taxa and their relative availability was found, and there were peaks in the occurrence of seasonally abundant taxa including cicadas (Cicadidae), and huhu beetles (Prionoplus reticularis). The tendency of moreporks to prey on abundant taxa indicates that they are unlikely to depress prey populations to low levels, and may have some degree of stabilising influence. A significant increase in the rodent component of the diet in April indicated that the risk to moreporks of secondary poisoning during mammalian pest control operations may vary considerably with the time of year. The secondary aims were to collect data on roost site characteristics and breeding success. Moreporks roosted at a mean height of 4m, and foliar cover at the 4-6m height tier appeared to be the most important characteristic of roost sites when compared with control sites. These findings suggested that moreporks were selecting roost sites with high overhead cover. Possible reasons for this include predator avoidance, avoidance of mobbing passerines, and the microclimate provided. None of three established pairs and two other birds were observed to establish a nest or breed successfully. Additionally, only three juvenile moreporks were sighted or heard across the 90ha study area suggesting low breeding success in 2007-08. This may have been influenced by a range of factors including 1), predation by the high densities of ship rats on Ponui, or other predators 2), a lack of suitable nest sites such as tree hollows in some areas or 3), competition for invertebrate prey with high densities of ship rats and North Island brown kiwi (Apteryx mantelli).

The sap beetle genus Carpophilus Stephens (Coleoptera: Nitidulidae) is a large genus consisting of over 200 species and are found worldwide. Several species are important pests of crops and stored products, and are frequently intercepted as part of biosecurity operations. The genus is poorly known taxonomically, and there are several species groups that are challenging to identify by morphological methods. In particular, two species found across the Pacific, C. maculatus Murray and C. oculatus Murray are frequently confused with each other. These two species are similar in size and colour, but differ primarily by the shape of the colour pattern on their elytra. However, this colour pattern is highly variable within both species, leading to ambiguity in the indentification of these species. Within C. oculatus, three subspecies have been described based on differences in the male genitalia and pronotal punctation: C. o. oculatus and C. o. gilloglyi Dobson are distributed widely across the Pacific, while C. o. cheesmani Dobson is known only from Vanuatu. A search of literature records and specimen collections revealed 32 species of Carpophilus recorded from the Pacific region. In addition there remain several unidentified specimens representing at least four species, two of which will be described subsequent to this research. A number of species recorded in the literature may have been misidentified, and these require further field collections and inspection of museum specimens to confirm their presence in the Pacific. To test the validity of the subspecies of C. oculatus, and its distinctiveness from C. maculatus, a phylogeny of available specimens of Carpophilus was inferred from one mitochondrial gene (cytochrome c oxidase subunit I (COI)), and two nuclear genes (28S ribsomal RNA (28S) and the internal transcribed spacer 2 (ITS2)). These data show large genetic distances between the three subspecies of C. oculatus of 7-12%. Given these distances are similar to those between other species in the genus, this indicates these subspecies may be elevated to full species. The data also consistently support a monophyletic relationship between C. o. oculatus and C. o. gilloglyi. Nuclear genes also support C. o. cheesmani as part of a clade with the other subspecies, but these relationships are unresolved in COI. Carpophilus maculatus was not supported as being the sister taxon of the C. o. oculatus and C. o. gilloglyi clade. Other relationships within Carpophilus were unresolved, possibly due to a combination of incomplete taxon sampling, and saturation of substitutions within the COI gene. Phylogeographic analysis of specimens collected from several localities within the range of C. oculatus showed that, with only one exception, there were no shared haplotypes between archipelagoes. This result suggests it may be possible to determine the provenence of intercepted specimens, providing further information regarding potential invasion pathways. A degree of geographic structuring was also present within C. o. gilloglyi, being separated into a western clade found in Fiji and Rotuma and an eastern clade distributed from the Kermadec Islands and Tonga to French Polynesia. This separation was most profound in COI data, with a mean pairwise distance between the clades of 7%. ITS2 data also demonstrates a degree of differentiation between the two clades, based on differences in the insertions and deletions between the clades. The variability in the shape and colour of the elytral pattern of C. oculatus was also investigated. Colour was quantified using a method based on Red-Green-Blue (RGB) colour values derived from digital photographs, while an outline analysis of the elytral pattern was conducted using elliptic Fourier analysis (EFA). Principal Components Analysis of the RGB values and EFA coefficients showed no clear separation between subspecies, nor were any trends correlated with host fruit or collection localities. Variation at all levels and all measures studied in this thesis show that this geographic region and this genus of beetles offer intruiging insights into speciation, biogeography and biological invasions. There is much scope for further research on the causes and consequences of this variation and the lives of these interesting insects.

The lens of the eye needs to be completely transparent in order to allow all light entering the eye to reach the retina. This transparency is maintained by the highly ordered structure of the lens proteins the crystallins. Any disruption to the lens proteins can cause an opacity to develop which is known as cataract. During cortical cataract formation there is increased truncation of the lens crystallins. It is believed that overactivation of calcium-dependent cysteine proteases, the calpains, is responsible for the increased proteolysis of the crystallins seen during cataractogenesis. Within the ovine lens there are three calpains, calpain 1, 2 and the lens specific calpain Lp82. The aim of this thesis was to determine the changes in the lens proteins during ageing and cataractogenesis, and to establish the role of the calpains in these processes. Calpain 1 and 2 were purified from ovine lung and Lp82 was purified from lamb lenses using chromatography. Activity and presence of the calpains was determined by using the BODIPY-FL casein assay, gel electrophoresis, Western blot and casein zymography. Changes in the lens proteins, specifically the crystallins, were visualised using two-dimensional electrophoresis (2DE). Lenses from fetal, 6 month old and 8 year old sheep were collected, as well as stage 0, 1, 3 and 6 cataractous ovine lenses. The proteins from the lenses were separated into the water soluble and urea soluble fractions and analysed by 2DE. Mass spectrometry was used to determine the masses and therefore modifications of the crystallins. Finally, the individual crystallins were separated using gel filtration chromatography and incubated with the purified calpains in the presence of calcium. The extent of the proteolysis was visualised using 2DE and truncation sites determined by mass spectrometry. Purification of the calpains resulted in samples that were specific for each calpain and could be used in further experiments. 2DE analysis showed that there were changes to the crystallins during maturation of the lens. The α-crystallins become increasingly phosphorylated as the lens ages and a small amount becomes truncated. The β-crystallins were also modified during ageing by truncation and deamidation. When crystallins from cataractous lenses were compared using 2DE there were changes to both the α- and β-crystallins. The α-crystallins were found to be extensively truncated at their C-terminal tail. Four of the seven β-crystallins, βB1, βB3, βB2 and βA3, showed increased truncation of their N-terminal extensions during cataract formation. All three calpains truncated αA and αB-crystallin at their C-terminal ends after incubation. Calpain 2 and Lp82 each produced unique αA-crystallin truncations. All three calpains truncated βB1 and βA3 and calpain 2 also truncated βB3. When the truncations from the calpain incubations were compared to those seen during cataract formation, many of the truncations were found to be similar. Both the unique truncations from calpain 2 and Lp82 were found in cataractous lenses, with the Lp82 more obvious in the 2DE. The β-crystallin truncations found after incubation with the calpains were similar to those found during cataractogenesis. In conclusion this study documents the changes to the ovine lens during maturation and cataractogenesis and indicates a role for the calpain family in the increased proteolysis observed in the ovine cataract.

Utilizing environmental DNA (eDNA) for the detection of species in the field is a potentially cost-effective and time-saving technology that may be useful in understanding the distribution and abundance of threatened or endangered species such as the Eastern Massasauga (Sistrurus catenatus). I describe the development of an eDNA assay for the species and evaluate its ability to detect eDNA in laboratory and field conditions. In the field samples, I also investigated the potential for abiotic conditions to influence eDNA detection. Species-specific primers and probe were designed to amplify a 152 bp segment of the massasauga COI gene. Target eDNA could be detected in samples containing as little as 100 copies of target DNA/μL. Water samples collected from laboratory housed snakes indicated that eDNA can be detected in water 56 days after massasauga removal. Field samples were taken from crayfish burrows, known overwintering habitat for the species, from four sites that vary in snake use as ascertained by traditional visual surveys. Of the 60 burrows sampled, seven had a positive detection for massasauga eDNA with no difference in detection rate between DNA extracted from burrow water and burrow sediment. Occupancy models fitted to burrow water indicated that larger amounts of total DNA in a sample may increase the probability of detection of a massasauga eDNA. Large confidence intervals in site occupancy (ѱ) and burrow detection (Θ) values suggest that a larger sample size is needed for more reliable occupancy models. Abiotic conditions within crayfish burrows varied among sites but correlation with eDNA detection was not supported. Estimates of qPCR detection within a burrow with eDNA (ρ) suggest that up to 10 qPCR replicates per burrow sample may be necessary. Further studies need to examine eDNA degradation in the field, improve upon the limit of detection, and sample a larger number of sites for eDNA sampling to be a stand-alone survey method for Eastern Massasaugas.

Environmental DNA (eDNA) is being used increasingly for biomonitoring of communities (e.g., microbes, macroinvertebrates, fish species) across terrestrial and aquatic ecosystems. Developing methods that combine eDNA approaches with metagenomic barcoded amplicon sequencing (eDNA-metabarcoding) are now providing a powerful noninvasive and cost-effective means for comprehensively surveying biodiversity in a wide range of habitats. Invasive species have a substantial impact on the ecology and economics of the Great Lakes region, and eDNAmetabarcoding methods have recently been applied in monitoring non-native, as well as native, fish populations in the freshwater systems there. In this research, we validated an eDNAmetabarcoding approach that uses established platforms, the MiFish/MitoFish pipeline, for fish community monitoring on Lake Michigan. For validation, we compared survey results from our eDNA-metabarcoding approach to those obtained using traditional surveys (e.g., electrofishing and seining). We also sampled a closed 180,000-gallon freshwater fish tank system to see how well our methods characterized a known native fish population that resided in the tank. Finally, we applied the approach to monitoring invasive and native fish populations in southern Lake Michigan at a site that is currently undergoing restoration to improve the aquatic habitats.. We were able to reliably capture the fish community structure of the native fish tank as well as those of open waters on the lake using our methods. Diversity patterns detected at the restoration site using our eDNA-metabarcoding approach accurately reflected those of the historical record, which have taken many years to establish by conventional means. Overall, this study suggests eDNAmetabarcoding is an efficient, credible, and powerful approach to biomonitoring.

Osteoarthritis (OA) is a leading cause of disability globally, with higher incidence in older people and lower socioeconomic status populations. The challenges health care systems face with management of the disease highlights the importance of OA research. Many studies examine possible risk factors of knee and hip OA including obesity, smoking, and alcohol consumption. Findings support that while obesity increases risk of knee OA, smoking is not a major risk factor. These extrinsic factors are, however, associated with lower socioeconomic status, and also with anxiety and depression disorders. Up to 30% of patients with chronic knee OA have described psychological stress and decreased quality of life due to debilitating pain, but the effects of psychological stress on development of knee OA has not been described.

At the cellular level, mechanosensitive cation channels in cartilage and bone, are involved with OA, but studies looking specifically at synovium and joint capsule are limited. Transient receptor potential (TRP) channels are upregulated in joint capsule in end-stage primary shoulder OA. We were unable to identify any previous studies evaluating Piezo channel expression in musculoskeletal soft tissues, but Piezo channel antagonism reduces chondrocyte death after mechanical injury. These findings suggest channels may help regulate joint responses to repetitive loading during training or work while also contributing to protective mechanisms within the musculoskeletal system. The overall objective of this research was to investigate factors that impact OA development or the disease phenotype. Two studies evaluated the following aims: 1) demonstrate the influence of chronic psychological stress on knee OA and overall systemic health, and 2) characterize the role of mechanosensitive channels in the joint capsule in OA. The first study used a mouse chronic social defeat model paired with destabilization of the medial meniscus (DMM) surgery to create a social stress scenario during OA development. We hypothesized chronic social defeat would exacerbate knee OA structural changes and systemic inflammation. The second study aimed to explore the role of mechanosensitive channels in joint capsule during OA development in the equine. Immunohistochemistry was performed on forelimb fetlock joint capsule from horses with varying degrees of lameness to first identify TRP and Piezo channel expression. Next, fibroblasts were isolated from the tissue to determine channel activity. We hypothesized that TRP and Piezo channels are required for normal homeostasis, but are dysregulated in OA and dysregulation contributes to fibrosis of the joint capsule. Joint capsule fibrosis leads to joint stiffening and reduced range of motion, two of the cardinal signs of OA.

The results of the first study showed OA was induced to a similar extent in both groups of mice that underwent DMM surgery. While anxiety- and depressive-like behaviors were exhibited by mice that underwent chronic social defeat episodes, unexpectedly, the majority of systemic inflammatory markers were not worse in mice with DMM and chronic social defeat compared to DMM alone. We were also able to show TRP and Piezo channel expression in one normal dorsal and palmar fetlock joint capsule sample, however, COVID-19 prevented further investigation. With our results we were able to conclude that while chronic social stress influences development of OA, in the current experiments, neither systemic inflammation nor structural signs of knee OA were worse with chronic social stress. We hope that exploration of OA through these two studies will help us understand how the disease contributes to overall systemic dysfunction while also providing a baseline for future development of TRP and Piezo channel modulators to prevent joint pathologies.

Many animal species invest in extended parental care for their offspring. Parental care is costly, and natural selection favors investment strategies which maximize reproductive success. Biparental care is relatively rare, but when it does occur it has been found to increase success in terms of offspring survival and growth and in terms of future reproductive opportunities. In burying beetles (Nicrophorus spp.), both male and female participate in extended parental care. However, the fitness benefits of biparental care in burying beetles have been difficult to establish, with some studies reporting significantly smaller broods produced when both male and female are present. Variation in environmental conditions, such as temperature, is an important part of the context in which biparental care evolves. I hypothesize that biparental care acts as a buffer against environmental variation. This hypothesis predicts that biparental care will lead to greater reproductive success compared to uniparental care when temperature is increased during a reproductive attempt. I also tested the load-lightening hypothesis, which holds that biparental care benefits future reproduction by lowering the costs of reproduction. This predicts that the additional care by the other parent will allow females to rear higher quality second broods. I conducted a male removal experiment at two temperature treatments, using the species Nicrophorus orbicollis. I measured reproductive success during manipulated first brood and during second broods which females reared without a male, regardless of prior experience. I found that, contrary to my hypothesis, biparental care at the higher temperature resulted in reduced reproductive success compared to uniparental care. I found no effect of biparental care on the success of second broods. Instead, I found evidence of reproductive restraint associated with the higher temperature treatment in delayed egg-laying and increased feeding during second broods.

Methods for the preparation of concentrates of factor VIII, factor IX, high purity factor IX, Cl esterase inhibitor, specific immunoglobulin and platelet factor XIII are described. These procedures were developed or modified with the aim of integration into an automated process that would allow sequential recovery of all the clinically significant trace proteins from a single plasma pool. Concomitant recovery of important proteins such as transferrin, alpha-1-antitrypsin and platelet-derived growth factor was considered. A high-purity factor VIII concentrate heat-treated at 80°C for 96 h was prepared by a process that incorporated heparin fractionation. This method was shown to be suitable for assimilation into an existing regional blood processing laboratory. Several ion-exchange procedures for the recovery of factor IX were evaluated and higher purification of a factor IX concentrate was achieved on a new cellulose-based chromatographic medium. A chromatographic procedure for the preparation of a heat-treated high-purity Cl esterase inhibitor concentrate was described and the performance of a new cellulose-based desalting medium was evaluated in comparison with ultrafiltration. A heat-treated specific immunoglobulin concentrate was prepared from side-stream fractions of an automated chromatographic process for the production of albumin concentrate, and a pilot study for the fractionation of outdated platelet concentrates was carried out with the aim preparing components of potential therapeutic value. See summary flow diagrams of fractionation processes included with references in the back of this thesis.

Pancreatic ductal adenocarcinoma (PDAC) is an incredibly lethal disease with a 5-year survival rate of less than 8 percent in the United States due to a lack of viable treatment options. The failures of chemo- and radiotherapies have been linked to the heterogeneous nature of the tumor microenvironment which forms a hypovascular, immunosuppressive and high coagulation activity tissue. Indeed, PDAC patients have one of the highest rates of thrombosis complications among all cancer types. The expression of two key coagulation factors, Tissue Factor (TF) and Protease Activated Receptor 1 (PAR-1), have been associated with poor patient prognosis and aggressive cancer progression. However, the molecular roles/mechanisms of TF and PAR-1 in PDAC progression are not known. To establish how clotting factors (PAR-1, TF) influence PDAC tumor progression, I utilized a genetically modified mouse model (KPC) where KRas^G12D and TRP53^R172H mutations were specifically introduced into mouse pancreas acinar cells to initiate PDAC progression. Multiple primary mouse PDAC cell lines were generated and characterized. TF and PAR-1 were highly expressed in primary KPC pancreatic lesions, in PDAC tumors, and in KPC-derived cell lines, an expression profile that is also observed in PDAC patient biopsies. In allograft studies, tumor growth and metastatic potential were significantly diminished by shRNA reduction of TF or PAR-1 in cancer cells or by genetic or pharmacological reduction of the coagulation zymogen prothrombin in mice. Notably, PAR-1 deleted KPC cells (KPC-Par-1^KO) failed to generate sizable tumors; a phenotype completely rescued by restoration of PAR-1 expression. To test the significance of targeting PAR-1 in a clinical setting, PAR-1 expression was withdrawn from established tumors to mimic a potential inhibitory effect of PAR-1 on solid PDAC tumors. Removal of PAR-1 from tumors (11 days post injection) yielded a diverse effect on tumor growth which can be categorized into (i) a decline in tumor growth; (ii) continued tumor growth; and (iii) stagnant tumor growth. Immunohistochemistry analysis of KPC2 shCon vs. shPar-1 subcutaneous allograft tumor samples revealed a massive immune cell infiltration in KPC2 shPAR-1 tumors when compared to KPC2 shCon control tumors. Accordingly, KPC-Par-1^KO cells failed to form tumors in immune-competent mice but displayed robust tumor growth in immune-compromised NSG mice, providing the first evidence of a PAR-1 mediated tumor immune evasion pathway operating in PDAC.

Together, these results demonstrate that PDAC disease is driven by activation of the coagulation system through tumor cell-derived TF, circulating prothrombin, and tumor cell-derived PAR-1. These studies also highlight a novel mechanism by which thrombin/PAR-1-mediated tumor growth involves suppression of anti-tumor immunity in the tumor microenvironment.

External representations (ERs) used in science education are multimodal ensembles consisting of design elements to convey educational meanings to the audience. As an example of a dynamic ER, an animation presenting its content features (i.e., scientific concepts) via varying the feature’s depiction over time. A production team invited the dissertation author to inspect their creation of a biochemistry animation about the role of the actin cytoskeleton in cell motility and the animation’s implication on learning. To address this, the author developed a four-step methodology entitled the Multimodal Variation Analysis of Dynamic External Representations (MVADER) that deconstructs the animation’s content and design to inspect how each content feature is conveyed via the animation’s design elements.

This dissertation research investigated the actin animation’s educational value and the MVADER’s utility in animation evaluation. The research design was guided by descriptive case study methodology and an integrated framework consisting of the variation theory, multimodal analysis, and visual analytics. As stated above, the animation was analyzed using MVADER. The development of the actin animation and the content features the production team members intended to convey via the animation were studied by analyzing the communication records between the members, observing the team meetings, and interviewing the members individually. Furthermore, students’ learning experiences from watching the animation were examined via semi-structured interviews coupled with post- storyboarding. Moreover, the instructions of MVADER and its applications in studying the actin animation were reviewed to determine the MVADER’s usefulness as an animation evaluation tool.

Findings of this research indicate that the three educators in the production team intended the actin animation to convey forty-three content features to the undergraduate biology students. At least 50% of the student who participated in this thesis learned thirty-five of these forty-three (> 80%) features. Evidence suggests that the animation’s effectiveness to convey its features was associated with the features’ depiction time, the number of identified design elements applied to depict the features, and the features’ variation of depiction over time.

Additionally, one-third of the student participants made similar mistakes regarding two content features after watching the actin animation: the F-actin elongation and the F-actin crosslink structure in lamellipodia. The analysis reveals the animation’s potential design flaws that might have contributed to these common misconceptions. Furthermore, two disruptors to the creation process and the educational value of the actin animation were identified: the vagueness of the learning goals and the designer’s placement of the animation’s beauty over its reach to the learning goals. The vagueness of the learning goals hampered the narration scripting process. On the other hand, the designer’s prioritization of the animation’s aesthetic led to the inclusion of a “beauty shot” in the animation that caused students’ confusion.

MVADER was used to examine the content, design, and their relationships in the actin animation at multiple aspects and granularities. The result of MVADER was compared with the students’ learning outcomes from watching the animation to identify the characteristics of content’s depiction that were constructive and disruptive to learning. These findings led to several practical recommendations to teach using the actin animation and create educational ERs.

To conclude, this dissertation discloses the connections between the creation process, the content and design, and the educational implication of a biochemistry animation. It also introduces MVADER as a novel ER analysis tool to the education research and visualization communities. MVADER can be applied in various formats of static and dynamic ERs and beyond the disciplines of biology and chemistry.