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<p>Atherosclerosis is a chronic inflammatory disease of the arterial wall and is the primary cause of coronary artery disease, the most common cause of death in western societies. Risk factors for cardiovascular disease include dyslipidemia, diabetes, smoking, and obesity. These risk factors have also been shown to promote vascular endoplasmic reticulum (ER) stress; a cellular response characterized by the accumulation of misfolded proteins in the ER. Thickening and decreased stability of arterial plaque can lead to thrombosis and subsequent clinical complications of myocardial infarction and stroke. However, the exact mechanisms that lead to the development of atherosclerosis remain unclear. Here we show that inhibition, as well as a deficiency of glycogen synthase kinase (GSK)-3α, can protect against accelerated atherosclerosis in a low-density lipoprotein receptor (LDLR) knockout mouse model. Compared to LDLR^-/- controls, mice deficient in GSK-3α showed a decrease in lesion volume in the aortic root as well as protection against diet-induced hepatic steatosis. In addition, necrotic core volume was significantly reduced in LDLR^-/-GSK-3α^-/- mice compared to controls, a characteristic indicative of advanced plaque formation. Furthermore, hepatic and vascular ER stress levels were unaffected by the deletion of GSK-3α, a result that is consistent with the hypothesis that GSK-3α functions downstream of ER stress. Macrophages isolated from GSK-3α deficient mice had a reduction in unesterified cholesterol accumulation as well as a significant increase in the expression of the anti-inflammatory cytokine IL-10. Finally, BMT experiments showed a significant decrease in plaque size in the aortic sinus of LDLR^-/-GSK-3α^+/+ mice transplanted with GSK-3α deficient bone marrow. These results demonstrate a possible link between ER stress-induced activation of GSK-3α and the downstream effects leading to atherogenic initiation and progression.</p> / Master of Science (MSc)

Cigarette smoke contributes to 90% of lung cancer cases and 80% of COPD cases. These concerns loom large as lung cancer represents 13% of all cancer deaths and estimates report by 2020 COPD will be the third leading cause of death in the world. The master regulator of the ER stress response, IRE-1, in the context of cigarette smoke exposure lacks study. Interestingly, its downstream pathways are activated. In fact, the 2014 Surgeon General’s report on the health consequences of smoking highlighted the endoplasmic reticulum (ER) stress response as a potential mechanism leading to the development of lung cancer and Chronic Obstructive Pulmonary Disorder (COPD). Following acute cigarette smoke exposure, mouse lung homogenates exhibited increased levels of XBP-1 along with downstream mediators of IRE-1 activation— GRP-78 and CHOP. Specifically observing macrophages, an important immune cell and source of acute inflammation, cigarette smoke induced activation of IRE-1/XBP-1 pathway through splicing of XBP-1 mRNA. However, upon assaying for pro-inflammatory cytokines we were unable to determine that cigarette smoke directly caused inflammation in vitro. Furthermore, cigarette smoke inhibited the activation of M2 macrophages, an anti-inflammatory and tissue healing subset seen through CCL18 inhibition. A majority of M2 and M1 macrophage markers were decreased from IRE-1/XBP-1 inhibition. This suggests a different phenotype than classical M1 or M2 polarization being induced by cigarette smoke. In addition, it suggests the IRE-1/XBP-1 pathway having a robust role in controlling gene expression and balance of cellular proteomics. / Thesis / Master of Science (MSc) / Cigarette smoke exposure damages the lungs and over time places the user at risk for increased infections, progressive decreases in lung function and cancer. A specific cell of the immune system and found in the lungs, macrophages or “Big Eater” cells, responds first by picking up debris and responding to harmful foreign substances by releasing proteins signaling the immune system to become activated. Within all animal cells, an organelle called the Endoplasmic Reticulum (ER) manufactures a third of proteins produced allowing the cell to adapt to foreign substances, including cigarette smoke. Cigarette smoke could cause the ER, a plastic organelle, to change in size and function at a heightened level due to activation of a sensing protein integrated in the ER, Inositol Requiring Enzyme-1 (IRE-1). Both activation of the ER and cigarette smoke causes macrophages to behave as “tissue-healing” or M2 subsets that release factors promoting reconstruction of the lungs; alternatively, M1 macrophages fight diseases and promote further inflammation. Using genetic analysis of macrophages exposed to cigarette smoke in culture dishes and analyzing the proteins secreted, we determined cigarette smoke inhibits M1 macrophages and the “tissue-healing” subset, while increasing adhesion molecule expression. Overall, cigarette smoke affected the polarization of M1 and M2 phenotype, analyzed through proteins and genes expression. We observed an increase in sXBP-1, indicative of IRE-1/XBP-1 pathway activation, from cigarette smoke extract exposure in macrophages. However, the use of IRE-1 inhibitors increased ER stress markers while affecting M1 and M2 markers. This suggests ER compensation from the use of inhibiting one arm of the ER stress response.

<p> The need for higher bandwidth and people's desire to be "always connected" have spurred a new era of silicon photonics. The traditional integrated electrical transmission lines have been an obstacle preventing ultra high speed communication. Using monolithic chips of integrated optoelectronic circuits from silicon provides an economic way to realize Tetra Byte/Second bandwidth in a variety of areas such as "fiber to the home" and the buses linking chips inside computer.</p> <p> The heart of such optoelectronics-silicon laser-is still in pursuit. One of the most promising approaches is the erbium doped silicon nanocrystals embedded in silica system. External photon or hot electrons injection excites the silicon nanocrystals, which then transfer their energies to nearby erbium ions to emit light at 1.55 μm wavelength range.</p> <p> In this thesis, we investigate the effects of cavity and electric field on energy transfer from Si nanocrystals (Si-nc's) to Er ions, and simulate material gain in such systems. Our results show that microcavity can enhance the Forster energy transfer and material gain, reducing the requirements for Si-nc pumping. The electric field will hinder the radiation decay of Si-nc, but we have to further explore the tunneling mechanism before concluding the overall effect of electric field. Some future work needs to be done, which will shine some light on the design of the silicon laser.</p> / Thesis / Master of Applied Science (MASc)

While the impacts of maternal obesity on placental development have been extensively studied, the role of the father’s health in regulating placentation is less understood. Paternal obesity is associated with offspring metabolic dysfunction, but the mechanism regulating this association is unclear. We investigated how paternal diet-induced obesity impacted placental vascular development, associated cellular stress pathways, and markers of placental endocrine function and macronutrient transport across gestation in a murine model. We found that paternal obesity is associated with placental hypoxia as measured by CAIX and HIF1α at E14.5 which persisted to E18.5. Hypoxia was associated with increased VEGF protein levels, as well as its pro-angiogenic receptor, VEGFR2 in male and female E14.5 placentae, although, this increase was apparent only in females at E18.5. The proportion of placental tissue that was immunopositive for the endothelial cell marker CD31 was increased in female but not male E18.5 placentae. Paternal obesity was associated with cellular stress as measured by the three branches of the unfolded protein response (UPR): ATF6, PERK, and IRE1α. However, despite increased phosphorylation of PERK and IRE1α in placental tissue derived from obese fathers, there was no impact on downstream signal transducers. Pro-apoptotic Bcl2 family members’ transcript levels were reduced at E18.5 in placentae from obese fathers, but this did not correspond to any changes in cleaved casp-3 protein levels. Placental lactogen and macronutrient transporter transcript levels were similar between groups across gestation, although Igf2 transcripts were increased in female placenta from obese fathers at both mid and late gestation. Thus, paternal obesity results in placental hypoxia and VEGF mediated sex specific changes in vascularization with a pro-angiogenic response occurring in females. Future studies will investigate whether paternal obesity impairs early placental implantation, resulting in poor vascularization and hypoxia at E18.5. / Thesis / Master of Science (MSc)

The global prevalence of chronic kidney disease (CKD) has risen at an accelerating rate, increasing the global healthcare burden for long-term and chronic care costs. Multiple risk factors including hypertension, diabetes, and dyslipidemia synergistically induce the progression of CKD. Chief among these factors are dyslipidemia and obesity; increased free fatty acid uptake due to excess consumption of lipid-rich diets has been shown to promote intra-renal lipid accumulation in several in vivo models and in patients in various stages of CKD. Furthermore, patients with renal disease are also at a substantially higher risk for atherosclerotic cardiovascular disease (CVD). In the general population, as well as in patients with renal disease, circulating low-density lipoprotein cholesterol (LDLc) is a well-established driver of atherosclerotic lesion development and CVD progression. In 2003, the proprotein convertase subtilisin/kexin type-9 (PCSK9) was identified as the third locus of familial hypercholesterolemia and was further characterized for its ability to enhance the degradation of the low-density lipoprotein receptor (LDLR). Since this seminal discovery, the development of monoclonal antibodies targeted against PCSK9 demonstrated a significant reduction in LDLc and subsequent CVD risk, establishing the remarkable ‘bench to bedside’ transition. However, the inherent role of PCSK9 in regulating lipid homeostasis remained unknown in different pathological conditions. In the first chapter of my thesis, I demonstrate that PCSK9 regulates the LDLR as a feedback mechanism to protect against non-alcoholic steatohepatitis (NASH) progression induced by a high-fat diet (HFD) challenge. Since its seminal discovery, PCSK9 was also characterized to modulate a wide variety of receptors known to play a crucial role in lipid metabolism including the cluster of differentiation 36 (CD36), the very low-density lipoprotein receptor (VLDLR), and the apolipoprotein E receptor 2 (ApoER2). Previously, we have demonstrated that the absence of PCSK9 promotes diet-induced non-alcoholic steatohepatitis and liver injury through increased surface expression of CD36. Given that these same receptors are well-expressed on renal epithelia, the second chapter of my thesis demonstrates that PCSK9 is also able to modulate renal lipid metabolism by attenuating tubular lipid accumulation and subsequent renal injury. Furthermore, when PCSK9 was first characterized by Seidah and colleagues in 2003, in situ hybridization of murine PCSK9 demonstrated that it was primarily expressed in the liver, but also well-expressed in the kidney cortex, cerebellum, and small intestines. Despite its expression in a wide range of tissues, the secretion of PCSK9 was exclusive to the liver, thus, questioning what the intracellular role of PCSK9 may be. Hence, my last chapter of my masters studies lies in establishing the role of intracellular PCSK9 expression in a cellular process known as endoplasmic reticulum (ER) stress in the kidney. ER stress is a phenomena which primarily occurs due to increased accumulation of misfolded polypeptides, and has been implicated in numerous metabolic diseases including hepatic steatosis, CKD, and neurodegenerative pathologies. Previously, we have demonstrated that overexpressing wild-type and variants of PCSK9 in a Pcsk9-/- mouse does not induce the activation of the unfolded protein response (UPR) and attenuates hepatic ER stress. Using a well-established CKD model, I show that Pcsk9-/- mice exhibit increased renal ER stress and injury relative to wild-type controls. Overall, my findings demonstrate for the first time that both extracellular and intracellular PCSK9 has the ability to modulate renal injury using two distinct mechanism to protect against CKD progression. / Thesis / Master of Health Sciences (MSc)

Mitochondria are required for a diverse array of cellular functions and processes. ATAD3 (ATPase family AAA domain containing protein 3) proteins are newly discovered mitochondrial membrane proteins in Arabidopsis thaliana. Homologous to ATAD3A in metazoans, Co-Immunoprecipitation/Mass spectrometry and genomic analysis identified a four ATAD3A homologues in A. thaliana. The four A. thaliana proteins are referred to as ATAD3A1 (At3g03060), ATAD3A2 (At5g16930), ATAD3B1 (At2g18330), and ATAD3B2 (At4g36580). Studies in metazoans indicate that ATAD3A localizes to Mitochondria-ER contact sites and is involved in a variety of processes required for proper mitochondrial function, but ATAD3A proteins are poorly defined in plants. ATAD3A is a mitochondrial membrane protein with unique topology. It comprises an N-terminal DUF (Domain of unknown function) domain that contains two transmembrane sequences the are inserted or interact with both the inner and outer mitochondrial membranes, two coiled-coil domains thought to help in oligomerization, and a region that is exposed to the cytosol, proposed to interact with the ER. It has a C-terminal AAA domain exposed to the mitochondrial matrix. ATAD3 proteins in A. thaliana have undergone two gene-duplication events, resulting in two clades, both of which are required for plant viability. I created artificial microRNA to knockdown expression of ATAD3A1 in the atad3b1 mutant background to assess the growth and mitochondrial phenotypes and found these plants displayed delayed and deficient growth and deformed mitochondria. I utilized Bi-Molecular Complementation Fluorescence and Laser-Scanning Confocal Microscopy to assess oligomeric patterns of A. thaliana ATAD3 proteins in vivo and discovered that ATAD3 proteins hetero-oligomerize with each other. I also created multiple constructs encoding ATAD3A1 fusion proteins to elucidate the amino acid sequence required to target ATAD3A1 to the mitochondria, and ATAD3A1 fusions with TurboID to identify protein-protein interactions using proximity-based labeling.

This project has been done on the lamp shop named Lampshopen in Falun. The goal of the project has been to create a web application that is designed towards the customer and the administrators. The web application is built with a SPA design which gives the customer the possibility to see the products and have the possibility to book and unbook products given the customer is registered and logged in. The administrators have the possibility to view, add, revise, and delete products and information correlated to the product table. Administrators can also handle categories, other users permission levels and also delete users. The result of the project is a webstore where the demand from the firm is upheld within the projects scope. / Detta arbete har utförts för Lampshopen i Falun. Målet med projektet var att skapa en webbutik som är till för både besökaren och för administratörer. Genom ett SPA ska besökaren kunna se utbudet och ha möjligheten att boka eller avboka produkten givet att hen är registrerad. För administratören ska hen kunna lägga till, revidera eller ta bort produkter samt kunna hantera kategorier och konton på ett lämpligt sätt. Resultatet av projektet är en fungerade webbutik där de efterfrågade kraven uppnåtts.

Plusieurs expériences et études cliniques ont démontré que l’activation du système rénine-angiotensine (RAS) peut induire l’hypertension, un facteur de risque majeur pour les maladies cardiovasculaires et rénales. L’angiotensinogène (Agt) est l’unique substrat du RAS. Cependant, il n’a pas encore été démontré si l’activation du RAS intrarénal peut à elle seule induire des dommages rénaux, indépendamment de l’hypertension systémique, et ainsi jouer un rôle prépondérant dans la progression de la néphropathie diabétique. Afin d’explorer le rôle du RAS intrarénal dans les dommages rénaux, un diabète a été induit par l’injection de streptozotocin chez des souris transgéniques (Tg) surexprimant l’Agt de rat dans les cellules des tubules proximaux du rein (RPTC). Les souris Tg diabétiques ont été traitées soit avec des inhibiteurs du RAS (perindopril et losartan), de l’insuline ou une combinaison des deux pour 4 semaines avant d’être euthanasiées. Pour une autre étude, des souris Tg non-diabétiques ont été traitées soit avec des inhibiteurs du RAS, l’hydralazine (vasodilatateur) ou l’apocynine (inhibiteur de la NADPH oxydase) pour une période de 8 semaines avant l’euthanasie. Des souris non-Tg ont été utilisées comme contrôles. Des cellules immortalisées de tubule proximal de rat (IRPTC) transfectées de manière stable avec un plasmide contenant l’Agt ou un plasmide contrôle ont été employées comme modèle in vitro. Nos résultats ont démontré que les souris Tg présentaient une augmentation significative de la pression systolique, l’albuminurie, l’apoptose des RPTC et l’expression de gènes pro-apoptotiques par rapport aux souris non-Tg. Les mêmes changements ont été observés chez les souris Tg diabétiques par rapport aux souris non-Tg diabétiques. L’insuline et/ou les inhibiteurs du RAS ont permis d’atténuer ces changements, sauf l’hypertension qui n’était réduite que par les inhibiteurs du RAS. Chez les IRPTC transfectées avec l’Agt in vitro, les hautes concentrations de glucose augmentent l’apoptose et l’activité de la caspase-3 par rapport aux cellules contrôles et l’insuline et/ou les inhibiteurs du RAS empêchent ces augmentations. En plus des changements physiologiques, les RPTC des souris Tg présentent aussi une augmentation significative de la production des espèces réactive de l’oxygène (ROS) et de l’activité de la NADPH oxydase, ainsi qu’une augmentation de l’expression du facteur de croissance transformant-beta 1 (TGF-β1), de l’inhibiteur activateur du plasminogène de type 1 (PAI-1), des protéines de la matrice extracellulaire, du collagène de type IV et de la sousunité p47 de la NADPH oxydase. Le traitement des souris Tg avec l’apocynine et le perindopril a permis d’améliorer tous ces changements, sauf l’hypertension qui n’était pas corrigée par l’apocynine. D’autre part, l’hydralazine a prévenu l’hypertension, sans modifier l’albuminurie, l’apoptose des RPTC ou l’expression des gènes pro-apoptotiques. Ces résultats montrent bien que l’activation du RAS intrarénal et l’hyperglycémie agissent de concert pour induire l’albuminurie et l’apoptose des RPTC, indépendamment de l’hypertension systémique. La génération des ROS via l’activation de la NADPH oxydase induit en partie l’action du RAS intrarénal sur l’apoptose des RPTC, la fibrose tubulo-interstitielle et l’albuminurie chez les souris Tg. D’autre part, une expérience en cours a tenté d’encore mieux délimiter les effets de l’activation du RAS intrarénal, tout en éliminant la néphrotoxicité du STZ. Pour cette étude, les souris Tg surexprimant l’Agt de rat dans leurs RPTC ont été croisées aux souris Ins2Akita, un modèle spontané de diabète de type I, afin de générer des souris Akita-rAgt-Tg. Les résultats préliminaires indiquent que le RAS intrarénal est activé dans les souris Akita et que la combinaison avec l’hyperglycémie induit du stress du réticulum endoplasmique (ER) dans les RPTC in vivo. Le stress du ER contribue à l’apoptose des RPTC observée dans le diabète, à tout le moins dans le modèle Akita. Le traitement avec des inhibiteurs du RAS permet d’atténuer certains des dommanges rénaux observés dans les souris Akita-rAgt-Tg. / Experimental and clinical studies have shown that renin-angiotensin system (RAS)activation may lead to hypertension, a major cardiovascular and renal risk factor. Angiotensinogen (Agt) is the sole substrate of the RAS. However, it is unclear whether intrarenal RAS activation alone could induce kidney injury independently of systemic hypertension and play an important role in the progression of diabetic nephropathy (DN). To explore the role of intrarenal RAS in kidney injury, transgenic (Tg) mice overexpressing rat Agt in their renal proximal tubular cells (RPTCs) were rendered diabetic by streptozotocin (STZ). Diabetic Tg mice were treated with RAS blockers (perindopril and losartan), insulin or a combination of both and then euthanized after 4 weeks of treatment. In a separate study, non-diabetic Tg mice were treated with RAS blockers or hydralazine (a vasodilator) or apocynin (an NADPH oxidase inhibitor) and then euthanized after 8 weeks of treatment. Non-Tg littermates served as controls in both studies. Immortalized rat proximal tubule cells (IRPTCs) stably transfected with Agt cDNA or control plasmid were used in the experiments as an in vitro model. Our results showed that non-diabetic Tg mice displayed a significant increase in systolic blood pressure (SBP), albuminuria, RPTC apoptosis, and proapoptotic gene expression. Diabetic Tg mice had a further increase of albuminuria, RPTC apoptosis, and proapoptotic gene expression, though the SBP of the diabetic Tg mice was similar to that of non-diabetic Tg mice. RAS blockers and/or insulin treatments markedly attenuated these changes, except that insulin had no impact on hypertension. In vitro, high-glucose melieu significantly increased apoptosis and caspase-3 activity in Agt stable transfectants compared to control cells, and these changes were attenuated by insulin and/or RAS blockers. Furthermore, non-diabetic Tg mice showed significantly elevated reactive oxygen species (ROS) production and NADPH oxidase activity, as well as enhanced expression of transforming growth factor-beta 1 (TGF-β1), plasminogen activator inhibitor-1 (PAI-1), extracellular matrix proteins, collagen type IV, and NADPH oxidase subunit p47 in their RPTC. Treatment with apocynin and perindopril ameliorated these changes, but apocynin had no effect on SBP. In contrast, hydralazine prevented hypertension but not albuminuria, RPTC apoptosis, or proapoptotic gene expression. These data indicate that intrarenal RAS activation and hyperglycemia act in concert to induce albuminuria and RPTC apoptosis independent of systemic hypertension. ROS generation via NADPH oxidase activation mediates, at least in part, intrarenal RAS action on RPTC apoptosis, tubulointerstitial fibrosis and albuminuria in Tg mice. On the other hand, in an on-going experiment, to avoid the nephro-toxic effects of STZ and further delineate the effects of intrarenal RAS activation, Tg mice overexpressing rat Agt in their RPTCs were crossbred with Ins2Akita mice, a spontaneous type I diabetes model, to generate Akita-rAgt-Tg mice. Preliminary data indicated that hyperglycaemia and intrarenal RAS activation induced endoplasmic reticulum (ER) stress in RPTC in vivo, and the ER stress pathway contributed to RPTC apoptosis in diabetes, at least in the Akita model. RAS blockade was effective in attenuating some parameters of renal injury in AkitarAgt-Tg mice.

A evidência científica da cariologia atual vem apontando para a promoção de saúde bucal, técnicas preventivas, diagnóstico precoce da doença cárie e suas lesões nos elementos dentais, remineralização de lesões de cárie incipientes e a intervenção restauradora célere como procedimentos minimamente invasivos. Durante o ato de remoção de lesão de cárie, é comum remover-se inadvertidamente também tecido dental hígido durante a fase final, por ser um tanto quanto difícil precisar clinicamente os limites entre tecido dental comprometido e tecido dental viável. Usando-se a tecnologia com lasers de Érbio, uma dica clínica subjetiva é a mudança da percepção do som emitido pela ablação do tecido dental ao passar-se de substrato cariado para hígido (de grave para agudo), como uma forma adicional de saber que a ablação naquele ponto deve ser interrompida. Este estudo visa classificar estas diferenças sonoras em dentina e torná-las um parâmetro objetivo para odontologia de mínima intervenção ao usar-se lasers de Érbio. Para tanto, foram realizadas três fases do estudo: primeiramente, utilizaram-se vinte dentes posteriores humanos, sendo dez cariados e dez hígidos. A dentina foi irradiada com laser de Er:YAG sob os mesmos parâmetros, distância e refrigeração e um microfone monodirecional foi posicionado a 10cm da área operatória para captação e gravação dos sons produzidos pela ablação ao se operar tanto em dentina hígida quanto cariada. Dez pulsos por arquivo foram então analisados em um software (200 análises). Foram encontradas diferenças entre os padrões sonoros produzidos dos grupos cariados e hígidos e encontrado um ponto de corte para estas freqüências sonoras, que seria testado a seguir. Em outra segunda etapa, foi desenvolvido, testado e aplicado um software limitador de freqüência sonora, para que, em tempo real, avisasse o operador se o tecido dentinário que estava sendo ablacionado pelo laser de Érbio (Er:YAG e Er,Cr:YSGG) era cariado ou viável, e este evitasse o sobrepreparo, ou seja, desgastar o tecido sadio inadvertidamente, de acordo com os preceitos de mínima intervenção. Finalmente, em uma terceira etapa, o método foi validado através dos critérios visual-tátil e de fluorescência a laser; ilustrações representativas dos espécimes testados foram obtidas através de microscopia de luz e de microscopia eletrônica de varredura. Conclui-se que o som da ablação dentinária é um parâmetro objetivo que pode ser utilizado como um recurso adicional de orientação no processo de remoção de dentina cariada com lasers de Érbio e que a ferramenta desenvolvida para tanto foi efetiva, devendo ser aprimorada. O método foi validado exitosamente pelos critérios propostos. / Studies in cariology have been struggling for the development of health promotion, caries prevention techniques, precocious diagnoses of lesions, re-mineralization of incipient carious lesions and early restorative intervention with minimally invasive procedures. When removing caries lesion, healthy dental structure is often removed inadvertently during its final phase, for being quite difficult to clinically precise the limits between viable and decayed dental tissues. With laser technologies, a subjective clinical hint, often used to indicate when tissue ablation should be stopped is that different sounds are perceptive whether in carious (bass) or in healthy (treble) dental structure; when sound produced by ablation turned treble it would mean that healthy tissue was reached. This study aims to classify those audio differences and to turn them into objective parameters for a conservative operative dentistry with minimally invasive tissue removal when using Erbium lasers. For so, three phases of this study were needed: at first, twenty freshly extracted posterior human teeth were used (10 decayed and 10 sound teeth). Dentine was irradiated with Er:YAG laser under the same parameters, distance and refrigeration and a mono directional microphone was set 10cm far from the operative area in order to capture and record the ablation produced sounds when working either on carious or healthy dentine. Ten pulses per file were then analyzed in a computer software (200 analyses).It was permitted to draw differences between decayed and healthy produced sounds and also to establish a cut-off value for these sound frequencies, that would be tested later on. On a second phase, a piece of software which was able to border the sound frequency was then developed and tested. This tool was meant to warn the operator, in real-time basis, if sound dentine was reached, while it was ablated by Erbium lasers (either Er:YAG or Er,Cr:YSGG) and so one could avoid over-treatment, which means not to remove sound tissue inadvertently, according to minimal intervention dentistry concepts. Finally, on a third part of the study, the proposed guiding method for dentine caries removal was validated through visuo-tactile and laser fluorescence criteria. Representative illustrations of the tested specimens were obtained by light microscopy and scanning electron microscopy. As a conclusion, audio analysis came out to be a technical reliable objective parameter to determine whether laser ablated dentine substrates are decayed or sound; therefore it can be proposed as an additional conservative parameter to guide the clinician during dentine caries removal process, and that the tool developed for so was effective and should be sharpened. This method was successfully validated by the proposed criteria.