Fungi are vital microbes present throughout the biosphere. Many species are essential decomposers in the ecosystem, breaking down organic materials and nourishing other lives. Moreover, some have directly influenced human civilization by providing beneficial products, such as edible mushrooms, brewer's yeast, baker's yeast, and antibiotics. However, it's important to note that this group of organisms can also have a "dark side". Each year, fungal pathogens cause approximately 150 million severe infections and 1.7 million deaths. The high rate of infection is compounded by the limited availability of antifungal drugs and the increasing prevalence of antifungal resistance. In response to the global burden of fungal diseases, the World Health Organization published a list of priority fungal pathogens in 2022 and highlighted strategies such as surveillance, sustainable research investments, and public health interventions to combat the increasing fungal threats. My PhD research has focused on surveillance and genomic analyses of several human fungal pathogens, particularly Candida auris. Candida auris is an emerging multidrug-resistant yeast that causes systemic infections with high mortality rates. While initially recognized as a nosocomial pathogen, our genomic analyses of strains isolated from clinical environments, tropical wetlands, fruit surfaces, and dog ears revealed potential transmission routes between diverse environments and patients, including a potential driver for the prevalence of antifungal resistance. Furthermore, our research indicated limited genetic exchange within and between lineages of Candida auris. Through genome-wide association analyses of global Candida auris strains, several known and novel genomic variants were identified associated with susceptibility to azoles, echinocandins, and amphotericin B. Overall, our studies underscore the importance of continuous surveillance to understand potential routes of Candida auris transmission and the urgent need for innovative approaches to treat multidrug-resistant Candida auris infections. / Thesis / Doctor of Philosophy (PhD)

Amphiphilic kanamycin is one of the promising class of compounds for the treatment of fungal infections in plants and animal. Factor that lead to the restricting of compounds for commercialization includes, the higher cost of production and poor stability of the compound. However, the new lead, identified from the synthesis and biological testing, can be synthesized on a large scale with a cost comparable to commercial antifungals. The newly reported lead is stable at the acidic and basic conditions. Additionally, this compound has an excellent activity towards Candida auris, a multidrug-resistant superbug. Heart disease is the leading cause of death in the United States most of which are caused by cardiac ischemia and arrhythmias. Abnormal opening of Cx43 hemichannel can damage the heart muscles and lead to these conditions. A compound which can selectively inhibit the opening of Cx43 hemichannel may pave the way to reducing the mortality rate of heart disease. A selective inhibitor towards Cx43 hemichannel is explored from the synthesis and biological testing of kanamycin derivatives. The synthesis of the new inhibitor is scalable and cost-effective.

Clinical genomics/metagenomics is a rapidly developing field and it makes genomic, transcriptomic, and epigenomic evaluations of clinically relevant samples possible due to decreasing sequencing costs and large volumes of sequence datasets. Applications of comprehensive protocols for clinical metagenomic analysis is rapidly moving from the research laboratories to the clinical laboratories in healthcare settings. It has not only improved medical interventions but also continue to contribute to precision treatments. In this dissertation, I am going to discuss (i) the applications of clinical genomics/metagenomics protocols in several clinical cases of infections and (ii) the application of comparative genomics in Mycobacterium riyadhense clinical isolates which provide insights into ancestry and adaptive evolution in MTBC (Mycobacterium tuberculosis complex); The research questions are systematically addressed in four chapters. Briefly, Chapter 1 provides a brief introduction of conventional clinical microbiology and clinical metagenomics and a research summary of the thesis; Chapter 2 presents an analysis of Mycobacterium riyadhense from an evolutionary genomics point of view, followed by functional genomics experiments to look for clues obtained from the comparative genome analysis of M. riyadhense and other mycobacteria including members of the MTBC. The third chapter describes an imported case of Mycobacterium leprae found in Riyadh, Saudi Arabia, revealed by metagenomic sequencing and bioinformatic analysis, which was challenging for the clinicians to treat due to lack of timely diagnosis and occurrence of drug resistance during the course of treatment. The fourth chapter describes how applications of NGS facilitate rapid pathogen discovery in an imported case of Naegleria fowleri from the Cerebrospinal fluid (CSF) of a resident in KSA with recent travel history to Pakistan. The fifth chapter presents how we have monitored an ongoing outbreak by drug-resistant strains of the human pathogenic yeast Candida auris in Saudi Arabia from King Faisal Medical City, Riyadh, Saudi Arabia. In this thesis, I have shown several applications of NGS and bioinformatic analysis protocols in the clinical genomics/metagenomics fields. I believe some of the main clinical applications of NGS will lead to the adoption of these methodologies in clinical settings in Saudi Arabia in the forthcoming future.

Several Fungal species have the potential to cause a broad spectrum of diseases in humans, ranging from mild superficial to disseminated invasive infections that involve the bloodstream and vital organs. Invasive fungal infections are severe, life-threatening diseases that result in the deaths of 1.5 million patients each year. The most common fungal species responsible for the majority of invasive fungal infections include Candida, Cryptococcus, and Aspergillus. The current treatment options for invasive fungal infections are restricted to three classes of antifungals: Azoles, polyenes, and echinocandins. The emergence of new fungal species, especially C. auris, marked by high resistance profiles and increased mortality rates (30-60%), has further exacerbated the limitations in its therapeutic options. This emphasizes the urgent need for effective alternatives to combat these deadly pathogens. C. auris isolates exhibited high resistance capability especially against azole (fluconazole) and polyene (amphotericin B) antifungals. Here, we utilized the combinatorial strategy to screen ~3400 FDA-approved drugs and clinical compounds to identify hits that can enhance/restore the antifungal activity of azoles and amphotericin B against resistant C. auris. The HIV protease inhibitors (lopinavir and ritonavir) were identified as potent enhancers to the antifungal activity of azole drugs (fluconazole, voriconazole and itraconazole). We confirmed that lopinavir and ritonavir have the capability to interfere with fungal efflux pump machinery. The in vivo efficacy of the combination of azole antifungals and HIV protease inhibitors was also evaluated to discover the best combination of itraconazole, lopinavir and ritonavir. Three drugs (lansoprazole, rolapitant and idebenone) were identified to effectively enhance the antifungal effects of amphotericin B and overcome its resistance in C. auris. Furthermore, the synergistic interactions of these combinations were applied on other medically important Candida, Cryptococcus, and Aspergillus species. In a comprehensive mechanistic study, we discovered that lansoprazole interferes with an essential target in the fungal mitochondrial cytochrome system, cytochrome bc1. This interference induces oxidative stress in fungal cells and subsequently enhances the antifungal activity of amphotericin B. For rolapitant, a transcriptomic analysis along with ATP luminescence assays confirmed that rolapitant at sub-inhibitory concentrations significantly interferes with ATP production in C. auris. For idebenone, checkerboard assays confirmed the synergistic interactions between amphotericin B and idebenone against a diversity of medically important fungal species. This combination exhibited a rapid fungicidal activity within 4 hours. Additionally, the cytotoxicity of this combination was assessed in a cell line model of kidney cells. Based on the potent in vitro and in vivo synergistic relationships observed for the identified combinations, it can be concluded that our approach offers a new hope to restore the antifungal activity of the existing antifungal drugs, even against resistant fungal infections. Additionally, it provides valuable insights into identifying novel targets to overcome resistance in multidrug-resistant fungal pathogens. / Doctor of Philosophy / Fungi comprise a diverse group of organisms that interact with humans in many good and bad aspects. Candida auris, a recently identified fungus, poses a significant threat to patients with weak immune systems. Infections with C. auris can be associated with mortality rates of up to 60%. Notably, this fungus is characterized by its powerful spreading capability and displays extraordinary resistance to antifungal agents, rendering many existing antifungal drugs ineffective. As a result, there is an unmet need to find efficient treatments for such deadly fungal infections. In this study, several drugs were identified with the potential to restore the activity of traditional antifungal drugs. The study identified four promising drugs (lopinavir, lansoprazole, rolapitant, and idebenone) with the potential to enhance the activity of the antifungal drugs against C. auris. lopinavir showed great potential to enhance the activity of azole antifungals, including fluconazole, voriconazole, and itraconazole. Furthermore, three other drugs (lansoprazole, rolapitant, and idebenone) were identified for their potential to enhance the activity of amphotericin B, which is considered a last-line antifungal therapy. We clarified the mechanisms by which these drugs could restore the activity of antifungal agents. Finally, we confirmed the effectiveness of these combinations in animal models, providing valuable insights into their potential for clinical applications. In summary, our research has opened promising avenues to overcome resistance and develop new treatments for hard-to-treat fungal infections.

Candida auris, a recently emergent fungal pathogen, has caused invasive infections in health care settings worldwide. Mortality rates approach 60% and hospital spread poses a public health threat. Compared to other Candida spp., C. auris avoids triggering the antifungal activity of neutrophils, innate immune cells that are critical for responding to many invasive fungal infections, including candidiasis. However, the mechanism underpinning this immune evasion has been largely unknown. Here, we show that C. auris cell wall mannosylation contributes to the evasion of neutrophils ex vivo and in a zebrafish infection model. Genetic disruption of mannosylation pathways (PMR1 and VAN1) diminishes the outer cell wall mannan, unmasks immunostimulatory components, and promotes neutrophil engagement, phagocytosis, and killing. Upon examination of these pathways in other Candida spp. (Candida albicans and Candida glabrata), we did not find an impact on neutrophil interactions. These studies show how C. auris mannosylation contributes to neutrophil evasion though pathways distinct from other common Candida spp. The findings shed light on innate immune evasion for this emerging pathogen. IMPORTANCE The emerging fungal pathogen Candida auris presents a global public health threat. Therapeutic options are often limited for this frequently drug-resistant pathogen, and mortality rates for invasive disease are high. Previous study has demonstrated that neutrophils, leukocytes critical for the antifungal host defense, do not efficiently recognize and kill C. auris. Here, we show how the outer cell wall of C. auris promotes immune evasion. Disruption of this mannan polysaccharide layer renders C. auris susceptible to neutrophil killing ex vivo and in a zebrafish model of invasive candidiasis. The role of these mannosylation pathways for neutrophil evasion appears divergent from other common Candida species.

<p>In the past few decades, invasive mycosis has become a growing threat to global health, afflicting millions of people and claiming the lives of more than 1.5 million patients every year. Moreover, the economic burden of mycotic infections has become increasingly exhausting especially with the recent increases in the number of the high-risk population, the immunocompromised individuals. In the USA, the cost incurred by mycotic infections was estimated to be of more than $7.2 billion only in 2017. Of particular concern, <i>Candida</i> species are the most common fungal pathogens that infect humans, resulting in considerable morbidities and mortality rates that often exceed 50%. Unfortunately, the antifungal drug discovery is currently unable to keep pace with the urgent demand for more effective therapeutic options. Further complicating the situation is the recent emergence of multidrug-resistant species such as <i>Candida</i> <i>auris</i>, triggering outbreaks of deadly Candidemia across the globe. Given the risks inherent to the traditional de-novo drug discovery, combinatorial therapeutics stands out as a promising tool to hamper drug resistance and extend the clinical utility of the existing drugs. In this study, we assembled and screened ~3147 FDA-approved drugs and clinical molecules against fluconazole-resistant <i>C. albicans</i> and <i>C. auris</i> isolates, for the aim of restoring the antifungal activity of azole antifungals against drug-resistant <i>Candida </i>species. The screen revealed five promising hits: pitavastatin (antihyperlipidemic), ospemifene (estrogen receptor modulator), sulfa antibacterial drugs, lopinavir (antiviral), and aprepitant (antiemetic).</p> <p>All identified hits demonstrated variable azole chemosensitizing activities depending on the tested <i>Candida</i> species and the azole drug. Pitavastatin displayed broad-spectrum synergistic interactions with both fluconazole and voriconazole against isolates of <i>C. albicans</i>, <i>C. glabrata</i>, and <i>C. auris</i>. Ospemifene was able to interact synergistically with itraconazole against multiple fungal isolates including <i>Candida</i>, <i>Cryptococcus</i>, and <i>Aspergillus</i> species. Sulfa drugs displayed potent synergistic activities with different azoles against <i>C. albicans</i>, however, a limited efficacy was observed against efflux-hyperactive isolates such as <i>C. auris</i>. On the other hand, both lopinavir and aprepitant exerted potent and broad-spectrum synergistic activities with itraconazole and were effective against multiple <i>Candida</i> species including <i>C. albicans</i>, <i>C. auris</i>, <i>C. glabrata</i>, <i>C. krusie</i>, <i>C. tropicalis</i>, and <i>C. parapsilosis</i>. Furthermore, using <i>Caenorhabditis elegans</i> as an infection model, all drug combinations significantly reduced the fungal burden in the infected nematodes and significantly prolonged their survival as compared to single-drug treatments. Multiple phenotypic and molecular assays indicted that the identified hit compounds use distinct mechanisms to enhance the antifungal activity of azole drugs. These mechanisms include efflux pump inhibition, interference with the folate biosynthesis and disturbance of iron homeostasis. Taken together, this study reveals novel and potent azole chemosensitizing agents effective against multiple azole-resistant isolates and opens the door for more investigations to assess their clinical potential in human medicine as promising antifungal adjuvants.</p>

The continuous emergence of drug-resistance pathogens is a global concern. As a result, substantial effort is being expended to develop new therapeutics and mechanisms for controlling microbial growth to avoid entering a "post-antibiotic" era in which commonly used antibiotics are no longer effective in treating infections. In this work, we investigate the efficacy and application of ceragenins as non-peptide mimics of antimicrobial peptides (AMPs). First, this work examines the susceptibility of drug-resistant Gram-negative bacteria. The susceptibility of colistin-resistant clinical isolates of Klebsiella pneumoniae to ceragenins and AMPs suggests that there is little to no cross-resistance between colistin and ceragenins/AMPs. Furthermore, Lipid A modifications are found in bacteria with modest changes in susceptibility to ceragenins and with high levels of resistance to colistin. Next, we investigated the potential for cross resistance between chlorhexidine, colistin, AMPs and ceragenins as repeated exposure of bacteria to chlorhexidine might result in cross resistance with colistin, AMPs or ceragenins. Furthermore, a proteomics study on the chlorhexidine-resistant strains showed that chlorhexidine resistance is associated with upregulation of proteins involved in the assembly of LPS for outer membrane biogenesis and virulence factors in Pseudomonas aeruginosa.Second, this dissertation describes the antifungal activity of ceragenins against an emerging multidrug-resistant fungus, Candida auris. We found that lead ceragenins displayed activities comparable to known antifungal agents against C. auris isolates. We also found that fungal cell morphology was altered in response to ceragenin treatment, that ceragenins exhibited activity against sessile organisms in biofilms, and that gel and cream formulations including CSA-44 and CSA-131 resulted in a significant log reduction against established fungal infections in ex vivo mucosal tissues. Finally, a hydrogel film containing CSA-131 was generated on endotracheal tubes (ETTs). ETTs provide an abiotic surface on which bacteria and fungi form biofilms that cause serious infections. In this study, the eluting ceragenin prevented fungal and bacterial colonization of coated ETTs for extended periods while uncoated tubes were colonized by bacteria and fungi. Coated tubes were well tolerated in intubated pigs. The ability of ceragenins to eradicate established biofilms make them attractive potential therapeutics for persistent infections in the lung, including those associated with cystic fibrosis. In ex vivo studies, we initially found that this ceragenin, at concentrations necessary to eradicate established biofilms, also causes loss of cilia function. However, by formulating CSA-131 in poloxamer micelles, cilia damage was eliminated and antimicrobial activity was unaffected. These findings suggest that CSA-131, formulated in micelles, may act as a potential therapeutic for polymicrobial and biofilm-related infections in the lung and trachea.

[ES] La presente tesis doctoral titulada "New nanostructured suports with signal amplification features for the detection of molecules and biomolecules of interest" se centra en el diseño y preparación de nuevos materiales híbridos orgánicos-inorgánicos constituidos por puertas moleculares soportadas sobre alúmina mesoporosa con el objetivo de desarrollar nuevos sistemas sensores con aplicaciones potenciales en el campo de la diagnosis y del control alimentario. En el primer capítulo de la tesis se introducen los conceptos en los que están basados los estudios realizados y los materiales preparados. A continuación, en el segundo capítulo se describen los objetivos generales de la tesis que serán abordados en los siguientes apartados. En el tercer capítulo se presenta el diseño y optimización de un nanodispositivo para la detección de la bacteria Mycoplasma fermentans. En primer lugar, los poros de una placa de alúmina mesoporosa se cargan con un indicador fluorescente (rodamina B). Seguidamente, la superficie es funcionalizada con una secuencia de ADN complementaria a una región altamente conservada de la subunidad ribosomal 16S de la bacteria Mycoplasma fermentans. El impedimento estérico generado por las secuencias de ADN ancladas al exterior de los poros impide la salida del indicador encapsulado. Únicamente en presencia de DNA de la bacteria Mycoplasma fermentans, se produce la apertura de los poros permitiéndose la difusión de la carga (rodamina B) que es posteriormente medida mediante espectroscopía de fluorescencia. En el capítulo cuatro se diseña de un nanodispositivo capaz de detectar de forma rápida, sensible y selectiva la bacteria Staphylococcus aureus. Para la preparación del material sensor, un soporte de alúmina mesoporosa es, en primer lugar, cargado con el indicador fluorescente rodamina B. A continuación, los poros del soporte son tapados mediante el anclaje de un aptámero que reconoce de forma específica la bacteria. Solamente en presencia de Staphylococcus aureus se produce la liberación del indicador encapsulado, que es posteriormente medido mediante espectroscopía de fluorescencia. Además, la respuesta obtenida es específica para Staphylococcus aureus. Este sistema ha sido ensayado en muestras reales. En el sexto capítulo, diseña un nanodispositivo híbrido orgánico-inorgánico consistente en un material de alúmina mesoporosa cubierto con una secuencia de ADN específica para la detección de ADN del hongo Pneumocystis jirovecii. En este caso, el soporte de alúmina cargado con rodamina B se recubre con una secuencia de ADN específica para el reconocimiento de este hongo. En presencia del organismo, la horquilla hibrida con el ADN del hongo, lo que resulta en una conformación triplex con elevada afinidad y estabilidad que induce, al mismo tiempo, el desplazamiento de este complejo de la superficie. Como consecuencia de este reconocimiento la carga se libera y es cuantificada mediante espectroscopía de fluorescencia. El sistema ha sido satisfactoriamente validado. En el séptimo capítulo, se diseña un sistema sensor con la capacidad de detectar gluten de forma rápida y sencilla en extractos de alimentos procesados y no procesados. Para ello, un soporte de alúmina mesoporosa se carga con rodamina B y los poros se recubren con un aptámero específicamente diseñado para la detección de la proteína gliadina, que constituye el 50 % del total del clúster de elementos que forman el gluten. La elevada afinidad y especificidad entre el aptámero y la proteína en cuestión hacen que en presencia de ésta se produzca un desplazamiento de la puerta molecular que permite la difusión del colorante encapsulado que es finalmente monitorizado mediante espectroscopía de fluorescencia. Finalmente, en el capítulo octavo se discuten de forma conjunta los resultados obtenidos en los capítulos anteriores y la potencial aplicación de los sistemas desarrollados en el actual sistem / [CA] La present tesi doctoral, titulada "New nanostructured supports with signal amplification features for the detection of molecules and biomolecules of interest", es centra en el disseny i preparació de nous materials híbrids orgànics-inorgànics constituïts per portes moleculars suportades sobre alúmina mesoporosa amb l'objectiu de desenvolupar nous sistemes sensors amb potencials aplicacions en el camp de la diagnosi i del control alimentari. En el primer capítol de la tesi s'introdueixen els conceptes en què estan basats els estudis realitzats i els materials preparats. A continuació, en el segon capítol es descriuen els objectius generals de la tesi que seran abordats en els següents apartats. En el tercer capítol es presenta el disseny i optimització d'un nanodispositiu per a la detecció de la bactèria Mycoplasma fermentans. Primerament, els porus d'una placa d'alúmina mesoporosa són carregats amb un indicador fluorescent (rodamina B). Seguidament, la superfície és funcionalitzada amb una seqüència d'ADN complementaria a una regió altament conservada de la subunitat ribosomal 16S de la bactèria Mycoplasma fermentans. L'impediment estèric generat per les seqüències d'ADN ancorades a l'exterior dels porus impedeix l'alliberament de l'indicador encapsulat. Únicament en presencia d'ADN de la bactèria Mycoplasma fermentans, es produeix l'obertura dels porus permetent la difusió de la càrrega (rodamina B) que és posteriorment mesurada mitjançant fluorescència. En el capítol quatre es dissenya un nanodispositiu capaç de detectar de forma ràpida, sensible i selectiva la bactèria Staphylococcus aureus. Per a la preparació del material sensor, el suport d'alúmina mesoporosa és, primerament, carregat amb l'indicador fluorescent rodamina B. A continuació, els porus del suport són tapats mitjançant l'ancoratge d'un aptàmer que reconeix de forma específica a la bactèria. Solament en presència de Staphylococcus aureus es produeix l'alliberament de l'indicador encapsulat, que és posteriorment mesurat mitjançant espectroscòpia de fluorescència. A més a més, la resposta obtinguda és específica per Staphylococcus aureus. Aquest sistema ha sigut validat amb mostres reals de pacients. En el sisè capítol, es dissenya un nanodispositiu híbrid orgànic-inorgànic consistent en un material d'alúmina mesoporosa cobert amb una seqüència d'ADN específica per a la detecció de l'ADN del fong Pneumocystis jirovecii. En aquest cas, el suport d'alúmina carregat amb l'indicador fluorescent rodamina B és recobert amb una seqüència d'ADN específica per al reconeixement d'aquest fong. En presència de l'organisme, la forquilla hibrida amb l'ADN del fong, resultant en una conformació triplex amb elevada afinitat i estabilitat, que indueix, al mateix temps, el desplaçament d'aquest complex de la superfície. Com a conseqüència d'aquest reconeixement la càrrega és alliberada i quantificada mitjançant espectroscòpia de fluorescència. El sistema ha sigut validat com a mètode diagnòstic mitjançant l'anàlisi de mostres reals de pacients. En el seté capítol, es dissenya un sistema sensor amb la capacitat de detectar gluten de forma ràpida i senzilla en extractes d'aliments processats i no processats. Per a això, un suport d'alúmina mesoporosa es carrega amb indicador fluorescent rodamina B i posteriorment és recobert amb un aptàmer específicament dissenyat per a la detecció de la proteïna gliadina, que constitueix el 50 % del total del clúster d'elements que formen el gluten. L'elevada afinitat i especificitat entre l'aptàmer i la proteïna en qüestió fa que en presència d'aquesta es produesca un desplaçament de la porta molecular que permet la difusió de la càrrega encapsulada i que serà finalment monitoritzada mitjançant espectroscòpia de fluorescència. Finalment, en el capítol vuité es discuteixen de manera conjunta els result / [EN] The PhD thesis hereby presented and entitled "New nanostructured supports with signal amplification features for the detection of molecules and biomolecules of interest", focuses in the design and preparation of new hybrid organic-inorganic materials constituted by molecular gates supported over mesoporous alumina with the aim of developing new sensor probes of potential applications in the fields of diagnosis and food control. In the first chapter, the concepts in which studies and prepared materials are based, are introduced. Next, the second chapter describes the general objectives of this thesis, which will be approached in the following sections. In the third chapter, it is presented in detail the design and optimization process of a nanodevice applied for the detection of Mycoplasma fermentans bacterium. First of all, mesoporous alumina porous films are charged with a fluorescent indicator (rhodamine B). Then, the surface is functionalized with a DNA sequence complementary to a highly conserved region of the 16S ribosomal subunit of the bacterium Mycoplasma fermentans. Steric hindrance generated by DNA sequences on the surface inhibits the release of the encapsulated indicator. Only in the presence of bacterium Mycoplasma fermentans DNA, molecular gates open, allowing payload diffusion to the solution, which is measured by fluorescence spectroscopy. In chapter four, it is carried out the design and optimization of a nanodevice able to detect Staphylococcus aureus bacterium in a fast, sensitive and selective way. For the sensor preparation, alumina mesoporous support is, first, loaded with the rhodamine B fluorescent dye. Then, the mesoporous are blocked through the attachment of an aptamer that recognises specifically this bacterium. Exclusively in the presence of Staphylococcus aureus it is accomplished the release of the encapsulated dye, which is later monitored by fluorescence spectroscopy. The response obtained is specific for Staphylococcus aureus. This system has been validated in real samples. In the sixth chapter, it is detailed the design and optimization process of a hybrid organic-inorganic nanodevice based on a capped mesoporous alumina material for the detection of Pneumocystis jirovecii fungus DNA. In this case, the mesoporous alumina support is loaded with a fluorescent dye and decorated with a specific oligonucleotide sequence designed for the recognition of Pneumocystis fungus. In the presence of the target organism, the fork-like oligonucleotide hybridises with the DNA of the fungus, which results in the adoption of a triplex conformation with high affinity and stability that induces, at the same time, the displacement of this complex from the surface. Consequently, the payload diffused to the solution is quantified through fluorescence spectroscopy. The system has been successfully validated. In the seventh chapter, it was developed a sensor system for gluten detection, in a quick and easy way, in processed and non-processed food extracts. For this, a mesoporous alumina support is loaded with the fluorescent dye rhodamine B, and later was functionalized with an aptamer specifically designed for the detection of gliadin, a protein that constitutes 50 % of average cluster elements that forms gluten. The protein-aptamer high affinity and specificity induce the displacement of the capping aptamer and cargo delivery, which is monitored through fluorescence spectroscopy. Finally, in the eighth chapter, the results obtained in the previous chapters and the potential application of the systems developed as health and food control system are discussed. / We thank the Spanish Government projects MAT2015-64139-C4-1-R, AGL2015-70235-C2-2-R, and TEC2015-71324-R (MINECO/FEDER, UE), the Generalitat Valenciana (project PROMETEOII/2014/047), the Catalan authority (project AGAUR 2014SGR1344), and ICREA under the 2014 ICREA Academia Award for support. This study was supported by the Spanish Government projects RTI2018-100910-B-C41 and SAF2017-82251-R (MCUI/AEI/FEDER, UE), the Generalitat Valenciana (project PROMETEO/2018/024), the Universitat Politècnica de València−Instituto de Investigación Sanitaria La Fe (B02-MIRSA project), CIBER-BBN (NANOPATH and valorization project CANDI-EYE) and co-financed by the EU through the Valencian Community ERDF PO 2014-2020. This research was funded by the Spanish Government, projects RTI2018-100910-B-C41 (MCUI/AEI/FEDER, UE) and CTQ2017-84415-R / Pla Blasco, L. (2021). New nanostructured supports with signal amplification features for the detection of molecules and biomolecules of interest [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/166500 / TESIS

<p>  </p> <p>Fungal pathogens are an increasing threat to humans, plants, and animals worldwide. Death and disease caused by fungal pathogens results in the loss of over 1.5 million lives, 12 million tons of crops, and even entire species every year. <em>Candida </em>species are the leading cause of invasive fungal species lead by <em>Candida albicans, </em>and <em>Candida glabrata </em>in second. <em>Candida glabrata </em>intrinsically has a low susceptibility to azole treatment, and multidrug resistant isolates are becoming more common. Additionally, new emerging <em>Candida </em>species have been found, and most clinical isolates are resistant to one or more drugs. There is a critical need to better understand drug resistance and pathogenesis to generate new therapies. </p> <p>Drug resistance can be caused by several different genetic factors, but until recently epigenetic factors have been frequently overlooked. Epigenetic research has revolutionized the treatment and detection of many cancers. And now, early research has shown epigenetic mechanisms play a role in drug resistance and pathogenesis in fungal species. Limited resources exist to combat fungal infections and understanding the epigenetic mechanisms that contribute to drug resistance and pathogenicity will provide new drug targets for future treatment.</p> <p>Previous publications from the Briggs’ lab showed Set1-mediated histone H3K4 methylation was necessary for proper ergosterol homeostasis and Brefeldin A resistance. One of the three classes of antifungals, azoles, target the ergosterol pathway. The ergosterol connection resulted into this thesis project, investigating the role of Set1-mediated histone H3K4 methylation in drug resistance and pathogenicity in <em>Saccharomyces cerevisiae, Candida glabrata, Candida albicans, </em>and <em>Candida auris. </em>This research was the first to characterize the Set1 complex in <em>C. glabrata </em>and show it is the sole histone H3K4 methyltransferase in <em>C. glabrata </em>and <em>C. auris. </em>Additionally, it shows loss of <em>SET1 </em>in <em>C. glabrata </em>and <em>C. auris </em>reduces pathogenicity and alters drug efficacy. Interestingly, although the loss of <em>SET1</em> seems to cause a similar pathogenic defect in all three <em>Candida </em>species, the role Set1 plays in drug efficacy including which drug and severity varies amongst species and isolates. Altogether, this research project provides new possible drug targets for fungal treatment and knowledge added to the scientific community on the role of epigenetics in fungal pathogens. </p>