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Bladder Cancer is the tenth most common malignancy globally, and is the thirteenth most common cause of tumor associate morbidity. Bladder cancer is largely stratified into two categories: Non-Muscle Invasive Bladder Cancer (NMIBC) and Muscle Invasive Bladder Cancer (MIBC). NMIBC represents disease localized to the urinary bladder, and can be stratified into low and high-grade disease. MIBC represents an aggressive class of bladder cancer, with invasion into the underlying muscle layers of the bladder. MIBC can be classified as either non-metastatic MIBC, with disease localized to the bladder corpus, or metastatic MIBC, with disease spreading to sites beyond the bladder corpus. High grade NMIBC presents significant risk for progression to MIBC, and collectively both high grade NMIBC and MIBC bladder cancers demonstrate poor prognostic outcomes in clinical settings in terms of responses to therapy, recurrence risks, and overall survival. Hexaminolevulinate is a precursor of Protoporphyrin IX (PpIX) in the heme biosynthetic pathway. Hexaminolevulinate has been FDA approved under the trade name Cysview for diagnostic usage in blue light cystoscopies for fluorescence mediated visualization of disease along the bladder wall. I demonstrate that in addition to its diagnostic utility, Cysview and blue light irradiation can be utilized clinical as a potential therapeutic modality. I demonstrate the significant selective cytotoxicity of Cysview in combination with blue light against patient derived organoids (PDOs) from primary bladder cancers. My results determine that Cysview and blue light induce a rapid cell death program mediated by an influx in Reactive Oxygen Species (ROS) production, resulting in less than 5% viability within 24 hrs of treatment. This massive loss in viability is observed in low and high grade NMIBC, as well as MIBC derived PDOs with diverse mutational profiles. The results of this work demonstrate that PDOs are a significant platform for assessing therapy responses for correlation with the large patient population. Furthermore, the work identifies photodynamic therapy with Cysview and blue light irradiation as a putative therapeutic modality for localized bladder cancers, with the potential for significant improvement in patient outcomes. The identification and characterization of the therapeutic effects of Cysview come at a critical time during a global shortage of conventional therapeutics for localized bladder cancer, and presents a pathway for patients affected by these shortages. Progression in bladder cancer has been understood to be driven by processes governing subpopulation and cell state and lineage transformation. Previous studies identify phenotypic plasticity within a subset of bladder cancers and have correlated this phenomenon with an increased risk for disease progression from NMIBC to MIBC. In previous work, a subset of PDOs derived from luminal primary tumors demonstrated significant degrees of luminal to basal plasticity in vitro. In my analysis of these PDOs using transcriptomic and chromatin accessibility data, I identified a transcriptomic and epigenetic signature unique to plastic PDOs. Furthermore, I identified HNF1B, GRHL2, GATA6, and SNAI2 as putative regulators of luminal to basal plasticity in bladder cancer. Using these molecular profiles, I correlated the plasticity phenotype with reduction in overall survival using data from published bladder cancer patient cohorts. Finally, I developed a novel transcriptomic subtypes classification scheme and an accompanying R package to classify epithelial heterogeneity in bladder cancer, based on the transcriptomic subtypes I identified in bladder cancer PDOs.

New advances in computational modeling and instrumentation in the past decade has enabled the use of electromagnetic radiation for non-invasive monitoring of the physio-logical state of biological tissues. The near infrared (NIR) light having the wavelength range of 600 nm -1000 nm has been the main contender in these emerging molecular imaging modalities. Assessment of accurate pathological condition of the tissue under investigation relies on the contrast in the molecular images, where the endogenous contrast may not be sufficient in these scenarios. The fluorescence (exogenous) contrast agents have been deployed to overcome these difficulties, where the preferential uptake by the tumor vasculature leads to high contrast,making this modality one of the biggest contenders in small-animal and soft-tissue molecular imaging modalities. In Fluorescence diffuse optical spectroscopy/imaging, this exogenous drug is excited by NIR laser light causing the emission of the fluorescence light. The emitted fluorescence light is typically dependent on the life time and concentration of the exogenous drug coupled with physiology associated with the tissue under investigation. As there is an excitation and emission of the light,the underlying physics of the problem is described by a coupled diffusion equations. These coupled diffusion equations are typically solved by advanced numerical methods, which tend to be computationally demanding. In this work, analytical solutions for these coupled partial differential equations (PDEs) for the regular geometries for both time-domain and frequency-domain cases were developed. Till now, the existing literature has not dealt with all regular geometries and derived analytical solutions were only for couple of geometries. Here a universally acceptable generic solution was developed based on Green’s function approach that is applicable to any regular geometry. Using this, the analytical solutions for the regular geometries that is encountered in diffuse fluorescence spectroscopy/imaging were obtained. These solutions can play an important role in determining the bulk fluorescence properties of the tissue, which could act as good initial guesses for the advanced image reconstruction techniques and/or can also facilitate the calibration of experimental fluorescence data by removing biases and source-detector variations. In the second part of this work, the developed analytical models for regular geometries were validated through comparison with the established numerical models that are traditionally used in the diffuse fluorescence spectroscopy/imaging. This comparison not only validated the developed analytical models, but also showed that analytical models are capable of providing bulk fluorescence properties with at least one order of magnitude less computational cost compared to the highly optimized traditional numerical models.

Sediment records of two Italian subalpine lakes (Lake Garda and Lake Ledro) were analyzed in order to reconstruct their ecological evolution over the past several hundred years. A multi-proxy and multi-site approach was applied in order to disentangle the effects of local anthropogenic forcings, such as nutrients, and climate impacts on the two lakes and their catchments. Biological indicators (sub-fossil pigments, diatoms and Cladocera) were used to reconstruct changes in the aquatic food web and to define the lake reference conditions, while geochemical methods, i.e. wavelength-dispersive X-ray fluorescence spectroscopy (WD-XRF), were used to provide quantitative information on the different physical or chemical processes affecting both lake and catchment systems. Sub-fossil pigments and diatoms, together with their respective inferred TP values, suggested very stable oligotrophic conditions in both lakes until the 1960s. The period following was affected by nutrient enrichment, which led to a drastic shift in the phytoplanktonic community. The response of sub-fossil pigments and diatoms to major climatic anomalies such as the Medieval Climatic Anomaly (MCA) and the Little Ice Age (LIA) were not pronounced, and the taxonomic composition remained relatively stable. On the contrary, these proxies showed an indirect response to climate variability since the beginning of the nutrient enrichment phase in the 1960s. In Lake Garda, the winter temperature regulates the water column mixing, which in its turn controls the degree of nutrient fertilization of the entire water column, and the related phytoplankton growth. In Lake Ledro a rapid reorganization of planktonic diatoms was observed only during the temperature recovery after the LIA, while recent temperature effects are masked by the prevailing nutrient effects. In Lake Garda, Cladocera remains responded in quantitative and qualitative terms to climatic changes, whereas in Lake Ledro they appeared to be mainly affected by variations in hydrological regimes, i.e. flood events. Cladocera remains corroborated the nutrient enrichment after the 1960s in both lakes as inferred by diatoms and pigments. In Lake Garda, the geochemical data showed a pronounced shift in elemental composition since the mid-1900s, when major elements and lithogenic tracers started to decrease, while some elements related to redox conditions and other (contaminant) trace elements increased. The general trends since the mid-1900s agree with the biological records. However, some differences recorded in the two different basins of Lake Garda reflected the effects of local conditions, both related to hydrology and sedimentation patterns. Lake Ledro showed higher short-term variability for most elements, even though some features were comparable to Lake Garda. The geochemical record of Lake Ledro revealed a major influence of human-induced lake-level fluctuations and catchment properties. This paleolimnological study allows us to place temporally restricted limnological surveys into a longer-term secular perspective, which is highly valuable for the definition of lake reference conditions. Because the restoration targets are usually based on the lake reference conditions, this study highlighted also the necessity to pay particular attention to the lake-specific sensitivity patterns. The multi-proxy and multi-site approach showed that the lake conditions of large and deep lakes in northern Italy, such as Lake Garda, are mainly driven by nutrient enrichment and/or climate change. In contrast, smaller lakes with larger catchment areas, such as Lake Ledro, are seemingly more impacted by conditions and processes occurring in the drainage basin.

Photodynamic therapy (PDT) is a multimodality cancer treatment available for the palliation or eradication of systemic and cutaneous malignancies. In this thesis, the application of PDT is for the treatment of non-melanoma skin cancer (NMSC). While PDT has a well-documented track record, there are, at this time no significant indicators to suggest the superiority of one treatment regime over the next. The motivation for this work is to provide additional evidence pertaining to PDT treatment variables, and to assist in optimising PDT treatment regimes. One such variable is the treatment light dose. Determining the light dose more accurately would assist in optimising treatment schedules. Furthermore, choice of photosensitiser pro-drug type and application times still lack an evidence base. To address issues concerning treatment parameters, fluorescence spectroscopy – a valuable optical diagnostic technique – was used. Monitoring the in vivo PpIX fluorescence and photobleaching during PDT was employed to provide information pertaining to the progression of treatment. This was demonstrated by performing a clinical study at the Photobiology Unit, Ninewells Hospital and Medical School, Dundee. Two different photosensitiser pro-drugs – either 5-aminolaevulinic acid (ALA) or its methyl ester (MAL) – were investigated and based on the fluorescence and pain data recorded both may be equally suitable for topical PDT. During PDT, surface fluorescence is observed to diminish with time – due to photobleaching – although cancerous cells may continue to be destroyed deep down in the tissue. Therefore, it is difficult to ascertain what is happening at depth in the tumour. This raised the questions; How long after surface PpIX fluorescence has diminished is the PDT treatment still effective and to what depths below the surface is effective treatment provided? In order to address these important questions, a three-dimensional (3D) Monte Carlo radiation transfer (MCRT) model was used to compute the light dose and the ¹O₂ production within a tumour, and the PpIX fluorescence emission from the tumour. An implicit dosimetry approach based on a single parameter – fluorescence photobleaching – was used in order to determine the ¹O₂ generation, which is assumed to be related to tissue damage. Findings from our model recommended administering a larger treatment light dose, advocating an increase in the treatment time after surface PpIX fluorescence has diminished. This increase may ultimately assist in optimising PDT treatment regimes, particularly at depth within tumours.

Les toxines formeuses de pore (PFTs) sont des protéines exogènes responsables d’un grand nombre de maladies infectieuses qui perméabilisent les membranes cellulaires de leur hôte. La formation des pores ou l’introduction d’une enzyme dans le cytoplasme peut entrainer l’apparition de symptômes de maladies connues (l’anthrax, le botulisme) et, dans le pire des cas, la mort. Les mécanismes d’infection et de destruction des cellules infectées sont bien caractérisés. Toutefois, l’aspect dynamique des changements de conformation durant le processus de perméabilisation reste à découvrir pour la majorité des toxines formeuses de pore. Le but de cette thèse est d’étudier les mécanismes d’oligomérisation des PFTs, ainsi que la formation des pores à la membrane lipidique grâce à la spectroscopie de fluorescence. Nous avons choisi la toxine Cry1Aa, un bio pesticide produit par le bacille de Thuringe et qui a été rigoureusement caractérisé, en tant que modèle d’étude. La topologie de la Cry1Aa à l’état actif et inactif a pu être résolue grâce à l’utilisation d’une technique de spectroscopie de fluorescence, le FRET ou transfert d’énergie par résonance entre un fluorophore greffé au domaine formeur de pore (D1) et un accepteur non fluorescent (le DPA ou dipicrylamine) localisé dans la membrane et qui bouge selon le potentiel membranaire. Le courant électrique, ainsi que la fluorescence provenant de la bicouche lipidique membranaire horizontale ont été enregistrés simultanément. De cette manière, nous avons pu localiser toutes les boucles reliant les hélices de D1 avant et après la formation des pores. Dans la forme inactive de la toxine, toutes ces boucles se trouvent du côté interne de la bicouche lipidique, mais dans sa forme active l’épingle α3-α4 traverse du côté externe, alors que toutes les autres hélices demeurent du côté interne. Ces résultats suggèrent que α3-α4 forment le pore. Nous avons découvert que la toxine change significativement de conformation une fois qu’elle se trouve dans la bicouche lipidique, et que la Cry1Aa attaque la membrane lipidique de l’extérieur, mais en formant le pore de l’intérieur. Dans le but de caractériser la distribution de toxines à chaque extrémité de la bicouche, nous avons utilisé une technique de double FRET avec deux accepteurs ayant des vitesses de translocation différentes (le DPA et l’oxonol) dans la membrane lipidique. De cette manière, nous avons déterminé que la toxine était présente des deux côtés de la bicouche lipidique durant le processus de perméabilisation. La dynamique d’oligomérisation de la toxine dans une bicouche lipidique sans récepteurs a été étudiée avec une technique permettant le compte des sauts de fluorescence après le photoblanchiment des fluorophore liés aux sous unités composant un oligomère présent dans la bicouche lipidique supportée. Nous avons confirmé de cette manière que la protéine formait ultimement des tétramères, et que cet état résultait de la diffusion des monomères de toxine dans la bicouche et de leur assemblage subséquent. Enfin nous avons voulu étudier le « gating » de la colicine Ia, provenant de la bactérie E.Coli, dans le but d’observer les mouvements que font deux positions supposées traverser la bicouche lipidique selon le voltage imposé aux bornes de la bicouche. Nos résultats préliminaires nous permettent d’observer un mouvement partiel (et non total) de ces positions, tel que le suggèrent les études de conductances du canal. / Pore forming toxins (PFTs) are exogenous often pathogenic proteins that permeabilize the host membrane. Permeabilization or subsequent introduction of an enzyme leads to health disorders and sometimes death. Although the fundamental infection and destruction mechanisms are known, the underlying molecular basis and their link to the structural information remains undetermined for many pore forming toxins. The purpose of this thesis was to study the mechanisms of oligomerization on the membrane and pore formation of PFTs using fluorescence spectroscopy in planar lipid bilayer. We chose Cry1Aa as the most intensively studied member of Bacillus thuringiensis’s toxins. In order to probe the topology both in inactive and active congformation, we used Förster resonance energy transfer (FRET) between a fluorophore site-directedly attached to different positions in the pore forming domain (D1) of Cry1Aa toxin and an acceptor compound dipicrylamine (DPA) in the membrane, which moves in response to the membrane potential. Electrical current and fluorescence emission from planar lipid bilayers in a horizontal configuration were simultaneously recorded. We probed all loops between the seven α helices of D1. All of them were located on the inner leaflet of the bilayer prior to pore formation. In the active form, the α3-α4 hairpin were found to translocate back to the outer leaflet of the bilayer, whereas all other positions remained in the inner leaflet, suggesting that α3-α4 are the pore lining helices. The toxins undergo significant conformational changes once they enter the host membrane, and we found Cry1Aa to attack from the exterior but translocate to the interior. To estimate the distribution of the toxins on either side of the membrane, we used the double-FRET technique. Here, two different acceptors (DPA and oxonol) with different dynamics (time constants) allowed us to determine that approximately equal amounts of the toxin were present on either leaflet during the permeabilization process. We also studied the oligomerization mechanism of Cry1Aa toxins inserted into supported lipid bilayers using a single subunit counting technique based on the step-wise photodestruction (bleaching) of the attached fluorophores. This system allowed determining the number of subunits composing each oligomer. We found that oligomerization is a highly dynamic process which occurs after insertion into the bilayer by lateral diffusion. The final (likely the pore forming) entity of the toxin is tetrameric. Finally, we used the same FRET approach to investigate the gating process of two positions of the pore forming domain of colicin Ia, an antibiotic toxin produced by E. coli. These positions were suspected to translocate reversibly from the outer to the inner leaflet during the gating process. In preliminary results, we found that these positions are moving between the two leaflets of the bilayer during pore formation.

Ces travaux de thèse s'inscrivent dans le cadre d'un vaste projet qui vise à construire des membranes hybrides constituée d'un support solide nanoporeux et de protéines canal-ionique biologiques. Nous nous intéressons ici à un film polymère nanoporeux en polycarbonate et à la Gramicidine-A. La membrane ainsi réalisée est étudiée par des mesures expérimentales. Ce travail peut être divisé en deux parties. Dans la première, nous rapportons l'étude du confinement de la protéine canal ionique, au sein des nanopores du film « track-etched » en polycarbonate. Après imprégnation de gA, la membrane est étudiée par Spectroscopie de Fluorescence Confocale. Les premiers résultats expérimentaux particulièrement encourageants montrent que la gA est localisée dans les nanopores et non pas à la surface de la membrane. Dans la deuxième, les propriétés de transport ionique de la membrane hybride sont caractérisées par le biais de deux grandeurs : d'une part les coefficients de diffusion mesurés à partir d'une cellule et d'autre part les conductivités via la Spectroscopie d'Impédance Complexe (S.I.C). Les électrolytes aqueux étudiées sont : XCl(2) où X=Na, K, Mg et Ca à des concentrations comprises entre 5.10-3 à 1M. Une étude statistique approfondie des données obtenues par la méthode de la variance permet de déterminer les effets relatifs des différentes variables : nature et concentration du sel, présence de la Gramicidine A et traitement à l'éthanol de la membrane. Cette analyse révèle clairement que la présence de Gramicidine A au sein des nanopores de 15nm modifie de façon positive le transport ionique. Il est, par contre, difficile de conclure sur la nature sélective du transport ionique en présence de cette protéine. Ce travail de thèse ouvre un champ de recherche très prometteur dans le domaine de la nanofiltration. / This project of thesis is to build of a bio-inspired hybrid membrane made of a thin nanoporous polymer film in which a biological ionic channel is confined. Thus, this work may be divided in two parts. First, we report the confinement of the biological ionic channel, i.e. Gramicidin A, inside the nanopore of nanoporous thin film, i.e. a track etched polycarbonate film (Whatman NucleoporeTM). After impregnation with Gramicidine-A, the membrane is studied by means of confocal fluorescence spectroscopy. The results show the ionic channel is well located into the nanopores and not at the surface of the membrane. Secondly, ionic transport properties are measured by means of two experiments: on the one hand, ionic diffusion coefficients are measured using a cell and on the other hand, ionic dc conductivity is measured via Complex Impedance Spectroscopy (SIC). Various aqueous electrolytes (XCl(2) where X=Na,K, Mg et Ca) at different concentrations ranging from 5.10-3 à 1M are carried out. A statistical analysis of the data so-obtained allows to determine the relative effects of the different parameters: the nature and concentration of the electrolytes, the presence of Gramicidine A and the membrane pre-treatment with ethanol treatment. It is thus clearly pointed out that the presence of Gramicidine A inside the 15nm nanopores improves ion permeability. However, it is difficult to conclude about ionic selectivity of the hybrid membrane. Nevertheless, this work which is the first attempt ever to build such a bio-inspired system opens an extremely promising field of research in the domain of nanofiltration.

Le photochromisme se définit comme une transformation réversible d'une espèce entre deux états dont les spectres d'absorption sont différents. La réversibilité de la réaction peut se faire thermiquement et/ou photochimiquement. Cet effet suscite un grand intérêt pour l'utilisation de ses propriétés dans différentes applications (matériaux à transmission optique variable, mémoires optiques…). Parmi les nombreuses familles de photochromes organiques, les chromènes (benzopyranes et naphtopyranes) et les diaryléthènes, dont le photochromisme procède par électrocyclisation, occupent une place privilégiée. Ces dernières années, l'enjeu est d'utiliser ces unités photochromiques pour élaborer de nouveaux systèmes multichromophores. Dans ce projet, des réactions de couplage de la chimie organométallique ont permis de préfonctionnaliser des unités photochromiques monomères, servant de briques moléculaires pour l'architecture de structures complexes. Ainsi, deux séries de nouveaux systèmes photochromiques ont été élaborés autour de la triphénylamine en modifiant la jonction. Ces systèmes ont ensuite été étudiés en spectroscopie d'absorption électronique et de fluorescence, puis des études préliminaires sous irradiation ont été réalisées afin d'évaluer leur propriétés photochromiques. En parallèle, un diaryléthène substitué par un atome de silicium a été synthétisé en utilisant les micro-ondes, ouvrant de nouvelles perspectives pour des systèmes photochromiques bistables. / The photochromism is defined as a reversible transformation between two states of the molecule. The reversibility can occur thermally and/or photochemically. The intrinsic properties make the photochromic molecular system highly promising for various applications such as optical memory and molecular switches. From the large family of organic photochromic compounds, chromenes (benzopyrans and napthopyrans) and diarylethenes, both involving a ring closure/opening electrocyclic isomerisation, are largely used. In the last decades, a renewal of interest in the synthesis of supramolecular structures have been observed in view of application for optoelectronic devices. In this project, the original idea was to design and synthesize novel star-shaped like molecular architecture having triphenylamine as core block bridging several thermoreversible photochromic units in one molecule. In this context, new series of supramolecular assemblies were synthesized by introducing chromenes units on the core and changing the linkage. Then, their spectroscopic properties were studied by absorption and fluorescence spectroscopy before evaluating their photochromic properties. In a second way, a diarylethene substituted by a silicon atom was synthesized under micro-wave to open an interesting perspective in order to access a new photochromic family.

Single-stranded DNA binding proteins (SSB) bind to single-stranded DNA (ssDNA) that is generated by molecular machines such as helicases and polymerases. SSBs play crucial roles in DNA translation, replication and repair and their importance is demonstrated by their inclusion across all domains of life. The homotetrameric E. coli SSB and the heterotrimeric human RPA demonstrate how SSBs can vary structurally, but all fulfil their roles by employing oligonucleotide/oligosaccharide binding (OB) folds. Nucleofilaments of SSB proteins bound to ssDNA sequester the ssDNA strands, and in doing so protect exposed bases, keep the ssDNA in conformations favoured by other proteins that metabolise DNA and also recruit other proteins to bind to ssDNA. This thesis focuses on the SSB from the archaeon S. solfataricus (SsoSSB), and has found SsoSSB to be a monomer that binds cooperatively to ssDNA with a binding site size of 4-5 nucleotides. Tagging ssDNA and SsoSSB with fluorescent labels allowed the real time observation of single molecule interactions during the initial nucleation event and subsequent binding of an adjacent SsoSSB monomer. This was achieved by interpreting fluorescent traces that have recorded combinations of FRET, protein induced fluorescent enhancement (PIFE) and quenching events. This novel analysis gave precise measurements of the dynamics of the first and second monomers binding to ssDNA, which allowed affinity and cooperativity constants to be quantified for this important molecular process. SsoSSB was also found to have a similar affinity for RNA, demonstrating a promiscuity not found in other SSBs and suggesting further roles for SsoSSB in the cell - possibly exploiting its capacity to protect nucleic acids from degradation. The extreme temperatures that S. solfataricus experiences and the strength of the interaction with ssDNA and RNA make exploring the application of SsoSSB for industrial uses an interesting prospect; and its rare monomeric structure provides an opportunity to investigate the action of OB folds in a more isolated environment than in higher order structures.