As part of a project designed to utilize common dairy waste products profitably, reconstituted dry whey permeate and delactosed whey were tested as growth substrates for mycelia of the edible mushroom Lentinus edodes. This mushroom was chosen because it is possible to profitably cultivate it due to its popular culinary appeal and perceived medical benefits. Growth experiments were performed in petri dishes containing either reconstituted dry whey permeate or delactosed whey as a growth substrate, and the measured response was the size of the growing mycelia colony. When reconstituted dry whey permeate was utilized as a growth substrate, the factors of substrate concentration, pH, and growth temperature were controlled in an effort to determine the optimal growth conditions for the mushroom mycelia. These conditions were determined by applying an analytical method known as response surface methodology (RSM). RSM is a collection of mathematical techniques that is able to determine optimal values for many variables run simultaneously in an experiment. Mycelia were also grown on delactosed whey at different substrate concentrations in an effort to determine if this substrate would be suitable for the growth of mushroom mycelia. Results: RSM was successfully utilized to determine the optimal growth conditions for L. edodes when grown on reconstituted dry whey powder. These conditions were 40 g/L substrate concentration, pH 4 .97, and temperature 23.6°C Delactosed whey was successfully utilized as a growth substrate for L. edodes. However, delactosed whey concentrations above 40% v/v were lethal to the mushroom mycelia, suggesting a possible use for delactosed whey as a fungicide.

Indiana University-Purdue University Indianapolis (IUPUI) / Stiffening and softening of extracellular matrix (ECM) are critical processes governing many aspects of biological processes. The most common practice used to investigate these processes is seeding cells on two-dimensional (2D) surfaces of varying stiffness. In recent years, cell-laden three-dimensional (3D) scaffolds with controllable properties are also increasingly used. However, current 2D and 3D culture platforms do not permit spatiotemporal controls over material properties that could influence tissue processes. To address this issue, four-dimensional (4D) hydrogels (i.e., 3D materials permitting time-dependent control of matrix properties) are proposed to recapitulate dynamic changes of ECM properties. The goal of this thesis was to exploit orthogonal enzymatic reactions for on-demand stiffening and/or softening of cell-laden hydrogels. The first objective was to establish cytocompatible hydrogels permitting enzymatic crosslinking and stiffening using enzymes with orthogonal reactivity. Sortase A (SrtA) and mushroom tyrosinase (MT) were used sequentially to achieve initial gelation and on-demand stiffening. In addition, hydrogels permitting reversible stiffening through SrtA-mediated peptide ligation were established. Specifically, poly(ethylene glycol) (PEG)-peptide hydrogels were fabricated with peptide linkers containing pendent SrtA substrates. The hydrogels were stiffened through incubation with SrtA, whereas gel softening was achieved subsequently via addition of SrtA and soluble glycine substrate. The second objective was to investigate the role of dynamic matrix stiffening on pancreatic cancer cell survival, spheroid formation, and drug responsiveness. The crosslinking of PEG-peptide hydrogels was dynamically tuned to evaluate the effect of matrix stiffness on cell viability and function. Specifically, dynamic matrix stiffening inhibited cell proliferation and spheroid formation, while softening the cell-laden hydrogels led to significant increase in spheroid sizes. Matrix stiffness also altered the expression of chemoresistance markers and responsiveness of cancer cells to gemcitabine treatment. markers and responsiveness of cancer cells to gemcitabine treatment.

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In this thesis is handled the problem of mushroom detection and recognition in natural environment. Convolutional neural networks are used. The beginning of this thesis is dedicated to the theory of neural networks. Further is solved the problem of object detection and classification. Using neural network trained for classification is solved also the task of localization. Results of trained CNNs are analised.

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The current anthropogenic practices of generating single-use waste streams in agriculture, forestry and manufacturing industries have created a host of environmental health problems. Humankind's reliance on non-renewable resources for the production of food and materials, and its current approach to product design and development, are clearly unsustainable. One mitigation strategy to reducing industrial and municipal solid waste, as well as environmental pollution, can be found in using white rot fungi to valorize our planet's most abundant and regenerative natural resource – plant biomass containing lignocellulose. From residual dry plant matter, white rot fungi can be employed through a solid-state fermentation process to produce a variety of edible, nutrient-dense saprotrophic mushrooms in addition to biologically augmented composite materials. Under the framework of the circular economy, agricultural and forestry byproducts with fibers containing lignin, cellulose and hemicellulose may be used as a feedstock for the production of both food and biomaterials – keeping plant biomass revolving through multiple cycles of use and reuse for a variety of product outputs that are biodegradable and help to sequester carbon. In this study, mushrooms were grown on a variety of lignocellulosic substrates derived from agricultural and forestry residues. Hemp-based substrates performed the best of the feedstocks with regard to mushroom yield and mycelium colonization time. Additionally, a number of mycelium composite products were designed and fabricated in this study using residual lignocellulosic plant biomass, including: insulation bricks, acoustical panels, and biodegradable planter pots. In particular, spent mushroom substrate containing hemp hurd and other agricultural and forestry residues showed significant potential in upcycling lignocellulosic plant biomass for the production of both mushrooms and mycelium materials. Regenerative design practices demonstrated how food and materials can be generated from the same lignocellulosic feedstock; therefore, reducing waste, circulating products and materials, and ultimately regenerating nature. / Master of Science / Environmental pollution and natural resource scarcity have encouraged exploration into using biologically based materials for the production of more ecologically friendly products. By valorizing the Earth's most abundant, renewable natural resource for the production of food and materials– dry plant matter containing lignocellulose – waste is reduced, carbon is stored, and materials can remain upcycled through multiple generations of production. Lignocellulosic residues – natural fibers containing the biopolymers lignin, cellulose and hemicellulose – have recently been given increased attention due to their ability to be aggregated and grown into low-cost, lightweight materials using white rot fungi. Mushroom farming has historically valorized lignocellulosic agricultural and forestry residues to grow an edible, nutrient-dense food crop. This thesis investigates the potential of various agricultural and forestry residues for the production of mushrooms and mycelium-based lignocellulosic composites. Furthermore, this study explores the utilization of spent mushroom substrate for the production of several mycelium-based composite products within the framework of the circular economy. Hemp-based substrates demonstrated significant potential in both mushroom production and mycelium composite fabrication, outperforming other agricultural residues in this study with regard to mushroom yield and speed of mycelial growth of Pleurotus ostreatus. More research into the tunable lignocellulosic substrate compositions will continue to help advance mushroom production and mycelium-based composite generation as environmentally friendly materials and production practices continue to gain interest.

Sufficient vitamin D is important for overall health. However, cutaneous production of vitamin D is limited by season and little vitamin D naturally occurs in food. Therefore, vitamin D supplementation is necessary. Vitamin D is available in pharmacies as vitamin D2 and vitamin D3, and can also be obtained by irradiating mushrooms to produce vitamin D2. Types of vitamin D supplementation were tested to compare their ability to increase vitamin D status and their effect on broad gene expression in human white blood cells. 2000 IU of vitamin D2, vitamin D3 or vitamin D2 in irradiated mushroom powder were given to subjects daily for twelve weeks. A placebo mushroom powder group was included in the second half of the study. To determine the effect of different supplementation on vitamin D status, whole blood was obtained weekly and serum was assayed for 25(OH)D2 and 25(OH)D3. Change in total 25(OH)D was determined from baseline to twelve weeks; 25(OH)D levels in the placebo mushroom powder group did not change significantly at 1.8 ± 1.8 ng/ml (9.6 ± 9.6%), the mushroom D2 group increased by 10.9 ± 10.2 ng/ml (53.2 ± 49.8%), the supplemental D2 group increased by 11.8 ± 7.4 ng/ml (60.2 ± 37.8%) and the supplemental D3 group increased by 21.7 ± 8.9 ng/ml (114.2 ± 46.8%). As expected, the total active form of vitamin D (1,25-dihydroxyvitamin D) showed no change in all groups because of its tight regulation. To determine the potential influence of vitamin D supplementation on differential gene expression in the immune system, white blood cells were isolated from whole blood samples taken before and after supplementation. RNA was extracted, and microarray assays were performed. Gene Set Enrichment Analysis was completed to determine strongly influenced pathways. However, due to the numerous variables between halves of the study, gene expression data was treated as separate studies. Even so, pathways involving RNA activation and degradation were significant between mushroom powder and mushroom D2 supplementation in both halves of the study, indicating the influence of compounds in mushrooms on RNA metabolism pathways. Supplemental vitamin D2 affected gene expression, though only two pathways showed significant change. Supplemental vitamin D3 was found to influence pathways involved in replication, transcription, and translation in both halves of the study. In conclusion, mushrooms powder, mushroom vitamin D2, supplemental vitamin D2, and supplemental vitamin D3 all influence differential gene expression in human white blood cells.

This thesis describes novel research using Portobello mushroom spores (PMS) as a biotemplate and reducing agent that may be used to produce nanocomposites and replicas with societal benefits. First, the use of PMS as a green eco-friendly bioreagent for the biosynthesis of Ag, Au and TiOx nanoparticles (NPs) and their hybrids is described. These have been characterised using SEM, TEM, XRD, FTIR, UV, SIMS, DLS and TGA (where a full list of acronyms is to be found on page iv). The spherical shape, location and mean diameter of the PMS-held (5-30nm) Ag and Au NPs were confirmed. PMS functional groups are comparable with those in plant sources and allow them to act as reducing/capping/stabilising agents. Second, the sequential biosynthesis of PMS-held Ca2+, CaCO3 and nanohydroxyapatite (nHAp) is described where surface nucleation and growth are facilitated by biotemplate surface hydrophilic polar groups (-OH and -COO-). XRD and HRTEM suggest that the nHAp so formed has lower crystallinity and greater directed growth in the (002) direction than commercial nHAp (e.g. Bio-Oss), making PMS-routes useful for the delivery of artificial bone (after subsequent PMS removal). Furthermore, PMS-held nHAp showed more rapid in-vitro mineralization in simulated body fluid (SBF). Third, it is shown that ascorbic acid (AA), haemoglobin (Hb) and insulin (In) can be loaded into/released by PMS through its cell walls faster than with synthetic hydrogels (PVA); the transdermal use of PMS nanocomposites is considered in the context of drug delivery nanotechnology, avoiding size-dependent toxicity. Fourth, it is shown by traditional diffusion and novel methods that the antimicrobial activity especially of Ag/PMS, but also TiOx/PMS and Ag-TiOx/PMS (but not Au/PMS) is good in the inhibition of the growth of E.coli and S.aureus bacteria. The highest activity of Ag/PMS is attributed to Agx+ release. Fifth, the synthesis of NPs/PMS and biomimetic TiOx/PMS has shown to lead to good photocatalysis for the removal of coloured organic pollutants (e.g. methyl orange) from water, with faster rates of removal in molecules/mg/s and turnover numbers (TON) (s-1) than commercial anatase-rutile TiO2 (P25) possibly as the texture of the titania replicas raises the number of multiple reflections of light.

Learning allows animals to adapt their behaviour to changes in the environment. In humans and other mammals, memories are stored in the hippocampus and cerebellum, whereas in insects, they are stored inside the mushroom bodies (MB). Here, MB-intrinsic Kenyon cells (KCs) form plastic synapses to MB output neurons (MBONs) that are modulated by the reinforcing action of dopaminergic neurons (DANs). Despite decades of research on the MB, the main neurotransmitter underlying the plastic KC → MBON synapse has remained a mystery. Here, I show that this synapse is cholinergic in the fruit fly Drosophila melanogaster. MBONs show fast excitatory responses to direct acetylcholine (ACh) application. KCs synthesise ACh-related proteins ChAT and VAChT. MBONs express and require nicotinic ACh receptors (nAChRs) to become fully activated by odour presentation. Lastly, artificial activation of KCs leads to MBON calcium responses that are blocked by nicotinic antagonists and genetic reduction of VAChT in KCs. Short neuropeptide F (sNPF) may play a role as a modulatory co-transmitter that can either excite or inhibit specific MBONs and DANs. The retrieval of memories is state-dependent and known to potentially change the original memory. Fruit flies need to be hungry to express appetitive memories. Hunger state depends on insulin signalling that activates the GABAergic MBON MVP2, while appetitive memory retrieval depends on decreased activity in M4/6 MBONs. Here, I show that optogenetic MVP2 activation acutely inhibits M4/6 odour responses, rendering MVP2 an inhibitory MBON interneuron. I also show that other MBONs are functionally connected to DANs, thus linking memory reinforcement and retrieval pathways in a way that enables the updating of the original memory. These findings show that associative memories in Drosophila are initially formed at cholinergic-MBON synapses, and can be retrieved and modified through an intricate KC-MBON-DAN network.