<p>The intention is to draw more specific connections between certain deconvolution methods and also to demonstrate the application of the statistical theory of estimation in the presence of measurement error. A parametric methodology for deconvolution when the underlying distribution is of the Pareto form is developed. Maximum likelihood estimation (MLE) of the parameters of the convolved distributions is considered. Standard errors of the estimated parameters are calculated from the inverse Fisher&rsquo / s information matrix and a jackknife method. Probability-probability (P-P) plots and Kolmogorov-Smirnov (K-S) goodnessof- fit tests are used to evaluate the fit of the posited distribution. A bootstrapping method is used to calculate the critical values of the K-S test statistic, which are not available.</p>

Infrared absorption spectra can provide analytically useful information on a large variety of compounds, ranging from small ions to large biological molecules. In fact, all analytes that possess a dipole moment that changes during vibration are infrared-active. The infrared (IR) spectrum can be subdivided into far-, mid- and near- regions. The focus of attention in this thesis is the mid-IR region, in which the fundamental vibrations of most organic compounds are located, thus providing scope for positive structural identification. However, while such near-ubiquitous signals can be very useful for monitoring simple molecules in simple systems, they can be increasingly disadvantageous as the number of analytes and/or the complexity of the sample matrix increases. Thus, hyphenation to a separation system prior to detection is desirable. Paper I appended to this thesis presents (for the first time) the on-line hyphenation between Fourier transform infrared spectroscopy, FTIR, and capillary zone electrophoresis, CZE. CZE is a highly efficient separation technique that separates ionic analytes with respect to their charge-to-size ratio. It is most commonly performed in aqueous buffers in fused silica capillaries. Since these capillaries absorb virtually all infrared light an IR-transparent flow cell had to be developed. In further studies (Paper II) the applicability of CZE is expanded to include neutral analytes by the addition of micelles to the buffer, and micellar electrokinetic chromatography, MEKC, was successfully hyphenated to FTIR for the first time. Paper III describes an application of the on-line CZE-FTIR technique in which non-UV-absorbing analytes in a complex matrix were separated, identified and quantified in one run. Measuring aqueous solutions in the mid-IR region is not straightforward since water absorbs intensely in this region, sometimes completely, leaving no transmitted, detectable light. For this reason, quantum cascade lasers are interesting. These lasers represent a new type of mid-IR semiconducting lasers with high output power due to their ingenious design. The laser action lies within one conduction band (intersubband) and can be tailored to emit light in the entire mid-IR region using the same semiconducting material. To investigate their potential to increase the optical path length in aqueous solutions, these lasers were used with an aqueous flow system (Paper IV), and the experience gained in these experiments enabled hyphenation of such lasers to a CZE system (Paper V).

Maraging steels have got high strength and good toughness. Maraging steel MDN 250A grade is used for slat tracks on the leading edge of the wing of Light Combat Aircraft (LCA). This application requires high strength and good wear resistance. Required wear resistance for this application can be achieved by ion-nitriding (also called as Plasma nitriding or Glow discharge nitriding) process of case hardening. In conventional gas nitriding process the nitriding temperature is 500 – 550 0C, which is above the aging temperature of maraging steel. Ion-nitriding at temperatures lower than 480 0C is thought to be suitable for surface hardening for application of this material in slat tracks of the leading edge of the wing of LCA. Besides this ion-nitriding is stated to improve fatigue & wear resistance properties without significantly affecting other properties. Also this is a process where the thickness requirement is minimum for the wear properties accomplished. Further, no distortion, warpage and such other dimensional inhomogeneity problems are encountered. In ion-nitriding method glow discharge technology is used to introduce nascent (elemental) nitrogen to the surface of a metal part for subsequent diffusion into the material. The plasma assisted surface modification techniques offer a great flexibility and are capable in tailoring desirable chemical and structural surface properties independent of the bulk properties. It has other advantages like nil or very thin white layer after nitriding and there is no machining or grinding involved for complex parts after the process. The hardened surface layers become an integral part of the basic material without any significant reduction in the properties of base material. Aim of the present work is to study ion-nitriding process on Maraging steels, to carry out ion nitriding of Maraging steel MDN 250A grade at different temperatures and evaluation of properties. Presently, the ion-nitrided slat tracks have been cleared for limited usage instead of full expected life. Studies are also needed to understand the fatigue behavior upon nitriding. The main aspects considered in this study are, optimization of thickness of the ion-nitrided layer and properties required for aircraft applications like slat tracks on the leading edge of the wing where wear resistance, corrosion resistance, mechanical properties like tensile strength, impact strength and fatigue strength are important. To establish the variation in the basic properties of maraging steel (MDN 250A Grade) because of ion nitriding. Aging of Maraging steel is done at 480 ± 5 0C with very close temperature control. In industrial ion-nitriding furnaces the temperature control is of the order of ± 10 0C. As a part of this research work ion-nitriding at three different temperatures which are well below 480 0C were chosen. These temperatures are 435 0C, 450 0C and 465 0C and soaking time i.e., ion-nitriding duration is 10 hours. Microstructure is characterized using optical microscope, scanning electron microscope and X-ray Diffraction. Properties evaluated include Hardness, Case depth, Tensile, Low Cycle Fatigue, Impact and Corrosion by Salt Spray. Optical microscopy and SEM examination don’t show change in core microstructure of the material after ion-nitriding. The iron compound formed in the hardened layer is Fe4N for all the three nitriding temperatures. Ion-nitrided specimens exhibit higher surface hardness and good case depth. Salt spray test results shows that there is animprovement in corrosion resistance after nitriding. The nitrided samples shown higher UTS and 0.2 % PS values than un nitrided ones, where as % Elongation and % Reduction in Area are lower for nitrided samples as compared to un nitrided samples. This can be attributed to the surface layer getting hardened because of ion nitriding. Impact strength of nitrided samples is lower compared to un nitrided samples. This is again due to the surface layer getting hardened because of ion nitriding. Low cycle fatigue properties obtained on ion-nitrided samples show improvement in the fatigue strength due to introduction of the residual compressive stresses. The mechanical properties satisfy the design requirements. Thus ion-nitriding of this Maraging steel (MDN 250A Grade) is suitable for parts which are subjected to constant wear and are complex in shape.

Šio darbo tikslas yra ištirti mažų koncentracijų matavimų vandeniniuose tirpaluose tikslumą, ypač monoizotopiniams elementams, taikant Element2 indukciniu būdu kuriamos plazmos masių spektrometrijos metodą, nustatyti pagrindines matavimų tikslumą ribojančias priežastis ir pabandyti sumažinti jų įtaką, įvertinti tokių matavimų taikymo galimybes medžiagų kilmės atpažinimui. / High resolution sector field ICP MS measurements of concentrations are widely used in modern analytical chemistry and applications. Two problems are concerned in the present work: improvement of accuracy of measurements of concentration of monoisotopic elements (As) and analysis of applicability of the multielement analytical data for classification of samples according to their origin. 66Zn, 77Se and 94Mo were used as internal standards to measure the concentration of As in water by the method of additions. Application of 77Se as internal standard resulted in the best accuracy of analysis. Drift of the measured signal ratios was identified as the main source of uncertainty limiting the accuracy of the mass fraction measurements at relative expanded uncertainty of 2% (k=2). The information as well as the level of inherent noise characterizes the quality of application of the multivariate analysis of multielement data for classification of samples. The role of noise increases if large number of parameters is needed. Stepwise approach is tested dividing the wine origin classification test into some steps to simplify the problem. Outcomes of the approach in comparison to the straightforward test are studied on the basis of the chemical analytical data obtained for 25 elements in 65 wines from 8 countries. Bivariate Gaussian distribution is used to evaluate the reproducibility of the discrimination between countries of origin and reliability of classification of the samples. In... [to full text]

The extent of devastation caused by a biological warfare attack is highly correlated to the time from release to detection. As a step towards lowering the detection time the international project TWOBIAS was launched. Here, the main goal is to develop an automated, specific and sensitive combined detection and identification instrument capable of identifying a biological threat within an hour. The identification unit is comprised of a sample preparation module, an amplification module and a detection module and utilizes a proximity ligation assay in combination with circle-to-circle amplification in order to detect a biological threat. This thesis describes the automation of the sample preparation steps of the assay and the integration with the downstream units. The functionality of the sample preparation module was verified by subjecting it to biological samples in a laboratory and at a real-life location. The results showed that the sample preparation module was capable of preparing a sample collected in a complex environment with the same results as a sample prepared in a laboratory.

<p>The intention is to draw more specific connections between certain deconvolution methods and also to demonstrate the application of the statistical theory of estimation in the presence of measurement error. A parametric methodology for deconvolution when the underlying distribution is of the Pareto form is developed. Maximum likelihood estimation (MLE) of the parameters of the convolved distributions is considered. Standard errors of the estimated parameters are calculated from the inverse Fisher&rsquo / s information matrix and a jackknife method. Probability-probability (P-P) plots and Kolmogorov-Smirnov (K-S) goodnessof- fit tests are used to evaluate the fit of the posited distribution. A bootstrapping method is used to calculate the critical values of the K-S test statistic, which are not available.</p>

<p>In South Africa, it is estimated that approximately 70% of the population frequently make use of traditional medicinal plants for their health care needs. The use of Lessertia frutescens by the&nbsp / various cultural groups in South Africa dates back to the earlier civilizations and continues to be used today to treat a multitude of ailments. To get the best results from a medicinal plant, one&nbsp / &nbsp / would need to ensure that the crude material is of good quality through interventions like being properly grown, well dried and correctly processed. This would add a measure of quality&nbsp / assurance, which will contribute towards the safety and efficacy aspect of herbal medicine. The aim of this study was to investigate what impact a particular season of harvest and the time in&nbsp / storage would have on the flavonoid and triterpenoid marker levels of Lessertia frutescens. To achieve this, the following was investigated: (1) storage variation of Lessertia frutescens leaves&nbsp / by comparing the results obtained from the High Performance Liquid Chromatography (HPLC) analysis of the flavonoids and triterpenoids, (2) seasonal variation of Lessertia frutescens&nbsp / leaves by comparing the results obtained from the HPLC analysis of the flavonoids and triterpenoids, (3) leaf and stem variation of Lessertia frutescens by comparing the results obtained from HPLC analysis of the flavonoids and triterpenoids. The hypotheses were: (1) the stored sample would indicate the same level of the biomarkers for the flavonoids and triterpenoids, as that of&nbsp / the freshly prepared sample, (2) the sample that was harvested during the summer season would indicate higher levels of the biomarkers of&nbsp / flavonoids and triterpenoids than the other three&nbsp / seasons, (3) the leaf sample would indicate the same level of the biomarkers for the flavonoids and triterpenoids, as that of the stem sample. An Agilent 1200 series HPLC was used for the&nbsp / determination of the flavonoids sutherlandin A and sutherlandin D as well as the triterpenoids sutherlandioside B and sutherlandioside D. Results show that for both sutherlandin A (summer:&nbsp / 3.67 &plusmn / 2.88 mg/ml / storage: 4.07 &plusmn / 2.88 mg/ml) and D (summer: 4.10 &plusmn / 1.06 mg/ml / storage: 4.25 &plusmn / 1.06 mg/ml) show significantly (P &lt / 0.0001) higher concentrations in the case of the storage&nbsp / amples. For both sutherlandioside B (summer: 3.01 &plusmn / 0.39 mg/ml / storage: 2.82 &plusmn / 0.39 mg/ml) and D (summer: 5.82 &plusmn / 0.42 mg/ml / storage: 4.66 &plusmn / 0.42 mg/ml) show significantly (P &lt / &nbsp / .0001)&nbsp / higher concentrations in the case of the fresh summer samples.For the seasonal comparison, results show that for sutherlandin A (summer: 3.67 &plusmn / 12.49 mg/ml / autumn: 4.75 &plusmn / &nbsp / 12.49 mg/ml / winter: 4.23 &plusmn / 12.49 mg/ml / spring: 6.56 &plusmn / 12.49 mg/ml) show significantly (P &lt / 0.0001) higher concentrations in the case of the spring sample. For sutherlandin D (summer: 4.10&nbsp / &nbsp / 10.32 mg/ml / autumn: 6.37 &plusmn / 10.32 mg/ml / winter: 5.25 &plusmn / 10.32 mg/ml / spring / 6.08 &plusmn / 10.32 mg/ml) show significantly (P &lt / 0.0001) higher concentrations in the case of the autumn sample. For both sutherlandioside B (summer: 3.01 &plusmn / 7.19 mg/ml / autumn: 2.15 &plusmn / 7.19 mg/ml / winter: 2.89 &plusmn / 7.19 mg/ml / spring: 1.47 &plusmn / 7.19 mg/ml) and D (summer: 5.82 &plusmn / 14.48 mg/ml / autumn: 3.33 &plusmn / 14.48 mg/ml / winter: 4.23 &plusmn / 14.48 mg/ml / spring: 2.50 &plusmn / 14.48 mg/ml) show significantly (P &lt / 0.0001) higher concentrations in the case of the autumn sample. For the summer&nbsp / leaf/stem comparison, results show that for sutherlandin A (leaf: 3.67 &plusmn / 8.18 mg/ml / stem: 4.67 &plusmn / 8.18 mg/ml) show significantly (P &lt / 0.0001) higher concentrations in the case of the stem&nbsp / sample. For the sutherlandin D (leaf: 4.10 &plusmn / 4.81 mg/ml / stem: 3.31 &plusmn / 4.81 mg/ml) show significantly (P &lt / 0.0001) higher concentrations in the case of the summer leaf sample. For both the&nbsp / sutherlandioside B (leaf: 3.01 &plusmn / 4.24 mg/ml / stem: 3.62 &plusmn / 4.24 mg/ml) and D (leaf: 5.82 &plusmn / 0.42 mg/ml / stem: 5.80 &plusmn / 0.42 mg/ml) show significantly (P &lt / 0.0001) higher concentrations in the&nbsp / case of the stem samples. Results demonstrate that the production of secondary metabolites are influenced by&nbsp / &nbsp / environmental factors like seasonal harvesting, as indicated by the variation in the chemical constituent composition of Lessertia frutescens depending on the season collected in. Moreover, the storage of Lessertia frutescens for a period of one year resulted in an&nbsp / increase of two of the four constituents being monitored. There was slight variations in the chemical constituents, depending on whether the leaf or stem material of Lessertia frutescens was being used. Finally, the type of chemical constituent being monitored was also important in the consideration of this study. Therefore, this study can be seen as a starting point to further&nbsp / &nbsp / investigations of these aspects, which are of clinical, pharmacological and economic</p>

Outcome dependent sampling may increase efficiency in observational studies. It is however not always obvious how to sample efficiently, and how to analyze the resulting data without introducing bias. This thesis describes a general framework for efficiency calculations in multistage sampling, with focus on what is sometimes referred to as ascertainment sampling. A method for correcting for the sampling scheme in analysis of ascertainment samples is also presented. Simulation based methods are used to overcome computational issues in both efficiency calculations and analysis of data. / At the time of doctoral defense, the following paper was unpublished and had a status as follows: Paper 1: Submitted.

Submitted by ANA CAROLINE SONCIN DA SILVA null (col_zinha@hotmail.com) on 2016-05-21T03:08:22Z No. of bitstreams: 1 Dissertação_Mestrado.pdf: 2242269 bytes, checksum: d87570a91a3ceb91484c2ca2920007e1 (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-05-24T13:46:03Z (GMT) No. of bitstreams: 1 silva_acs_me_ilha.pdf: 2242269 bytes, checksum: d87570a91a3ceb91484c2ca2920007e1 (MD5) / Made available in DSpace on 2016-05-24T13:46:03Z (GMT). No. of bitstreams: 1 silva_acs_me_ilha.pdf: 2242269 bytes, checksum: d87570a91a3ceb91484c2ca2920007e1 (MD5) Previous issue date: 2016-03-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O mexilhão dourado (Limnoperna fortunei) tem provocado transtornos ambientais e econômicos nas localidades onde foi introduzido. Dessa forma, o uso do seu resíduo moído como reparador da defasagem de nutrientes e de acidez em solos agrícolas pode vir a ser uma boa aplicação para esse molusco bioinvasor. Entretanto, apesar dessa ação reparadora, é necessária uma averiguação prévia da ausência de elementos potencialmente tóxicos no resíduo. Neste contexto, o presente trabalho foi realizado com o intuito de quantificar, em amostras do Limnoperna fortunei, os elementos chumbo, cádmio, alumínio, ferro e manganês, os quais dependo de suas quantidades podem ser tóxicos aos vegetais e aos demais seres vivos ao longo da cadeia alimentar. Para quantificação desses analitos foi utilizada a Espectrometria de absorção atômica com fonte contínua e de alta resolução. Foram desenvolvidos e otimizados programas de aquecimento para a determinação dos elementos traço chumbo e cádmio, empregando amostragem direta de sólidos e atomização com forno de grafite. A determinação direta de chumbo só foi possível com a utilização da calibração com o material de referência certificado para tecido de mexilhão (Mussel Tissue – 2976), empregando 5 µL da mistura 0,1% (m/v) Pd (NO3)2 + 0,05% (m/v) Mg (NO3)2 em presença de 0,05% (m/v) de Triton X-100 como modificador químico. As temperaturas de pirólise e atomização foram 1000°C e 2200°C, respectivamente. O estudo de massa permitiu a utilização de massas de amostras dentro da faixa de 0,150 a 0,850 mg. Para o cádmio só foi realizada calibração com solução padrão 12,5 µg/L, também com o uso de 5 µL do modificador químico Pd+Mg. Assim, foram alcançadas temperaturas de pirólise de 900 °C e de 1600 °C para a atomização. O estudo de massa precisou ser desenvolvido com uma das amostras coletadas e demonstrou que puderam ser usadas, para as quantificações, massas próximas a 0,250 mg. Os limites de detecção e quantificação foram, nessa ordem, 0,009 mg/kg e 0,03 mg/kg para o chumbo, e 0,0004 e 0,001 mg/kg para o cádmio. No caso dos elementos alumínio, ferro e manganês, as quantificações foram realizadas utilizando a atomização com chama. Os limites de detecção e quantificação foram, respectivamente, 0,15 mg/kg e 0,50 mg/kg para o alumínio, 0,050 mg/kg e 0,15 mg/kg para o ferro, e 0,00067 mg/kg e 0,0023 mg/kg para o manganês. Os resultados para todos os elementos quantificados nesse estudo apresentaram concordância de 95% com o test-t (Student) não pareado, o que valida e viabiliza os métodos desenvolvidos para as quantificações de chumbo e cádmio, bem como comprova a exatidão dos demais resultados obtidos. Portanto, as técnicas analíticas selecionadas foram eficientes para as quantificações dos elementos desejados no resíduo moído desse mexilhão dourado. / The golden mussel (Limnoperna fortunei) has caused environmental and economic disruption in the locations where it was introduced. Thus, the use of their ground waste as repairer of the gap of nutrients and acidity in soils may prove to be a good application for this mollusk bio invader. However, despite of this remedial action, a prior test of the absence of potentially toxic elements in the residue is required. In this context, the present study was performed in order to quantify, in samples of the Limnoperna fortunei, the elements lead, cadmium, aluminum, iron and manganese, which depending on their quantities they can be toxic to plants and other living beings throughout the food chain. To quantify these analytes was used high-resolution continuum source atomic absorption spectrometry. Heating programs were developed and optimized for the determination of lead and cadmium trace elements, using direct solid and graphite furnace atomization. The direct determination of lead was only possible with the use of calibration with certified reference material for mussel tissue (Mussel Tissue - 2976), using 5 uL of the mixture 0.1% (m/v) Pd (NO3)2 + 0.05% (w/v) Mg (NO3)2 in the presence of 0.05% (w/v) Triton X-100 as a chemical modifier. The temperatures of pyrolysis and atomization were 1000 ° C and 2200 ° C respectively. The mass study allowed the use of samples of mass within the range of 0.150 to 0.850 mg. For cadmium it was only performed calibration with standard 12.5 g/L solution, also using 5 uL chemical modifier Pd + Mg. Thus, pyrolysis temperatures were reached 900 ° C and 1600 ° C for atomization. The mass study needed to be developed with one of the samples collected and it showed that they could be used for the measurements, masses close to 0,250 mg. The limits of detection and quantification were, in this order, 0.009 mg/kg and 0.03 mg/kg to lead, and 0.0004 and 0.001 mg/kg to cadmium. In case of the elements aluminum, iron and manganese, the measurements were carried out using atomization with a flame. The limits of detection and quantification were, respectively, 0.15 mg/kg and 0.50 mg/kg to aluminum, 0.050 mg/kg and 0.15 mg/kg for iron, and 0.00067 mg/kg and 0.0023 mg/kg to manganese. The results for all quantified elements of this study showed agreement of 95% with the t-test (Student) unpaired, which validates and enables the methods developed for lead and cadmium quantifications and proves the accuracy of the other results. Therefore, the selected analytical techniques were efficient to the quantifications of the desired elements in the ground waste this golden mussel.

Spermatogenesis is a complex and coordinated process of formation of sperms from the precursor spermatogonia, occurring inside the unique environment existing in the seminiferous epithelium. This process of development, characterized by concomitant changes in the cellular morphology, metabolism and differential gene expression, can be divided into 3 distinct phases: i) proliferation of the spermatogonia through mitosis; ii) meiosis or reduction division, which commences with transformation of the type B spermatogonia into primary spermatocytes and their subsequent entry into the meiotic prophase I. These primary spermatocytes, divide to form secondary spermatocytes, and then divide again to form haploid round spermatids; (iii) spermiogenesis or differentiation and maturation of the round spermatids without further division to form the unique spermatozoa (Kerr and De Kretser, 2006, Clermont, 1966, Heller and Clermont, 1964). This complex process of division and differentiation is regulated at three distinct levels: i) The extrinsic level where the gonadotropins and testosterone regulate gene expression in the germ cells sustaining their survival and differentiation (French, 2012); ii) The interactive regulation that involves interactions between the somatic cells such as the Sertoli cells and the germ cells; iii) The intrinsic gene expression associated with each step of development of the germ cells (Eddy, 2002) wherein each stage of differentiation is accompanied by precise stage-specific differential gene expression. (Kleene, 1996, Kierszenbaum et al., 2003, Sassone-Corsi, 2002, Kleene, 2001, Sassone-Corsi, 1997). Any alterations in this gene expression pattern leads to disruption and/or arrest of spermatogenesis at various stages, causing male infertility (Zorrilla and Yatsenko, 2013, Krausz et al., 2015). Many studies have been focused on investigating the underlying molecular mechanisms governing the process of germ cell development such as self-renewal, meiotic recombination and differentiation (Hecht, 1998, Grootegoed et al., 2000, Robles et al., 2017). Analysis of differential gene expression in isolated and purified populations of different germ cells have been very useful in the understanding of the genetic regulation of human spermatogenesis by providing information about the cell type-specific gene expression and regulation. (Meistrich et al., 1973, Bellvé, 1993, Meistrich et al., 1981, Chalmel et al., 2007). However, these methods are limited by the large amounts of tissue required, which is difficult to obtain in the case of humans (Schultz et al., 2003). Large-scale gene expression studies and the “omics revolution” have also helped in identifying some of the regulators of spermatogenesis (Carrell et al., 2016). In spite of advances in the current understanding of the regulation of spermatogenesis, the exact molecular mechanisms of how the genetic and epigenetic alterations affect human spermatogenesis are still unclear (Neto et al., 2016). The present study is an attempt to investigate the human testicular gene expression pattern in the germ cells of patients with various types of azoospermia, and correlate the same to infertility. Comparative analysis of the testicular transcriptomes of infertile individuals (with arrested spermatogenesis) with the control, fertile individuals (with normal spermatogenesis) would allow identification of the cell type-specific altered genes. Analysis of these genes would provide an insight into the genetic regulation of the progress of spermatogenesis as well as allow identification of the crucial genes responsible for the arrest. The first step in this study was to ascertain the exact status of spermatogenesis in patients’ testes. Forty-four azoospermic patients were classified clinically into two major groups – obstructive (OA) and non-obstructive (NOA) azoospermia and further classified using flow cytometric analysis of the germ cells. The patients with OA exhibited presence of the diploid, tetraploid and haploid cells indicating complete spermatogenesis (Group I: DTH). The patients with NOA showed incomplete spermatogenesis with arrest at either the meiotic stage showing the presence of diploid and tetraploid cells, but not the haploid cells (Group II: DT), or at the pre-meiotic stage with only diploid cells (Group III: D). This was further verified by RT-PCR analyses for specific markers for different testicular cells. The Group I patients showed expression of markers specific for the Leydig cell (LHCGR, HSD3B2 and HSD17B3), the Sertoli cell (FSHR, KITL), spermatogonia (KIT), tetraploid cells (CCNA1, LDHC) and haploid cells (PRM1). The Group II patients showed expression of CCNA1 and LDHC, but not of PRM1. The Group III patients did not express any of the haploid or tetraploid specific markers. The germ cell pattern was further confirmed by histology where a clear difference was seen across the groups in accordance with their flow cytometric profiles. Subsequent to grouping of the patient samples based on their testicular germ-cell pattern, microarray analysis was carried out with representative samples from each group leading to identification of diploid-/tetraploid-/haploid-specific (D/T/H) genes. The enrichment, probable pathways and network interactions of these identified genes were analyzed and found to be in agreement with the classification made in this study. Further, based on their network interactions, the genes that were under multiple modes of regulation and the transcription factors that regulated multiple pathways were selected for further analysis. In absence of an in-vitro system to study germ cell differentiation, the importance of the selected genes in the progression of human spermatogenesis was analyzed from the data extrapolated from information available in the literature about expression of each gene in the human testes (wherever available), known function of the genes in various somatic cells, function in developing and adult testes of model organisms and the data from the knockout or transgenic animals where disruption of the gene/s resulted in an arrest or disruption of spermatogenesis. Expression of all the putative crucial genes was analyzed in all the patients including the control patients at the transcript level and three selected genes (one from each group- D, T and H) were further validated at the protein level using immunohistochemistry. All the genes showed a similar pattern of amplification in the different groups of patients to the pattern observed from the microarray. The diploid-specific genes (selected based on the available literature) were mainly the inhibitors or regulators of the cell cycle (CDKN1A, GADD45A, FOXM1) (Xiong et al., 1991, Jin et al., 2002, Laoukili et al., 2005) and regulators of cellular proliferation (KLFs, FOS, SRF, ATFs, SMADs) (Garrett-Sinha et al., 1996, Persengiev and Green, 2003, Angel and Karin, 1991, Ten Dijke et al., 2002). Six diploid-specific genes that were potential regulators of spermatogenesis were identified to be probable causes for the arrest of spermatogenesis at the pre-meiotic stage. CDKN1A showed elevated expression at the transcript level which suggested that DNA-damage induced proliferation check (mediated through CDKN1A) in the diploid cells probably prevented these cells from entering meiosis. This was further verified at the protein level by immuno-staining for CDKN1A. Further, GADD45A, KLF4, FOS, MCL1 and SERPINE1 were identified as genes crucial for transition from the diploid to the tetraploid stage and their aberrant expression correlated to the arrest of spermatogenesis in the Group D patients. Six tetraploid-specific genes and four haploid-specific genes were identified to be potential regulators of the tetraploid-haploid transition and responsible for the meiotic arrest. Over expression of the pro-inflammatory genes such as CCL3, IL1B and IL8 (Guazzone et al., 2009) was seen in the testis of the arrested patients which suggested that there was a potential alteration of the normal testicular micro-environment. Expression of EGR2 (a spermatogonial-maintenance gene controlling mitosis (Joseph et al., 1988)) was seen in the nucleus of spermatocytes in group DT patients which indicated its role in the meiotic arrest. To understand the role of the haploid-specific genes in the context of spermatocyte differentiation, only those genes whose expression are reported in the spermatocytes and persist till the spermatid stage were selected. Lack of expression of CST8 was identified to be potentially responsible for loss of germ cell integrity, and the loss of GGN expression in the Group DT patients seemed to be a significant contributor to the genotoxic stress in these patients. In the arrested patients RFX2 (reported to be master regulator of spermiogenesis (Wu et al., 2016)) was seen to be down regulated at the transcript level which indicated its role in the control of meiosis. This was further confirmed by IHC, where expression of RFX2 was seen to be present in the tetraploid cells of the Group DTH patients while no expression was seen in the tetraploid cells of Group DT patients. Thus, this study identified a role for RFX2 in the regulation of meiosis in humans, similar to the findings reported in rats (Horvath et al., 2009). The study also identified autophagy as a mechanism for the clearance of the arrested cells in NOA patients. IHC data using αLC3B showed that autophagy was up regulated in the arrested patients as compared to the Group DTH patients suggesting its role in cell survival and recycling of nutrients. Further, in-situ TUNEL labeling of tissue sections from the different groups (DTH, DT and D) revealed that there were no difference in the status of apoptosis in the azoospermic patients. The latter observation further corroborated with the elevated expressions of CDKN1A, GADD45A, MCL1, TNFAIP3 (reported to ensure cell survival by negatively modulating apoptosis) as seen in the NOA patients. In conclusion, this study identifies several genes that control the progression of spermatogenesis, including the genes whose alterations contribute towards an arrest in spermatogenesis, especially in azoospermia. These identified genes may be used as novel markers in the diagnosis of male infertility. The study opens up the possibility of using the identified genes as future therapeutic targets using small molecular regulators for treatment of infertility as well as targets for male contraception. The study also identifies a novel role for autophagy in patients with NOA which opens up new avenues for further investigation. Thus, this study is the beginning of understanding the complex events that regulate spermatogenesis.