Computational Modeling of Facial Response for Detecting Differential Traits in Autism Spectrum Disorders

This dissertation proposes novel computational modeling and computer vision methods for the analysis and discovery of differential traits in subjects with Autism Spectrum Disorders (ASD) using video and three-dimensional (3D) images of face and facial expressions. ASD is a neurodevelopmental disorder that impairs an individual’s nonverbal communication skills. This work studies ASD from the pathophysiology of facial expressions which may manifest atypical responses in the face. State-of-the-art psychophysical studies mostly employ na¨ıve human raters to visually score atypical facial responses of individuals with ASD, which may be subjective, tedious, and error prone. A few quantitative studies use intrusive sensors on the face of the subjects with ASD, which in turn, may inhibit or bias the natural facial responses of these subjects. This dissertation proposes non-intrusive computer vision methods to alleviate these limitations in the investigation for differential traits from the spontaneous facial responses of individuals with ASD. Two IRB-approved psychophysical studies are performed involving two groups of age-matched subjects: one for subjects diagnosed with ASD and the other for subjects who are typically-developing (TD). The facial responses of the subjects are computed from their facial images using the proposed computational models and then statistically analyzed to infer about the differential traits for the group with ASD. A novel computational model is proposed to represent the large volume of 3D facial data in a small pose-invariant Frenet frame-based feature space. The inherent pose-invariant property of the proposed features alleviates the need for an expensive 3D face registration in the pre-processing step. The proposed modeling framework is not only computationally efficient but also offers competitive performance in 3D face and facial expression recognition tasks when compared with that of the state-ofthe-art methods. This computational model is applied in the first experiment to quantify subtle facial muscle response from the geometry of 3D facial data. Results show a statistically significant asymmetry in specific pair of facial muscle activation (p\u3c0.05) for the group with ASD, which suggests the presence of a psychophysical trait (also known as an ’oddity’) in the facial expressions. For the first time in the ASD literature, the facial action coding system (FACS) is employed to classify the spontaneous facial responses based on facial action units (FAUs). Statistical analyses reveal significantly (p\u3c0.01) higher prevalence of smile expression (FAU 12) for the ASD group when compared with the TD group. The high prevalence of smile has co-occurred with significantly averted gaze (p\u3c0.05) in the group with ASD, which is indicative of an impaired reciprocal communication. The metric associated with incongruent facial and visual responses suggests a behavioral biomarker for ASD. The second experiment shows a higher prevalence of mouth frown (FAU 15) and significantly lower correlations between the activation of several FAU pairs (p\u3c0.05) in the group with ASD when compared with the TD group. The proposed computational modeling in this dissertation offers promising biomarkers, which may aid in early detection of subtle ASD-related traits, and thus enable an effective intervention strategy in the future

Comparative Live-Cell Imaging Analyses of SPA-2, BUD-6 and BNI-1 in Neurospora crassa Reveal Novel Features of the Filamentous Fungal Polarisome

A key multiprotein complex involved in regulating the actin cytoskeleton and secretory machinery required for polarized growth in fungi, is the polarisome. Recognized core constituents in budding yeast are the proteins Spa2, Pea2, Aip3/Bud6, and the key effector Bni1. Multicellular fungi display a more complex polarized morphogenesis than yeasts, suggesting that the filamentous fungal polarisome might fulfill additional functions. In this study, we compared the subcellular organization and dynamics of the putative polarisome components BUD-6 and BNI-1 with those of the bona fide polarisome marker SPA-2 at various developmental stages of Neurospora crassa. All three proteins exhibited a yeast-like polarisome configuration during polarized germ tube growth, cell fusion, septal pore plugging and tip repolarization. However, the localization patterns of all three proteins showed spatiotemporally distinct characteristics during the establishment of new polar axes, septum formation and cytokinesis, and maintained hyphal tip growth. Most notably, in vegetative hyphal tips BUD-6 accumulated as a subapical cloud excluded from the Spitzenkörper (Spk), whereas BNI-1 and SPA-2 partially colocalized with the Spk and the tip apex. Novel roles during septal plugging and cytokinesis, connected to the reinitiation of tip growth upon physical injury and conidial maturation, were identified for BUD-6 and BNI-1, respectively. Phenotypic analyses of gene deletion mutants revealed additional functions for BUD-6 and BNI-1 in cell fusion regulation, and the maintenance of Spk integrity. Considered together, our findings reveal novel polarisome-independent functions of BUD-6 and BNI-1 in Neurospora, but also suggest that all three proteins cooperate at plugged septal pores, and their complex arrangement within the apical dome of mature hypha might represent a novel aspect of filamentous fungal polarisome architecture

Bacteria from freshwater ecosystems: structural aspects and programmed cell death

Bacteria are important components of the food web structure in aquatic ecosystems in which they influence the flow of carbon and energy. Populations of bacteria in these ecosystems comprise a diverse spectrum of individual cells able to respond to many factors such as nutrient supply, temperature and virus infection, which regulate bacterial life and death. Bacterial death is a key cellular event involved in the control and production of bacteria in aquatic ecosystems with functional meaning in the carbon and nutrient cycles. Therefore, the study of bacterial structural features and cellular mechanisms underlying bacterial death is crucial to understand processes affecting the entire population. However, both bacterial structure and cellular events of death in aquatic ecosystems are still poorly understood. In the present work, we used single cell approaches to study the structural organization of bacteria as well as to characterize cellular processes of death in these organisms. First, by using fluorescence and transmission electron microscopy (TEM), we provided a general panorama of how microscopy techniques, especially TEM, are powerful tools to understand bacterial structure and their responses to environmental stresses. We showed that bacteria from aquatic ecosystems have remarkable ultrastrutural diversity with components such as bacterial envelope of individual cells differing in structure within the same population. Second, we sought to identify and characterize mechanisms of bacterial cell death. Because our TEM analyses revealed morphological signs of apoptosis, a type of program cell death (PCD), in aquatic bacteria directly collected from natural ecosystems, we applied different techniques to detect apoptosis in bacteria cultured from natural samples. We used TEM as well as different probes to detect this type of PCD in cultured bacteria exposed to increased temperature and viral infection, which are recognized inducers of bacterial death. TEM showed, in both situations, ultrastructural changes indicative of apoptosis, such as cell retraction and condensation, similar to those reported for eukaryotic cells. Assays for membrane permeability, DNA fragmentation, phosphatidilserine exposition and caspase activation were significantly increased in treated bacteria compared to the control group. Altogether, our data demonstrate, for the first time, that PCD occur in aquatic bacteria, and that this event may be a basic mechanism for regulation of bacterial communities in these ecosystems.

Engineering a microwell duct-on-chip technology to translate exocrine pancreatic organoids to a cancer model

Das duktale Adenokarzinom der Bauchspeicheldrüse (PDAC) ist eine der tödlichsten Erkrankungen der exokrinen Bauchspeicheldrüse, für die uns relevante Frühdiagnosemarker fehlen. Um PDAC-Marker zu identifizieren, werden in vitro kultivierte exokrine Pankreasmodelle aus dem frühestmöglichen, präkanzerösen Stadium benötigt. Die Übertragung der Pankreasgang-Differenzierung von humanen pluripotenten Stammzellen (hiPSCs) in in vitro-Krankheitsmodelle erfordert ein umfassendes Verständnis der Entwicklungsbahnen von pankreasspezifischen Zelltypen. In dieser Arbeit wurde eine Microwell-Chip-Technologie mit definierten mikrostrukturierten Strukturen entwickelt, um aus hiPSC differenzierte Vorläuferzellen des Pankreas (PP) in einer 3-dimensionalen Zellkultur zu assemblieren. Die Vorteile der Chip-Plattform sind i) die parallele Bildung von Hunderten gleichgroßer 3D-Zellaggregate, ii) eine Matrigel-freie Mikroumgebung, iii) die Kompatibilität mit hochauflösender Bildgebung, iv) die einfache Anwendbarkeit für verschiedene nachfolgende Analysen mit minimaler Störung und v) die Möglichkeit, Ko-Kulturen zu etablieren. Der Chip wurde verwendet, um in weniger als 6 Stunden tausende von 3D-Zellaggregaten aus etwa 600 PPs zu bilden. In den folgenden 14 Tagen wurden die 3D-PP-Kulturen mit einem definierten Wachstumsfaktorprotokoll in pankreatische dukt-ähnliche Organoide differenziert. Zeitaufgelöste Einzelzell-Transkriptionsprofile und Immunfluoreszenz von gereinigten dukt-ähnlichen Organoiden der Bauchspeicheldrüse zeigten die Entstehung von zwei Arten von duktalen Vorläufern, Zwischenstufen, und reifen duktalen Zellen und wenigen nicht-duktalen Zelltypen. Entsprechende dynamische Transkriptionsstadien wiesen auf definierte Differenzierungsrouten der duktalen Zellen hin, die in zwei entweder CFTR+ oder Mucin+ Subpopulationen resultieren. Diese Subpopulationen wurden bereits in primären Einzelzelltranskriptomen des Pankreas gefunden[4]. Die Integration unseres Einzelzelldatensatzes mit drei primären Pankreasdatensätzen[4-6] zeigte, dass unsere dukt-ähnlichen Zellen zusammen mit primären duktalen Zellen zu den beiden Subpopulationen clustern. Außerdem konnten die Marker der Subpopulationen in einem reanalysierten Primärdatensatz[5] erneut identifiziert und in menschlichem Primärgewebe angefärbt werden. Darüber hinaus wurde die Duct-on-Chip-Plattform genutzt, um Organoid-Ko-Kulturen mit humanen Stellat-Zellen zu etablieren. Als zusätzliche Anwendung ermöglichte die Matrigel-freie Chip-Technologie die Entnahme des Sekretoms und Proteoms der Organoide. In Verbindung mit dem Einzelzell-Transkriptom und der klinischen Validierung ermöglichten uns diese Sekretomstudien die Entdeckung eines beispielhaften frühen PDAC-Marker namens FLNB, welcher sowohl in Biopsien als auch im peripheren Blut von Patienten im Frühstadium nachweisbar ist. Zusammenfassend zeigt diese Arbeit die erfolgreiche Herstellung von Pankreas dukt-ähnlichen Organoiden aus hiPSCs, die ein Reifestadium aufweisen, welches mit dem des fötalen Pankreas vergleichbar ist. Durch die Kombination von zeitaufgelöster Einzelzelltranskriptomik mit verschiedenen Analysemethoden, Sekretomstudien, Proteomstudien und klinischer Validierung auf unserem Microwell-Chip wurde ein patientenspezifisches Duktmodell und ein potenzielles Krebsdiagnoseinstrument entwickelt.Pancreatic ductal adenocarcinoma (PDAC) is one of the most severe diseases of the exocrine pancreas, for which relevant early diagnostic markers are still missing. To identify PDAC biomarkers, experimental models employing in vitro cultivation of exocrine pancreas models require as early as possible precancerous stages. The translation of pancreatic ductal differentiation of human pluripotent stem cells (hiPSCs) into in vitro disease models requires a comprehensive understanding of the developmental trajectories of pancreas-specific cell types. In this study, a microwell chip technology exhibiting defined microstructured patterns to assemble hiPSC-derived pancreatic progenitor cells (PP) into a 3-dimensional cell culture was developed. The advantages of the chip platform are i) the parallel formation of hundreds of equally sized 3D cell aggregates, ii) a Matrigel-free microenvironment, iii) the compatibility with high-resolution imaging, iv) simple applicability for several downstream analyses with minimal perturbation, and v) the possibility to establish co-cultures. The chip was used to generate thousands of 3D cell aggregates from approximately 600 PPs, in less than six hours. For the following 14 days, the 3D PP cultures were differentiated towards pancreatic ductal-like organoids by employing defined growth factor protocols. Time-resolved single-cell transcriptional profiling and immunofluorescence of cleared pancreatic duct-like organoids revealed the emergence of two types of ductal progenitors, intermediates, mature duct-like cells, and a few non-ductal cell types. Corresponding dynamic transcriptional stages indicated defined differentiation routes of duct-like cells, cumulating in two either CFTR+ or mucin+ subpopulations, which have been found before in primary single-cell transcriptomes of the pancreas[4]. The integration of the PDLO single-cell dataset into three primary pancreas datasets[4-6] showed that the duct-like cells clustered together with primary ductal cells into the two subpopulations. Furthermore, the markers of the subpopulations could be reidentified in a reanalyzed primary dataset[5] and subjected to confirmation by immunofluorescence in primary human tissue. Additionally, the duct-on-chip platform was exploited to establish organoid co-cultures with stellate cells. As an additional application, the Matrigel-free chip technology allowed the characterization of secretome and proteome. Together with the single-cell transcriptome and clinical validation, these secretome studies revealed an exemplary early PDAC marker, called FLNB, which is detectable in biopsies and early-stage patients' peripheral blood. In conclusion, this study reports the successful engineering of pancreatic duct-like organoids from hiPSCs, which show a maturation stage comparable to the fetal pancreas. By combining time-resolved single-cell transcriptomics with different analysis methods, secretome, proteome and clinical validation on our microwell chip, a patient-specific duct model and a potential cancer diagnostic tool was developed

Pathways that regulate renal development, fibrosis, and metabolic disease in mouse models

The kidney is an essential organ that maintains homeostasis, maintains water and mineral balance, and removes metabolic waste products from the body. In mammals, the kidney derives from the intermediate mesoderm (IM) and develops through a multistep process where undifferentiated mesenchyme is converted into a highly complex organ. Several transcriptional regulators, including the Pax2 gene, have been identified in the specification and maintenance of this multistep process. The Pax2 gene marks the IM shortly after gastrulation, when the mesoderm becomes compartmentalized into paraxial, intermediate, and lateral plate. Pax2 expression in the IM distinguishes all of the cells fated to become epithelia in the urogenital tract and is necessary to establish and maintain this phenotype. Pax2 null mutants do develop a nephric duct (Brophy et al., 2001; Soofi et al., 2012), but the duct is completely absent in a Pax2/8 double mutant, suggesting that these Pax genes function redundantly in this early IM domain; however, in Pax2 homozygous mutant mice, the metanephric mesenchyme neither responds to inductive signals nor does the mutant mesenchyme aggregate into early renal vesicles resulting in a lack of kidneys, ureters, and genital track. We describe two new alleles of Pax2 created by inserting the Enhanced Green Fluorescent Protein coding region into the 5' untranslated leader sequence. One allele is a hypomorph that generates less protein and exhibits structural defects in kidneys and ureters upon homozygosity. A second allele is a true null that can be used to image Pax2 expressing cells in a mutant background. Organ culture and embryo analyses point to a loss of epithelial cell polarity and increased mobility in cells that have deleted Pax2 function. These experiments provide new insight into the role of Pax2 protein levels in determining correct renal architecture and cell fate. The prevalence of chronic kidney disease (CKD) worldwide is reflected by the increasing number of people with end stage renal disease (ESRD) requiring some form of renal replacement therapy. The overall incidence of ESRD is increasing at an alarming rate and is correlated with the rise of diabetes, obesity, and hypertension. Yet, effective therapies for chronic fibrosis in the kidney and other tissues are still awaited. Among the most extensively studied signaling pathways in renal fibrotic disease are those of the TGFb superfamily (TGFb and BMPs). Given the critical roles for TGFb and BMP proteins in enhancing or suppressing renal interstitial fibrosis, respectively, the results of this thesis will show how the expression of this secreted protein KCP could diminished renal fibrosis in mouse models of chronic and acute kidney disease. In vivo, KCP-KO mice are viable and fertile but are more sensitive to tubular injury and exhibit significant pathology after recovery. Also, deletion of KCP sensitized mice to developing obesity and associated complications such as liver steatosis and glucose intolerance. In contrast, transgenic mice that expressed KCP in the kidney, liver, and brown adipose tissues were resistant to developing high fat diet induced obesity and had significantly reduced white adipose tissue. This data demonstrates that modulation of the TGFβ signaling with secreted inhibitors or enhancers can alter the profile of adipose tissue, which reduces obesity and impaired the progression of metabolic disease. The Metabolic Syndrome is reaching epidemic proportions in the developed world, primarily due to the increased availability of high caloric foods and the decrease in daily physical activity. Energy balance is critical for maintaining normal body weight and homeostasis. When caloric intake chronically exceeds energy expenditure, white adipose tissue stores excess energy in the form of triglycerides, leading to obesity and related complications such as type-2 diabetes, a condition also referred to as metabolic syndrome which is a condition of chronic sub-clinical inflammation. In mice, the TGFβ superfamily has been implicated not only in the development and differentiation of white and brown adipose tissues, but also in the induction of the pro-inflammatory state that accompanies (Tseng et al., 2008). The work outlined in this thesis suggests that altering the TGFβ superfamily signaling pathway by a secreted protein (KCP) can attenuate renal fibrosis and the negative effects of obesity-associated metabolic syndrome. Providing a conceptual basis for the use of small molecule analogues of KCP to attenuate profibrotic pathways that depend on continued TGFβ signaling and/or counteraction by BMPs may potentially provide a novel approach to translating the protective role of specific BMPs (e.g. BMP-7) into clinical benefit

The influence of genome size and limitation of nitrogen and phosphorus on photosynthesis efficiency

Genome size varies 2,400-fold in angiosperms and is an important trait influencing cellular and physiological parameters. One of the major drivers of the astonishing genome size (GS) diversity in angiosperms is polyploidisation and most flowering plant lineages have undergone multiple rounds of polyploidy in their ancestry. Because of the frequency of ancestral polyploidy, one might expect angiosperm genomes to be larger than other eukaryotes, where polyploidy is less frequent. But this is not the case, where GS in angiosperms is skewed towards small genomes, suggesting that, following polyploidy, there is selection over time to reduce GS. It is possible that one selection pressure that acts to reduce the size of the genome is the efficiency of photosynthesis, which may be enhanced in species with small genome sizes. This is because there is a positive correlation between the size of the nucleus and the guard cells across species, which can in turn influence the rate of gas exchange through stomata pores. Photosynthesis may also be influenced by nitrogen (N) and phosphorus (P) availability. These macronutrients are limiting nutrients to plants and play an important role for them, because they are the main constituents of the nucleic acids and they play crucial role in photosynthesis in many processes. Nitrogen is needed to build photosynthetic proteins, but especially for the RuBisCO enzyme and chlorophylls, which are N demanding. Phosphorus is used as ATP and NADPH to give the chemical energy necessary for the fixation of CO2. Both these N and P demands for photosynthesis may compete with the N and P demands of the nucleus, which may be higher in species with large genomes than species with smaller genomes. Thus in considering the selection constraints on genome size in plants it is necessary to consider the effects of GS and nutrient availability on photosynthesis. The overall aim of this PhD project is to determine how GS and nutrient availability impacts photosynthesis. To do that three experimental systems are exploited. These are: (1) The effects of GS on the efficiency of photosynthesis in plant genus Fritillaria, selected because it has particularly large genome sizes, and it has the largest range in genome size, all at the diploid level, for any genus (70 Gb/1C range). These materials enable determination of the impact of GS on cell size, gas exchange and light harvesting properties of photosynthesis. Surprisingly, no effect of GS on cell size 5 was observed, contrary to published expectation, but there was a significant correlation between GS and photosynthesis readings. (2) The effects of GS on the efficiency of photosynthesis in plant genus Nymphaea, selected because it has small genome sizes and polyploidy variants. The polyploid variants enable the effect of step changes in GS associated with polyploidy to be determined. This enables the determination of the impact of polyploidy on photosynthesis and to determine the efficiency of photosynthesis across species in an aquatic plant genus. Exceptionally low non-photochemical quenching (NPQ) was observed in these species, indicative of highly efficient light energy use, perhaps associated with small genome sizes overall and an aquatic habit. There was a relationship between GS and cell size in this genus, despite the range of GS being smaller than for Fritillaria. (3) The effect of nutrient availability and photosynthesis in wheat, selected because of its agricultural importance, its large genome size, and relatives at different ploidy levels with which the data could be compared in future studies. This material enables the effects of nutrient limitation on photosynthesis to be determined, and which components of photosynthesis are most impacted. The results revealed that some components of photosynthesis were significantly impacted by P alone (photochemical quenching (qP, negatively), non-photochemical quenching capacity (NPQ, positively)), others by N alone (maximum rate of carboxylation by RuBisCO (Vcmax, negatively)), whilst both N and P limitation and their interactions reduced biomass. The data show that interactions between photosynthesis, N and P and GS play a role in influencing plant biomass. What we now need to know in future studies is if there are N and P trade-offs between the nucleic acid sink represented by the plant genome and proteins and pigments (chlorophyll) needed for photosynthesis. For example, RuBisCO, essential for the dark reaction of photosynthesis, is likely to compete with the nucleic acid sinks for N, whilst metabolic processes, which require for example ATP, NADPH or protein phosphorylation, are likely to compete with the nucleic acid sinks for P